K - P Realtime qRT - PCR revealed the expression profiles of four zebrafish chs genes in different tissues ( K ) , different gut sections ( L ) , and different developmental stages ( M - P ) . One representative example of three repeat experiments is shown . Data were the mean ± SEM of n = 3 technical replicates . n . s . no significant , no label P < 0 . 05 by student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K-P Realtime qRT-PCR revealed the expression profiles of four zebrafish chs genes in different tissues (K), different gut sections (L), and different developmental stages (M-P). One representative example of three repeat experiments is shown. Data were the mean ± SEM of n=3 technical replicates. n.s. no significant, no label P < 0.05 by student's t-test.


F - M Compared with wild - types ( F - I ) , mutants ( J - M ) completely lost the CM ( red ) in the gut of 7dpf larva ( F , J ) , as well as in the intestinal bulb ( G , K ) , middle intestine ( H , L ) and posterior intestine ( I , M ) of adults . Red , SNAP - CBD stain ; blue , DAPI stain ; WT , wild - type ; MU , chs1 - / - mutant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O

F-M Compared with wild-types (F-I), mutants (J-M) completely lost the CM (red) in the gut of 7dpf larva (F, J), as well as in the intestinal bulb (G, K), middle intestine (H, L) and posterior intestine (I, M) of adults. Red, SNAP-CBD stain; blue, DAPI stain; WT, wild-type; MU, chs1-/- mutant.


A , B The survival rates of wild - types and mutants in the first eight days ( A ) and the first six months ( B ) . Two independent batches ( 1 and 2 ) were conducted for each experiment . n , the initial numbers of zebrafish . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by long - rank ( Mantel - Cox ) test . Please see Table EV1 for data on another three batches of independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, B The survival rates of wild-types and mutants in the first eight days (A) and the first six months (B). Two independent batches (1 and 2) were conducted for each experiment. n, the initial numbers of zebrafish. * P < 0.05, ** P < 0.01, *** P < 0.001 by long-rank (Mantel-Cox) test. Please see Table EV1 for data on another three batches of independent experiments.


E - J 6 / 7 dpf larva mutants were fed with 6 - μm polystyrene beads . The beads jammed in the intestinal bulb and middle intestine ( E , F , H , I ) . In some cases , the beads escaped the bead cord and stuck between villi ( white triangles ) ( E , G , I , J ) . Blue rectangle indicates the intestinal bulb . Right panels were magnification of the red rectangle area in left panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-J 6/7 dpf larva mutants were fed with 6-μm polystyrene beads. The beads jammed in the intestinal bulb and middle intestine (E, F, H, I). In some cases, the beads escaped the bead cord and stuck between villi (white triangles) (E, G, I, J). Blue rectangle indicates the intestinal bulb. Right panels were magnification of the red rectangle area in left panels.


B Comparison of 3 - month - old wild - types and mutants in the pairwise microbiota compositional dissimilarity ( Jaccard distance ) between the digesta ( niche1 ) and the epithelia ( niche2 ) . This metric represents the radial gradient of the gut bacterial composition . The comparison was done on four taxonomy levels . Data of n = 14 individuals were represented in box ( interquartile range ) and whisker ( min to max ) plots . Individual data points were plotted as black dots . * P < 0 . 05 , * * P < 0 . 01 by the conservative , nonparametric Kruskal - Wallis test . Please see Appendix Fig S10 for another way of data presentation , and Dataset EV1 for the details of the sample information and raw statistics . Note that each 16S - rRNA library contains only one sample from one animal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Comparison of 3-month-old wild-types and mutants in the pairwise microbiota compositional dissimilarity (Jaccard distance) between the digesta (niche1) and the epithelia (niche2). This metric represents the radial gradient of the gut bacterial composition. The comparison was done on four taxonomy levels. Data of n=14 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05, ** P < 0.01 by the conservative, nonparametric Kruskal-Wallis test. Please see Appendix Fig S10 for another way of data presentation, and Dataset EV1 for the details of the sample information and raw statistics. Note that each 16S-rRNA library contains only one sample from one animal.


Comparison of the bacterial community richness of the intestine samples ( S3 - S6 ; including both the digesta and the epithelia ) between six 3 - month - old wild - types and six 3 - month - old mutants . Three alpha diversity metrics were used : Ace ( abundance - based coverage estimator ) , Chao1 Data of n = 6 individuals were represented in box ( interquartile range ) and whisker ( min to max ) plots . Individual data points were plotted as black dots . * P < 0 . 05 by Kruskal - Wallis test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of the bacterial community richness of the intestine samples (S3-S6; including both the digesta and the epithelia) between six 3-month-old wild-types and six 3-month-old mutants. Three alpha diversity metrics were used: Ace (abundance-based coverage estimator), Chao1 Data of n=6 individuals were represented in box (interquartile range) and whisker (min to max) plots. Individual data points were plotted as black dots. * P < 0.05 by Kruskal-Wallis test.


E Immunohistochemical detection of BrdU - labeled cells in the intestine bulb ( section S2 ) of two 3 - month - old adult wild - types and two 3 - month - old adult mutants to measure the rate of cell proliferation ( new cells were stained bright green ) and migration . Compared with wild - types ( WT ) , the mutants had more green spots along the villi 2 hours after treatment , and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments . The violin plots ( interquartile range with mean line ) show the relative numbers of green puncta from tip to base of villi , which were calculated from 3 - 5 villi from the raw version of these images . Please see Appendix Fig S19 for the images of more animals and time points .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Immunohistochemical detection of BrdU-labeled cells in the intestine bulb (section S2) of two 3-month-old adult wild-types and two 3-month-old adult mutants to measure the rate of cell proliferation (new cells were stained bright green) and migration. Compared with wild-types (WT), the mutants had more green spots along the villi 2 hours after treatment, and mutants also have more green spots concentrated in the tip of villi 12 hours after treatments. The violin plots (interquartile range with mean line) show the relative numbers of green puncta from tip to base of villi, which were calculated from 3-5 villi from the raw version of these images. Please see Appendix Fig S19 for the images of more animals and time points.


