C of total numbers of generated types ( BrdU + ; rNSCs , IPCs + neuroblasts , astrocytes ) per in DGs of 8 week - , 14 month - , 21 month - old hGFAPeGFP . rNSCs : * * * p = 0 . 00076 , * * p = 0 . 0014 ; IPCs / neuroblasts * * * * p < 0 . 0001 ; astrocytes : * * * p = 0 . 0008 ; * p = 0 . 0403 ; one - way ANOVA with Tukey ' s post hoc . D of percentages of generated types out of generated ( rNSCs , IPCs + neuroblasts , astrocytes ) in DG of 8 week - , 14 month - , 21 month - old . rNSCs : not significant ; IPCs / neuroblasts * * * * p < 0 . 0001 , * * * p = 0 . 0001 ; astrocytes : * * p < 0 . 0019 , * * * * p < 0 . 0001 ; one - way ANOVA with Tukey ' s post hoc . Data information : data are represented as mean ± ; number of experimental ( indicated by red dots ) : ( C , D ) n = 5 in 8 week - old mice , n = 6 in 14 month - old mice , n = 5 in 21 month - old

O O O O O O O O B-SMALL_MOLECULE O O O O O O B-CELL_TYPE O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O 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-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O B-ORGANISM O O O O O O O O O

C Quantification of total numbers of generated cell types (BrdU+; rNSCs, IPCs + neuroblasts, astrocytes) per area in the DGs of 8 week-, 14 month-, and 21 month-old hGFAPeGFP animals. rNSCs: *** p = 0.00076, ** p = 0.0014; IPCs/neuroblasts **** p < 0.0001; astrocytes: *** p = 0.0008; * p = 0.0403; one-way ANOVA with Tukey's post hoc test. D Quantification of the percentages of generated cell types out of all generated cells (rNSCs, IPCs + neuroblasts, astrocytes) in the DG of 8 week-, 14 month-, and 21 month-old animals. rNSCs: not significant; IPCs/neuroblasts **** p < 0.0001, *** p = 0.0001; astrocytes: ** p < 0.0019, **** p < 0.0001; one-way ANOVA with Tukey's post hoc test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (C,D) n = 5 in 8 week-old mice, n = 6 in 14 month-old mice, n = 5 in 21 month-old mice


E Representative of proliferating ( MCM2 + ; magenta ) : rNSCs [ SOX2 + nuclei ( cyan ) residing in SGZ with a NESTIN + radial process ( ) ] ; IPCs [ SOX2 + nuclei ( cyan ) located to SGZ ] , astrocytes [ SOX2 + ( cyan ) in hilus , GZ , ML ; arrowheads ] . F of total numbers of proliferating ( MCM2 + ) types per in DGs of 8 week - , 14 month - , 21 month - old . rNSCs : not significant ; IPCs * * * * p < 0 . 0001 ; astrocytes : * p = 0 . 0216 ; * p = 0 . 0266 ; one - way ANOVA with Tukey ' s post hoc . Data information : data are represented as mean ± ; number of experimental ( indicated by red dots ) : ( F ) n = 4 in 8 week - old mice , n = 6 in 14 month - 21 month - old mice dotted lines border SGZ GZ ; scale bars = 50 µm .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-TISSUE O O B-CELL_TYPE O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-TISSUE O O O O O O 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-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O B-ORGANISM O O O B-TISSUE I-TISSUE O O O O O O O

E Representative images of proliferating cells (MCM2+; magenta): rNSCs [SOX2+ cell nuclei (cyan) residing in the SGZ with a NESTIN+ radial process (white)]; IPCs [SOX2+ nuclei (cyan) located to the SGZ], and astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML; arrowheads]. F Quantification of total numbers of proliferating (MCM2+) cell types per area in the DGs of 8 week-, 14 month-, and 21 month-old animals. rNSCs: not significant; IPCs **** p < 0.0001; astrocytes: * p = 0.0216; * p = 0.0266; one-way ANOVA with Tukey's post hoc test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): (F) n = 4 in 8 week-old mice, n = 6 in 14 month- and 21 month-old mice dotted lines border SGZ and GZ; scale bars = 50 µm.


G Confocal of 8 week - 14 month - old NestinCreERT2 ; GFP , which received tamoxifen for five days ( 10 times , every 12 h ) were killed 12 days after last tamoxifen shot . majority of recombined were rNSCs [ SOX2 + nuclei ( cyan ) residing in SGZ with a NESTIN + radial process ( ) ] IPCs / neuroblasts [ SOX2 + ( cyan ) nuclei located to SGZ , DCX + ( magenta ) in SGZ GZ , respectively ] , only very few astrocytes [ SOX2 + ( cyan ) in hilus , GZ , ML ] were generated by initially recombined rNSCs . H of total numbers of recombined ( GFP + ) rNSCs , IPCs / neuroblasts , astrocytes per in DGs of 8 week - 14 month - old . rNSCs : * p = 0 . 0460 ; IPCs / neuroblasts : * * * * < 0 . 0001 ; astrocytes : not significant ; unpaired t - . Data information : data are represented as mean ± ; number of experimental ( indicated by red dots ) : ; ( H ) n = 3 in 8 week - old mice , n = 5 in 14 month - old mice ; dotted lines border SGZ GZ ; scale bars = 50 µm .