Comparison of the body length , weight and external appearance of five wild - types and five mutants after the alternative treatments of two sets of antibiotic mixtures . The treatments were started with 8 - week - old fish and continued for six weeks . The measurements and photoshoots were conducted five weeks later . Data of n = 5 are represented in box ( min to max with mean line ) plots . Individual data points for each animal were plotted as grey dots . n . s . , no significant , * * P < 0 . 01 by student ' s t - test . The raw statistics was listed in Table EV3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Comparison of the body length, weight and external appearance of five wild-types and five mutants after the alternative treatments of two sets of antibiotic mixtures. The treatments were started with 8-week-old fish and continued for six weeks. The measurements and photoshoots were conducted five weeks later. Data of n=5 are represented in box (min to max with mean line) plots. Individual data points for each animal were plotted as grey dots. n.s., no significant, ** P < 0.01 by student's t-test. The raw statistics was listed in Table EV3.


( C - D ) Snapshots from confocal live imaging movies of a representative embryo ( N = 7 ) during cellularisation co - expressing opto - Notch , Mito - Zdk , MCP : : GFP and sim - MS2 . Continuous photo - activation from the onset of cellularisation with a stack size of z = 25μm ( λ = 488 nm ) was alternated with mCherry excitation ( λ = 561 nm ) at 1 min intervals . Single confocal z - slices of the mCherry channel are shown to record opto - Notch localization before , and 10 minutes after sustained photo - activation . Magnified insets show opto - Notch bound to the mitochondrial anchor in the dark ( C ) and in the nucleus upon photo - activation ( D ) . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Snapshots from confocal live imaging movies of a representative embryo (N=7) during cellularisation co-expressing opto-Notch, Mito-Zdk, MCP::GFP and sim-MS2. Continuous photo-activation from the onset of cellularisation with a stack size of z= 25μm (λ=488 nm) was alternated with mCherry excitation (λ=561 nm) at 1 min intervals. Single confocal z-slices of the mCherry channel are shown to record opto-Notch localization before, and 10 minutes after sustained photo-activation. Magnified insets show opto-Notch bound to the mitochondrial anchor in the dark (C) and in the nucleus upon photo-activation (D). Scale bar, 10 μm.


( F - H ) Maximum intensity z - projections ( z = 25 μm ) of the same embryo simultaneously photoactivated and imaged in the GFP channel ( λ = 488 nm ) to record sim expression at a time resolution of 30 s . Shown are the first cycle of photo - activation , which approximates the dark state ( F ) , 15 min ( G ) , and 35 min ( H ) after continuous photo - activation . Magnified insets show that sim is not expressed in the dark at this stage ( F ) , but it is expressed in lateral ectodermal cells after 15 min of photo - activation ( G ) , and appeared to be shut off in most nuclei after 35 mins ( H ) . Scale bar , 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F-H) Maximum intensity z-projections (z = 25 μm) of the same embryo simultaneously photoactivated and imaged in the GFP channel (λ=488 nm) to record sim expression at a time resolution of 30 s. Shown are the first cycle of photo-activation, which approximates the dark state (F), 15 min (G), and 35 min (H) after continuous photo-activation. Magnified insets show that sim is not expressed in the dark at this stage (F), but it is expressed in lateral ectodermal cells after 15 min of photo-activation (G), and appeared to be shut off in most nuclei after 35 mins (H). Scale bar, 10 μm.


The number of sim - expressing nuclei as counted by automated spot detection over time ( A ) Continuous opto - Notch activation initially induces sim expression across a large number of nuclei . Over time , the majority of nuclei adapt to the input and terminate expression .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

The number of sim-expressing nuclei as counted by automated spot detection over time (A) Continuous opto-Notch activation initially induces sim expression across a large number of nuclei. Over time, the majority of nuclei adapt to the input and terminate expression.


( D - E ) Quantification of nuclear localisation of mCherry : : NICD during opto - Notch photo - activation . Raw data was bleaching - corrected with an exponential fit and shown is the nuclear / total ratio , which further normalises bleaching and embryo - to - embryo variability . NICD displays persistent nuclear enrichment during continuous activation ( D ) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol ( E ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E) Quantification of nuclear localisation of mCherry::NICD during opto-Notch photo-activation. Raw data was bleaching-corrected with an exponential fit and shown is the nuclear/total ratio, which further normalises bleaching and embryo-to-embryo variability. NICD displays persistent nuclear enrichment during continuous activation (D) and shuttles back and forth between nucleus and cytoplasm under the pulsatile protocol (E).


( D - E ) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant ( Hairless H2 ) and under overexpression of Twist ( Twist OE ) The observed outcomes are most consistent with the predictions from the state - dependent inactivation model .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(D-E) Results of continuous activation experiments in a Hairless heterozygous hypomorphic mutant (Hairless H2) and under overexpression of Twist (Twist OE) The observed outcomes are most consistent with the predictions from the state-dependent inactivation model.


( D ) Absolute cell numbers of the indicated cell types were determined by flow - cytometric analysis of the bone marrow from 3 - week - old Pax5 + / + and Pax5Jak2 / + mice . Average cell numbers are shown with SEM and were statistically analyzed by multiple t - tests ( unpaired two - tailed with Holm - Šídák ' s correction ) ; ns , not significant ( P > 0 . 05 ) . One of 3 independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown.


( F ) Kaplan - Meier survival analysis of Pax5Jak2 / + ( black ) and control Pax5 + / + ( grey ) mice . n , number of mice analyzed . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test.


( H ) Eosin - hematoxylin - stained sections of the lung and liver of a Pax5Jak2 / + tumor mouse . Infiltrating and blasting tumor cells are indicated by an arrow .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.


( I ) Flow - cytometric analysis of lymph node cells from a control Pax5 + / + mouse and a 10 - week - old Pax5Jak2 / + tumor mouse . ( J ) Flow - cytometric analysis of B220lowCD19 + B cells from the bone marrow of a 4 - week - old Pax5Jak2 / + mouse .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.


( D - G ) GSEA analysis of 327 repressed ( D ) or 330 activated ( F ) Pax5 target genes identified in pro - B cells as compared with the ranked log2 - fold gene expression changes in Pax5Jak2 / + ( PJ ) B - ALLs versus control ( Ctrl ) Pax5 + / - Cdkn2ab + / - B - ALLs ( left ) . NES , normalized enrichment score . The expression of five genes that are upregulated in Pax5Jak2 / + B - ALL cells ( upper row ) and Pax5 - / - ( KO ) pro - B cells ( lower row ) is shown in ( E ) . Likewise , the expression of five genes that are upregulated in control B - ALL cells ( upper row ) and Pax5 + / + ( WT ) pro - B cells ( lower row ) is shown in ( G ) . TPM , transcripts per million . Mean TPM values with SEM are shown for the following RNA - seq experiments : 3 ( WT pro - B ) , 2 ( KO pro - B ) , 2 ( Ctrl B - ALL ) , and 4 ( PJ B - ALL ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-G) GSEA analysis of 327 repressed (D) or 330 activated (F) Pax5 target genes identified in pro-B cells as compared with the ranked log2-fold gene expression changes in Pax5Jak2/+ (PJ) B-ALLs versus control (Ctrl) Pax5+/- Cdkn2ab+/- B-ALLs (left). NES, normalized enrichment score. The expression of five genes that are upregulated in Pax5Jak2/+ B-ALL cells (upper row) and Pax5-/- (KO) pro-B cells (lower row) is shown in (E). Likewise, the expression of five genes that are upregulated in control B-ALL cells (upper row) and Pax5+/+ (WT) pro-B cells (lower row) is shown in (G). TPM, transcripts per million. Mean TPM values with SEM are shown for the following RNA-seq experiments: 3 (WT pro-B), 2 (KO pro-B), 2 (Ctrl B-ALL), and 4 (PJ B-ALL).


( H ) Expression of the Ptprc ( CD45 ) gene from exon 3 to exon 8 , as determined by RNA - seq of Pax5Jak2 / + and Pax5 + / - Cdkn2ab + / - B - ALL cells . The alternatively spliced exons 4 ( A ) , 5 ( B ) and 6 ( C ) are indicated in orange in the respective exon - intron structure of the Ptprc gene . Ptprc transcripts of B220 + Pax5 + / - Cdkn2ab + / - B - ALL cells contain all three exons , thus giving rise to expression of the CD45 isoform RABC ( known as B220 ) . In contrast , reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2 / + B - ALL cells , which likely gives rise to the CD45 isoforms RBC and

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O

(H) Expression of the Ptprc (CD45) gene from exon 3 to exon 8, as determined by RNA-seq of Pax5Jak2/+ and Pax5+/- Cdkn2ab+/- B-ALL cells. The alternatively spliced exons 4 (A), 5 (B) and 6 (C) are indicated in orange in the respective exon-intron structure of the Ptprc gene. Ptprc transcripts of B220+ Pax5+/- Cdkn2ab+/- B-ALL cells contain all three exons, thus giving rise to expression of the CD45 isoform RABC (known as B220). In contrast, reads at exon 4 are barely detectable and reads at exon 6 are strongly reduced in the mRNA of Pax5Jak2/+ B-ALL cells, which likely gives rise to the CD45 isoforms RBC and RB


( D ) Flow - cytometric analysis of bone marrow cells from 4 - week - old Pax5ihCd2 / + and Pax5Jak2 / ihCd2 mice . The expression of human ( h ) CD2 from the Pax5ihCd2 allele is shown for CD19 + B220 + ( grey ) and CD19 + B220low ( black ) B cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O

(D) Flow-cytometric analysis of bone marrow cells from 4-week-old Pax5ihCd2/+ and Pax5Jak2/ihCd2 mice. The expression of human (h) CD2 from the Pax5ihCd2 allele is shown for CD19+B220+ (grey) and CD19+B220low (black) B cells.


( G ) Kaplan - Meier survival analysis of Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + ( black ) and Cd79a - Cre Pax5LSL - Jak2 / + ( grey ) mice . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; * * * * P < 0 . 0001 . n , number of mice analyzed .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Kaplan-Meier survival analysis of Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ (black) and Cd79a-Cre Pax5LSL-Jak2/+ (grey) mice. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ****P < 0.0001. n, number of mice analyzed.


( H ) Flow - cytometric analysis of B220 and CD19 expression in B - ALL tumor cells from the lymph node of a Cd79a - Cre Ikzf1neo / + Pax5LSL - Jak2 / + mouse ( black ; left ) . Pax5 expression in these B - ALL tumor cells ( black line ) and control Pax5 + / + lymph node B cells ( grey filled ) was determined by intracellular Pax5 staining ( right ) .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O

(H) Flow-cytometric analysis of B220 and CD19 expression in B-ALL tumor cells from the lymph node of a Cd79a-Cre Ikzf1neo/+ Pax5LSL-Jak2/+ mouse (black; left). Pax5 expression in these B-ALL tumor cells (black line) and control Pax5+/+ lymph node B cells (grey filled) was determined by intracellular Pax5 staining (right).


( B , Flow - cytometric analysis of bone marrow and spleen from Pax5Jak2 - KD / + ( black ; n = 11 ) and control Pax5 + / + ( grey ; n = 7 ) mice at the age of 6 - 8 weeks . The frequencies of the indicated B cell types are shown for each organ ( B ) . The data are presented as mean percentages with SEM and were statistically analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) ; ns ( P > 0 . 5 ) , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, Flow-cytometric analysis of bone marrow and spleen from Pax5Jak2-KD/+ (black; n = 11) and control Pax5+/+ (grey; n = 7) mice at the age of 6-8 weeks. The frequencies of the indicated B cell types are shown for each organ (B). The data are presented as mean percentages with SEM and were statistically analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction); ns (P > 0.5), *P < 0.05, **P < 0.01.


( D , Tumor progression in mice , transplanted with Pax5Jak2 - Luc / + tumor cells , in the presence or absence of the JAK1 / 2 inhibitor ruxolitinib . At day 14 after cell transfer , the transplanted mice were treated twice daily with ruxolitinib or vehicle , and the tumor mass was monitored by bioluminescence measurements . Images of three representative mice with or without ruxolitinib treatment are

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D, Tumor progression in mice, transplanted with Pax5Jak2-Luc/+ tumor cells, in the presence or absence of the JAK1/2 inhibitor ruxolitinib. At day 14 after cell transfer, the transplanted mice were treated twice daily with ruxolitinib or vehicle, and the tumor mass was monitored by bioluminescence measurements. Images of three representative mice with or without ruxolitinib treatment are shown


( F ) Kaplan - Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib ( black ) and vehicle ( grey ) . Statistical analysis of the survival curves ( F ) was performed with the log - rank ( Mantel - Cox ) test ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Kaplan-Meier survival analysis of the transplanted mice upon prolonged treatment with ruxolitinib (black) and vehicle (grey). Statistical analysis of the survival curves (F) was performed with the log-rank (Mantel-Cox) test; **P < 0.01.


( B ) Genome - wide binding of Pax5 - Jak2 in in vitro cultured pro - B cells from Pax5Jak2 / + Rosa26BirA / + mice at the age of 3 weeks ( expressing Pax5 ) and ex vivo Pax5Jak2 / + Rosa26BirA / + B - ALL tumors ( lacking Pax5 ) , as determined by Bio - ChIP - seq analysis The DNA - binding pattern of full - length Pax5 was determined by Bio - ChIP - seq analysis of ex vivo sorted Pax5Bio / Bio pro - B cells , which carried a C ‐ terminal biotin acceptor sequence together with an IRES ‐ BirA gene insertion in the 3 ' untranslated region of Pax5 Two independent Bio - ChIP - seq experiments were performed for each cell type . Representative binding patterns of Pax5 and Pax5 - Jak2 in the three B cell types are shown for a selected genomic region , with horizontal bars indicating Pax5 or Pax5 - Jak2 peaks that were identified by MACS peak calling ( left ) . The number of Pax5 ( white ) and Pax5 - Jak2 ( grey or black ) peaks , which were defined by stringent MACS peak calling with a P value of < 10 - 10 in the three B cell types , are shown to the right .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Genome-wide binding of Pax5-Jak2 in in vitro cultured pro-B cells from Pax5Jak2/+ Rosa26BirA/+ mice at the age of 3 weeks (expressing Pax5) and ex vivo Pax5Jak2/+ Rosa26BirA/+ B-ALL tumors (lacking Pax5), as determined by Bio-ChIP-seq analysis The DNA-binding pattern of full-length Pax5 was determined by Bio-ChIP-seq analysis of ex vivo sorted Pax5Bio/Bio pro-B cells, which carried a C‐terminal biotin acceptor sequence together with an IRES‐BirA gene insertion in the 3' untranslated region of Pax5 Two independent Bio-ChIP-seq experiments were performed for each cell type. Representative binding patterns of Pax5 and Pax5-Jak2 in the three B cell types are shown for a selected genomic region, with horizontal bars indicating Pax5 or Pax5-Jak2 peaks that were identified by MACS peak calling (left). The number of Pax5 (white) and Pax5-Jak2 (grey or black) peaks, which were defined by stringent MACS peak calling with a P value of < 10-10 in the three B cell types, are shown to the right.


( F , G ) Flow - cytometric analysis of the bone marrow and spleen from Pax5Prd * - Jak2 / + ( black ; n = 6 ) and control Pax5 + / + ( grey ; n = 5 ) mice at the age of 6 - 7 weeks . The frequency of the indicated B cell types in each organ is indicated in ( F ) , and the flow - cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in ( G ) . Statistical data are shown as mean percentages with SEM and were analyzed by multiple t - tests ( unpaired and two - tailed with Holm - Šídák ' s correction ) : ns > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F, G) Flow-cytometric analysis of the bone marrow and spleen from Pax5Prd*-Jak2 /+ (black; n = 6) and control Pax5+/+ (grey; n = 5) mice at the age of 6-7 weeks. The frequency of the indicated B cell types in each organ is indicated in (F), and the flow-cytometric analysis of CD19 and B220 expression on bone marrow B cells is shown in (G). Statistical data are shown as mean percentages with SEM and were analyzed by multiple t-tests (unpaired and two-tailed with Holm-Šídák's correction): ns > 0.05; *P < 0.05; **P < 0.01. Each dot corresponds to one mouse.


( H ) Kaplan - Meier survival analysis of Pax5Prd * - Jak2 / + ( black ) and Pax5 + / + ( grey ) mice . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test . n , number of mice analyzed .

O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Kaplan-Meier survival analysis of Pax5Prd*-Jak2 /+ (black) and Pax5+/+ (grey) mice. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test. n, number of mice analyzed.


( C ) Specificity of the anti - H3Y41ph antibody , as shown by immunoblot analysis . One phosphorylated ( pY41 ) or non - phosphorylated peptide was coupled to ubiquitin ( Ubi , 8 . 5 kDa ) , followed by separation of the protein conjugates on SDS - PAGE and immunoblot detection with the anti - H3Y41ph antibody present in the 8B2 - C1 cell supernatant . Following treatment with calf intestinal alkaline phosphatase ( CIP ) , the dephosphorylated Ubi - pY41 peptide conjugate could no longer be detected with the anti - H3Y41ph antibody .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O

(C) Specificity of the anti-H3Y41ph antibody, as shown by immunoblot analysis. One phosphorylated (pY41) or non-phosphorylated peptide was coupled to ubiquitin (Ubi, 8.5 kDa), followed by separation of the protein conjugates on SDS-PAGE and immunoblot detection with the anti-H3Y41ph antibody present in the 8B2-C1 cell supernatant. Following treatment with calf intestinal alkaline phosphatase (CIP), the dephosphorylated Ubi-pY41 peptide conjugate could no longer be detected with the anti-H3Y41ph antibody.


( D ) Immunoblot analysis of whole - cell extracts prepared from Pax5Jak2 / + and control Pax5Etv6 / + Cdkn2ab + / - B - ALL cells as well as from the human HEL , TMD8 and K1106 cell lines . Phosphorylated ( p ) STAT5 was detected with an anti - STAT5 ( pY694 ) antibody and the pY41 - peptide with the purified anti H3Y41ph antibody One to five pY41 - peptides were coupled to ubiquitin , which was added in the range of 50 ng per well . The Gapdh and histone H3 proteins were analyzed as loading control . An unspecific protein is denoted by an asterisk . One representative of 5 immunoblot experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunoblot analysis of whole-cell extracts prepared from Pax5Jak2/+ and control Pax5Etv6/+ Cdkn2ab+/- B-ALL cells as well as from the human HEL, TMD8 and K1106 cell lines. Phosphorylated (p) STAT5 was detected with an anti-STAT5 (pY694) antibody and the pY41-peptide with the purified anti H3Y41ph antibody One to five pY41-peptides were coupled to ubiquitin, which was added in the range of 50 ng per well. The Gapdh and histone H3 proteins were analyzed as loading control. An unspecific protein is denoted by an asterisk. One representative of 5 immunoblot experiments is shown.


( A , Tumor progression in Il7 + / + ( grey ) , Il7 + / - ( dashed ) and Il7 - / - ( black ) mice transplanted with Pax5Jak2 - Luc / + tumor cells , as determined by bioluminescence measurements at the indicated days after cell transfer . Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell

O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O

(A, Tumor progression in Il7+/+ (grey), Il7+/- (dashed) and Il7-/- (black) mice transplanted with Pax5Jak2-Luc/+ tumor cells, as determined by bioluminescence measurements at the indicated days after cell transfer. Images of three transplanted mice for each Il7 genotype are shown at the indicated days after cell transfer


( C ) Kaplan - Meier survival analysis of Il7 + / + , Il7 + / - and Il7 - / - mice transplanted with Pax5Jak2 - Luc / + tumors cells . Statistical analysis of the survival curves was performed with the log - rank ( Mantel - Cox ) test ; ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Kaplan-Meier survival analysis of Il7+/+, Il7+/- and Il7-/- mice transplanted with Pax5Jak2-Luc/+ tumors cells. Statistical analysis of the survival curves was performed with the log-rank (Mantel-Cox) test; ns P > 0.05, *P < 0.05, **P < 0.01.


( E ) Quantification of the median fluorescence intensity ( MFI ) values of the p - STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5 + / + ( grey ) , Pax5Jak2 / + ( black ) and Pax5Jak2 - KD / + ( white ) mice at the age of 3 - 10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Quantification of the median fluorescence intensity (MFI) values of the p-STAT5 levels determined by intracellular staining of splenic FO B cells from Pax5+/+ (grey), Pax5Jak2/+ (black) and Pax5Jak2-KD/+ (white) mice at the age of 3-10 weeks Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( F ) Flow cytometric analysis of p - STAT5 levels in pro - B cells ( B220 + CD19 + Kit + IgM - ) and leukemic B220low B cells of Pax5Jak2 / + mice ( black ) as well as in control pro - B cells of Pax5 + / + ( WT ) mice ( grey ) at the age of 4 - 5 weeks . A representative flow cytometric analysis of the bone marrow of a Pax5Jak2 / + mouse ( left ) and representative intracellular p - STAT5 stainings ( middle ) are shown . The p - STAT5 levels in pro - B and leukemic B220low B cells ( right ) are quantified as MFI values relative to that of Pax5 + / + pro - B cells . Statistical data are presented as mean values with SEM and were analyzed by the two - tailed unpaired Student ' s t - test : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Each dot corresponds to one mouse .

O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Flow cytometric analysis of p-STAT5 levels in pro-B cells (B220+CD19+Kit+IgM-) and leukemic B220low B cells of Pax5Jak2/+ mice (black) as well as in control pro-B cells of Pax5+/+ (WT) mice (grey) at the age of 4-5 weeks. A representative flow cytometric analysis of the bone marrow of a Pax5Jak2/+ mouse (left) and representative intracellular p-STAT5 stainings (middle) are shown. The p-STAT5 levels in pro-B and leukemic B220low B cells (right) are quantified as MFI values relative to that of Pax5+/+ pro-B cells. Statistical data are presented as mean values with SEM and were analyzed by the two-tailed unpaired Student's t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.


( I ) Upregulation of the genes Cxcr5 , Syndig1l and Sema4a in Pax5Jak2 / + ( PJ ) B - ALL cells . Mean TPM values with SEM are shown for the following RNA - seq experiments : 2 ( Ctrl B - ALL ) , 4 ( PJ B - ALL )

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Upregulation of the genes Cxcr5, Syndig1l and Sema4a in Pax5Jak2/+ (PJ) B-ALL cells. Mean TPM values with SEM are shown for the following RNA-seq experiments: 2 (Ctrl B-ALL), 4 (PJ B-ALL)


K ) Identification of Cxcr5 , Syndig1l and Sema4a as activated STAT5 target genes . ChIP - seq analysis identified STAT5 - binding regions at all three loci ( K ) . Horizontal bars below the ChIP - seq track indicate STAT5 - binding regions identified by MACS peak calling . RPM , reads per million .

O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

K) Identification of Cxcr5, Syndig1l and Sema4a as activated STAT5 target genes. ChIP-seq analysis identified STAT5-binding regions at all three loci (K). Horizontal bars below the ChIP-seq track indicate STAT5-binding regions identified by MACS peak calling. RPM, reads per million.


( A , Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + and Nlrp3A350V / wt CreT - mice were incubated with LPS ( 3 hr ) before culture media were analysed for secreted IL - 1β ( A ) Cytokine values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA and P - values were corrected for multiple comparisons ( Bonferroni ) . * * , p ≤ 0 . 01 .

O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS (3 hr) before culture media were analysed for secreted IL-1β (A) Cytokine values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA and P-values were corrected for multiple comparisons (Bonferroni). **, p ≤ 0.01.


B ) . Neutrophils isolated from tamoxifen - treated Nlrp3A350V / wt CreT + and Nlrp3A350V / wt CreT - mice were incubated with LPS and cell lysates and culture media were immunoblotted for β - actin , caspase - 1 and IL - 1β ( B ) .

O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O

B). Neutrophils isolated from tamoxifen-treated Nlrp3A350V/wt CreT+ and Nlrp3A350V/wt CreT- mice were incubated with LPS and cell lysates and culture media were immunoblotted for β-actin, caspase-1 and IL-1β (B).


( C ) Representative pictures of Nlrp3A350V / wt MRP8 - CreTg and Nlrp3A350V / wt MRP8 - Cre - mice on days 4 and 8 after birth . Left side in each picture : Nlrp3A350V / wt MRP8 - Cre - mice ; right side in each picture : Nlrp3A350V / wt MRP8 - CreTg . Abscesses ( day 4 ) and scaling erythema ( day 8 ) are observed in Nlrp3A350V / wt MRP8 - CreTg mice .

O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O

(C) Representative pictures of Nlrp3A350V/wt MRP8-CreTg and Nlrp3A350V/wt MRP8-Cre- mice on days 4 and 8 after birth. Left side in each picture: Nlrp3A350V/wt MRP8-Cre- mice; right side in each picture: Nlrp3A350V/wt MRP8-CreTg. Abscesses (day 4) and scaling erythema (day 8) are observed in Nlrp3A350V/wt MRP8-CreTg mice.


( D ) Survival curves of Nlrp3A350V / wt MRP8 - CreTg ( n = 22 ) and littermate Nlrp3A350V / wt MRP8 - Cre - ( n = 21 ) mice . Statistical significance was analysed by the log - rank Mantel - Cox test . * * * * , p ≤ 0 . 0001 .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Survival curves of Nlrp3A350V/wt MRP8-CreTg (n=22) and littermate Nlrp3A350V/wt MRP8-Cre- (n=21) mice. Statistical significance was analysed by the log-rank Mantel-Cox test. ****, p ≤0.0001.


( E ) Representative H & E - stained sections of spleen ( top , scale bar : 100 µM ) and liver ( bottom , scale bar : 200 µM ) of Nlrp3A350V / wt MRP8 - CreTg and littermate Nlrp3A350V / wt MRP8 - Cre - mice . Spleens of Nlrp3A350V / wt MRP8 - Cre - control mice ( top left ) display characteristic white pulp ( WP ) regions ( example shown surrounded by dashed line ) composed of lymphoid follicles and periarteriolar lymphoid sheaths ( PALS ) , which at this magnification are recognized by dark - purple staining , large aggregates of lymphocytes surrounding the central artery ( CA ) and their distinctive separation from the red - staining red pulp ( RP ) . WP was largely absent in spleens of Nlrp3A350V / wt MRP8 - CreTg ( top middle ) . Megakaryocytes ( MK ) are indicated for easier recognition of areas of extramedullary hematopoiesis ( EMH ) . Most of the spleen parenchyma of Nlrp3A350V / wt MRP8 - CreTg animals was replaced by EMH of mostly myeloid lineage , which can be recognized by the medium - sized cells with more cytoplasm , giving a lighter appearance ( inset ) . In both the overview picture ( top middle picture ) and inset ( top right ) of Nlrp3A350V / wt MRP8 - CreTg spleen , a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker - stained , smaller cells ( asterix , * ) . Liver sections of both Nlrp3A350V / wtMRP8 - Cre - control mice and diseased Nlrp3A350V / wt MRP8 - CreTg pups displayed comparable EMH of the erythroid lineage ( bottom left picture , indicated with yellow arrows ) . Liver sections of Nlrp3A350V / wt MRP8 - CreTg pups also showed a modest increase in EMH of the myeloid lineage ( bottom middle picture and inset ( scale bar : 100 µM ) in bottom right , indicated with yellow arrowheads ) , as well as hepatocyte enlargement . ( F - I ) H & E - stained histological sections of spleen ( F , G ) or liver ( H , I ) of Nlrp3A350V / wt MRP8 - Cre - ( Cre - , n = 8 ) and Nlrp3A350V / wt MRP8 - CreTg ( CreTg , n = 6 ) mice were assigned a score of 0 - 5 for lymphoid depletion ( F ) or EMH ( G , H ) or hepatocyte enlargement ( I ) by a board - certified pathologist . Values represent mean ± SEM of n = 6 - 8 biological repeats . Statistical significance was analysed by the Mann - Whitney U - test . * * * , p ≤ 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative H&E-stained sections of spleen (top, scale bar: 100 µM) and liver (bottom, scale bar: 200 µM) of Nlrp3A350V/wt MRP8-CreTg and littermate Nlrp3A350V/wt MRP8-Cre- mice. Spleens of Nlrp3A350V/wt MRP8-Cre- control mice (top left) display characteristic white pulp (WP) regions (example shown surrounded by dashed line) composed of lymphoid follicles and periarteriolar lymphoid sheaths (PALS), which at this magnification are recognized by dark-purple staining, large aggregates of lymphocytes surrounding the central artery (CA) and their distinctive separation from the red-staining red pulp (RP). WP was largely absent in spleens of Nlrp3A350V/wt MRP8-CreTg (top middle). Megakaryocytes (MK) are indicated for easier recognition of areas of extramedullary hematopoiesis (EMH). Most of the spleen parenchyma of Nlrp3A350V/wt MRP8-CreTg animals was replaced by EMH of mostly myeloid lineage, which can be recognized by the medium-sized cells with more cytoplasm, giving a lighter appearance (inset). In both the overview picture (top middle picture) and inset (top right) of Nlrp3A350V/wt MRP8-CreTg spleen, a small amount of EMH of the erythroid lineage is present that can be recognized by small aggregates of darker-stained, smaller cells (asterix,*). Liver sections of both Nlrp3A350V/wtMRP8-Cre- control mice and diseased Nlrp3A350V/wt MRP8-CreTg pups displayed comparable EMH of the erythroid lineage (bottom left picture, indicated with yellow arrows). Liver sections of Nlrp3A350V/wt MRP8-CreTg pups also showed a modest increase in EMH of the myeloid lineage (bottom middle picture and inset (scale bar: 100 µM) in bottom right, indicated with yellow arrowheads), as well as hepatocyte enlargement. (F-I) H&E-stained histological sections of spleen (F, G) or liver (H, I) of Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) mice were assigned a score of 0-5 for lymphoid depletion (F) or EMH (G, H) or hepatocyte enlargement (I) by a board-certified pathologist. Values represent mean ± SEM of n=6-8 biological repeats. Statistical significance was analysed by the Mann-Whitney U-test. ***, p ≤0.001.


( J - L ) Cytokine secretion analysis ( Luminex ) of serum samples obtained at day 7 from Nlrp3A350V / wt MRP8 - Cre - ( Cre - , n = 8 ) and Nlrp3A350V / wt MRP8 - CreTg ( CreTg , n = 6 ) pups . Values represent mean ± SEM of n = 6 - 8 biological repeats . Statistical analysis of serum cytokine levels was performed by the Welch ' s t - test . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-L) Cytokine secretion analysis (Luminex) of serum samples obtained at day 7 from Nlrp3A350V/wt MRP8-Cre- (Cre-, n=8) and Nlrp3A350V/wt MRP8-CreTg (CreTg, n=6) pups. Values represent mean ± SEM of n=6-8 biological repeats. Statistical analysis of serum cytokine levels was performed by the Welch's t-test. *, p ≤0.05; **, p ≤0.01.


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with LeTx ( 3 hr ) ( A , B ) IL - 1β secretion levels were determined in culture media ( A , and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) ( B , Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with LeTx (3 hr) (A, B) IL-1β secretion levels were determined in culture media (A, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) (B, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) ( C , D ) IL - 1β secretion levels were determined in culture media C , and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) D , Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (C, D) IL-1β secretion levels were determined in culture media C, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) D, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or infected with F . tularensis ( 18 hr ) ( E , F ) IL - 1β secretion levels were determined in culture media E , and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) F , Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or infected with F. tularensis (18 hr) (E, F) IL-1β secretion levels were determined in culture media E, and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) F, Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with Flatox ( 3 hr ) ( G , H ) . IL - 1β secretion levels were determined in culture media G ) ; and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) H ) . Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with Flatox (3 hr) (G, H). IL-1β secretion levels were determined in culture media G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) H). Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with LeTx ( A ) , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( B ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (B) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with ATP ( C ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with ATP (C) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( D ) . PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (D). PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


( E ) LPS - primed B6 BMNs were left untreated ( LPS ) or subsequently stimulated with FlaTox ( LPS + FlaTox ) for 3 hr . Data are representative of n = 3 biological repeats . Culture media ( Sup ) and whole cell lysates ( Lys ) were immunoblotted for release of cytosolic GAPDH into culture media . Data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(E) LPS-primed B6 BMNs were left untreated (LPS) or subsequently stimulated with FlaTox (LPS+FlaTox) for 3 hr. Data are representative of n=3 biological repeats. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.


( F ) B6 BMNs were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . Culture media ( Sup ) and whole cell lysates ( Lys ) were immunoblotted for release of cytosolic GAPDH into culture media . Data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

(F) B6 BMNs were left untreated (Mock) or infected with S. Typhimurium at MOI 25. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) ( A Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of the antibody . All data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (A Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) ( B , Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of the antibody . All data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) (B, Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) ( C , Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of the antibody . All data are representative of n = 3 biological repeats .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) (C, Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) D , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) D, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) E , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) E, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) F , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) F, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) G ) , IL - 1β secretion levels were determined by Luminex assay . Values represent mean ± SEM of n = 3 ( G , biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) G), IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 (G, biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) , H ) IL - 1β secretion levels were determined by Luminex assay . Values represent mean ± SEM of n = 4 ( H ) biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) , H) IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=4 (H) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) I ) . IL - 1β secretion levels were determined by Luminex assay . Values represent mean ± SEM of n = 3 I ) biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) I). IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 I) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) ( A ) * with Western blots indicates non - specific bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30) (A) * with Western blots indicates non-specific bands.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) ( B , PMP was assessed by measurement of SYTOX Green incorporation over time . Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * * , p ≤ 0 . 001 ; ns = non - significant . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) (B, PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for extracellular release of β - actin and

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with nigericin ( 100 mins ) Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for extracellular release of β - actin and

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with nigericin ( 100 mins ) G ) PMP was assessed by measurement of SYTOX Green incorporation over time . Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * * , p ≤ 0 . 001 ; ns = non - significant . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) G) PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with TcDA ( 3 hr ) ( H ) . Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for extracellular release of β - actin and GAPDH H ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with TcDA (3 hr) (H). Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH H).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( A ) Representative confocal micrographs taken at different time points were stained with DAPI ( dark blue nuclei ) and SYTOX Green ( light blue ) . Scale bar : 20 μM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs taken at different time points were stained with DAPI (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM.


( B ) Representative confocal micrographs of untreated ( Mock ) , S . Typhimurium - infected or PMA - stimulated wildtype BMNs stained for neutrophil elastase ( Red ) and SYTOX Green ( Green ) ( Scale bar : 10 μM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

(B) Representative confocal micrographs of untreated (Mock), S. Typhimurium-infected or PMA-stimulated wildtype BMNs stained for neutrophil elastase (Red) and SYTOX Green (Green) (Scale bar: 10 μM).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( C , Cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for the indicated proteins at 1h and 3 hr post - infection . Data information : S . Typh . : S . Typhimurium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (C, Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . D ) Cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for the indicated proteins at 1h and 3 hr post - infection . Data information : S . Typh . : S . Typhimurium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. D) Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( E ) Cell death was measured by LDH assay 3 h post - infection . Data information : In E , values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (E) Cell death was measured by LDH assay 3 h post-infection. Data information: In E, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( F ) IL - 1β secretion levels were determined by Luminex assay 1 hr post - infection . Data information : In F , values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (F) IL-1β secretion levels were determined by Luminex assay 1 hr post-infection. Data information: In F, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( G ) Representative confocal micrographs of neutrophils stained with DAPI ( dark blue nuclei ) and SYTOX Green ( light blue ) taken at different time points post - infection ( scale bar : 20 μM ) . ( H ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : In H , values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (G) Representative confocal micrographs of neutrophils stained with DAPI (dark blue nuclei) and SYTOX Green (light blue) taken at different time points post-infection (scale bar: 20 μM). (H) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: In H, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


( A ) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec ‐ 4 ( d ) or deg ‐ 3 ( d ) alleles in SNT ‐ 1 ‐ deficient mutants . For mec ‐ 4 ( d ) mutant animals , bars denote touch receptor neuron corpses . For deg ‐ 3 ( d ) mutants , bars denote inner labial neuron 1 ( L1 ) sensory neuron and PVC interneuron corpses .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in SNT‐1‐deficient mutants. For mec‐4(d) mutant animals, bars denote touch receptor neuron corpses. For deg‐3(d)mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses.


( B ) Expression of a full ‐ length MEC ‐ 4 : : GFP reporter fusion under the control of the mec ‐ 4 promoter in touch receptor neurons of wild ‐ type , snt ‐ 1 and unc ‐ 57 mutant animals . Representative photos of touch receptor neuron cell bodies are shown . Bar denotes 6 μm . The quantification ( % ) of GFP signal intensity from the animals examined is graphed below ( n = 100 neurons per assay ; P0 . 05 compared with wild type ; t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, snt‐1 and unc‐57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).


( C ) Percentage of SNT ‐ 1 ‐ deficient animals that survive near ‐ lethal treatment with sodium azide . This chemical inhibits the activity of the respiratory electron transport complex IV ( cytochrome C oxidase ) and simulates hypoxia .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia.


( D ) Number of touch receptor neuron corpses , at the L1 stage of development , per 100 animals carrying the neurotoxic mec - 4 ( d ) or deg ‐ 3 ( d ) alleles in UNC ‐ 57 ‐ deficient animals .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O

(D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg‐3(d) alleles in UNC‐57‐deficient animals.


( E ) Percentage of UNC ‐ 57 ‐ deficient animals that survive after the hypoxia ‐ inducing treatment with sodium azide .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

(E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide.


( F ) Number of touch receptor neuron corpses , at the L1 stage of development , per 100 animals carrying the neurotoxic mec ‐ 4 ( d ) or deg ‐ 3 ( d ) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin ‐ mediated endocytosis . UNC ‐ 11 and DPY ‐ 23 are adaptor proteins required for the formation of clathrin ‐ coated pits , DYN ‐ 1 is a GTPase necessary for fission of clathrin ‐ coated vesicles and UNC ‐ 26 participates in the uncoating of vesicles . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with wild ‐ type animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin‐mediated endocytosis. UNC‐11 and DPY‐23 are adaptor proteins required for the formation of clathrin‐coated pits, DYN‐1 is a GTPase necessary for fission of clathrin‐coated vesicles and UNC‐26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with wild‐type animals, unpaired t‐test).


( A ) Confocal images of touch receptor neurons expressing a pmec ‐ 17APS ‐ 2GFP reporter transgene . During early necrosis , the number of APS ‐ 2 : : GFP puncta , corresponding to clathrin ‐ coated pits and vesicles , in touch receptor neurons ofmec ‐ 4 ( d ) animals does not change significantly , whereas at later stages it decreases . A similar decrease is also observed in unc ‐ 57 ( e1190 ) mutants , defective for clathrin ‐ mediated endocytosis .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O

(A) Confocal images of touch receptor neurons expressing a pmec‐17APS‐2GFP reporter transgene. During early necrosis, the number of APS‐2::GFP puncta, corresponding to clathrin‐coated pits and vesicles, in touch receptor neurons ofmec‐4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc‐57(e1190) mutants, defective for clathrin‐mediated endocytosis.


( B ) Confocal images of touch receptor neurons expressing a pmec ‐ 7GFP : : RAB ‐ 5 reporter transgene . The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec ‐ 4 ( d ) animals significantly increases during early necrosis , whereas it declines later .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O

(B) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec‐4(d) animals significantly increases during early necrosis, whereas it declines later.


( C ) Confocal images of touch receptor neurons expressing a pmec ‐ 7GFP : : RAB ‐ 11 reporter transgene . The number of GFP : : RAB ‐ 11 puncta that correspond to recycling endosomes in touch receptor neurons of mec ‐ 4 ( d ) animals increases early during cell death , while it declines as degeneration proceeds . Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus . Bars denote 4 μm . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐11 reporter transgene. The number of GFP::RAB‐11 puncta that correspond to recycling endosomes in touch receptor neurons of mec‐4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).


Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death . ( A ) Depletion of synaptotagmin ( SNT ‐ 1 ) or endophilin ( UNC ‐ 57 ) does not further suppress MEC ‐ 4 ( d ) ‐ induced necrosis in cad ‐ 1 mutants with reduced cathepsin activity or with V ‐ ATPase dysfunction ( B , C ) .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death. (A) Depletion of synaptotagmin (SNT‐1) or endophilin (UNC‐57) does not further suppress MEC‐4(d)‐induced necrosis in cad‐1 mutants with reduced cathepsin activity or with V‐ATPase dysfunction (B, C).


Similarly , no synthetic reduction of necrotic cell death is observed in calpain ‐ deficient animals that also carry lesions in the synaptotagmin ( D ) or endophilin ( E ) genes . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Similarly, no synthetic reduction of necrotic cell death is observed in calpain‐deficient animals that also carry lesions in the synaptotagmin (D) or endophilin (E) genes. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( A ) Synaptotagmin ( SNT ‐ 1 ) deficiency further suppresses MEC ‐ 4 ( d ) ‐ induced necrosis in animals with impaired autophagy .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(A) Synaptotagmin (SNT‐1) deficiency further suppresses MEC‐4(d)‐induced necrosis in animals with impaired autophagy.


( B ) Endophilin dysfunction enhances suppression of necrosis in mec ‐ 4 ( d ) ; lgg ‐ 1 ( RNAi ) animals , where autophagy is impaired . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 01 , compared with single mutant control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Endophilin dysfunction enhances suppression of necrosis in mec‐4(d);lgg‐1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P0.01, compared with single mutant control animals, unpaired t‐test).


( A ) Depletion of the kinesin 1 heavy chain UNC ‐ 116 suppresses necrosis induced by both mec ‐ 4 ( d ) and deg ‐ 3 ( d ) toxic alleles .

O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O

(A) Depletion of the kinesin 1 heavy chain UNC‐116 suppresses necrosis induced by both mec‐4(d) and deg‐3(d) toxic alleles.


( B ) Expression of a full ‐ length MEC ‐ 4 : : GFP reporter fusion under the control of the mec ‐ 4 promoter in touch receptor neurons of wild ‐ type , unc ‐ 116 and unc ‐ 104 mutant animals . Representative photos of touch receptor neuron cell bodies are shown . Bar denotes 6 μm . The quantification ( % ) of GFP signal intensity from the animals examined is graphed below ( n = 100 neurons per assay ; P0 . 05 compared with wild type ; t ‐ test ) .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, unc‐116 and unc‐104 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).


( C ) Percentage of unc ‐ 116 animals that survive after treatment with sodium azide .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O

(C) Percentage of unc‐116 animals that survive after treatment with sodium azide.


( D ) Depletion of the monomeric kinesin UNC ‐ 104 protects neurons against MEC ‐ 4 ( d ) ‐ induced degeneration .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

(D) Depletion of the monomeric kinesin UNC‐104 protects neurons against MEC‐4(d)‐induced degeneration.


( E ) unc ‐ 104 mutant animals are more resistant to hypoxic death induced by sodium azide .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O

(E) unc‐104 mutant animals are more resistant to hypoxic death induced by sodium azide.


( F ) Combined inactivation of endocytosis and kinesin ‐ mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC ‐ 4 ( d ) ‐ induced degeneration . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Combined inactivation of endocytosis and kinesin‐mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC‐4(d)‐induced degeneration. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).


Genetic interaction between kinesin ‐ mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death . Dysfunction of either kinesin 1 heavy chain ( UNC ‐ 116 ) or the monomeric kinesin UNC ‐ 104 does not significantly enhance suppression of neurodegeneration in aspartyl protease ‐ deficient mutant animals ( A , B ) , in animals with compromised lysosomal acidification ( C , D ) , or in calpain protease ‐ deficient mutants animals ( E , F ) . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genetic interaction between kinesin‐mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death. Dysfunction of either kinesin 1 heavy chain (UNC‐116) or the monomeric kinesin UNC‐104 does not significantly enhance suppression of neurodegeneration in aspartyl protease‐deficient mutant animals (A, B), in animals with compromised lysosomal acidification (C, D), or in calpain protease‐deficient mutants animals (E, F). Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( A ) Kinesin 1 heavy chain ( UNC ‐ 116 ) deficiency .

O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O

(A) Kinesin 1 heavy chain (UNC‐116) deficiency.