O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O B-TISSUE O O B-GENEPROD O O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O B-SUBCELLULAR O O B-TISSUE O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O O B-CELL_TYPE O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O 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-ORGANISM O O O O O O O O O B-ORGANISM O O O O B-TISSUE I-TISSUE O O O O O O O

G Confocal images of 8 week- and 14 month-old NestinCreERT2; GFP animals, which received tamoxifen for five days (10 times, every 12 h) and were killed 12 days after the last tamoxifen shot. The majority of recombined cells were rNSCs [SOX2+ cell nuclei (cyan) residing in the SGZ with a NESTIN+ radial process (white)] and IPCs/neuroblasts [SOX2+ (cyan) nuclei located to the SGZ, DCX+ cells (magenta) in the SGZ and GZ, respectively], and only very few astrocytes [SOX2+ cells (cyan) in the hilus, GZ, and ML] were generated by initially recombined rNSCs. H Quantification of total numbers of recombined (GFP+) rNSCs, IPCs/neuroblasts, astrocytes per area in the DGs of 8 week- and 14 month-old animals. rNSCs: * p = 0.0460; IPCs/neuroblasts: **** < 0.0001; astrocytes: not significant; unpaired t-test. Data information: All data are represented as mean ± SEM; number of experimental animals (indicated by red dots): ; (H) n = 3 in 8 week-old mice, n = 5 in 14 month-old mice ; dotted lines border SGZ and GZ; scale bars = 50 µm.


A , Western blot of 293T treated with 20 μM erastin for indicated times .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O

A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.


B , H1299 transfected with a control siRNA ( siCont . ) or an RNF20 - specific siRNA ( siRNF20 ) a wild - type H2B ( H2BWT ) or a K120R - mutated H2B ( H2BK120R ) were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast were recorded ( magnification , × 20 ) , surviving were counted . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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, H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


C , Lipid ROS were assessed by flow cytometry after C11 - BODIPY in treated Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Lipid ROS levels were assessed by flow cytometry after C11-BODIPY staining in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


D , H1299 treated were subjected to transmission electron microscopy . Representative are shown .

O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O

D, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown.


E , Control H1299 ( transfected with siCont . or H2BWT ) H2Bub1 - depleted H1299 ( transfected with siRNF20 or H2BK120R ) were treated with erastin either with or without a ferroptosis - specific inhibitor , ferrostatin - 1 ( 2 μM ) , for 24 h . Representative phase - contrast were recorded ( magnification , × 20 ) , surviving were counted . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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, Control H1299 cells (transfected with siCont. or H2BWT) and H2Bub1-depleted H1299 cells (transfected with siRNF20 or H2BK120R) were treated with erastin either with or without a ferroptosis-specific inhibitor, ferrostatin-1 (2 μM), for 24 h. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


A , qRT - PCR ( left ) Western blot ( right ) analyses of H1299 transfected with a control siRNA ( siCont . ) or an RNF20 - specific siRNA ( siRNF20 ) a wild - type H2B ( H2BWT ) or a K120R - mutated H2B ( H2BK120R ) for 24 h . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, qRT-PCR (left) and Western blot (right) analyses of H1299 cells transfected with a control siRNA (siCont.) or an RNF20-specific siRNA (siRNF20) and a wild-type H2B (H2BWT) or a K120R-mutated H2B (H2BK120R) for 24 h. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


B , immunoprecipitation ( ChIP ) was carried out with anti - H2Bub1 antibodies in H1299 ( left ) or 293T either untreated or treated with 20 μM erastin for 24 h ( right ) . intergenic region was used as a negative control for occupancy of H2Bub1 . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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, Chromatin immunoprecipitation (ChIP) assay was carried out with anti-H2Bub1 antibodies in H1299 cells (left) or 293T cells either untreated or treated with 20 μM erastin for 24 h (right). The intergenic region was used as a negative control for the occupancy of H2Bub1. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


C , Intracellular GSH were examined in H1299 treated as indicated , bar graphs are shown . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Intracellular GSH levels were examined in H1299 cells treated as indicated, and bar graphs are shown. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


D , H1299 transfected as indicated were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h . Representative phase - contrast were recorded ( magnification , × 20 ) . E , Surviving from shown in ( D ) were counted . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O 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, H1299 cells transfected as indicated were treated with 12 μM erastin (+) or untreated (-) for 24 h. Representative phase-contrast images were recorded (magnification, ×20). E, Surviving cells from the assay shown in (D) were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


G , Affected metal ion - binding genes were selected subjected to cluster .

G, Affected metal ion-binding genes were selected and subjected to cluster analysis.


H , Labile iron were assessed by flow cytometry with a standard method in H1299 .

O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O

H, Labile iron levels were assessed by flow cytometry with a standard method in H1299 cells.


I , Labile iron examined were quantified . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 , * * : p < 0 . 01 .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Labile iron levels examined were quantified. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05, **: p<0.01.


Western blot was performed with H1299 or Hep3B transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O

Western blot analysis was performed with H1299 or Hep3B cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.


Western blot was performed with H1299 transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) for 48 h .

B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O

Western blot analysis was performed with H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) for 48 h.


C , Immunofluorescence ( IF ) for H1299 transfected with a Flag - tagged p53 ( Flag - p53 ) or an empty plasmid ( Cont . ) for 48 h . Representative are shown ( magnification , × 40 ) . Arrowhead indicates a Flag - p53 overexpressing .

O O B-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O 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-GENEPROD O B-GENEPROD O O

C, Immunofluorescence (IF) assay for H1299 cells transfected with a Flag-tagged p53 (Flag-p53) or an empty plasmid (Cont.) for 48 h. Representative images are shown (magnification, ×40). Arrowhead indicates a Flag-p53 overexpressing cell.


D , Western blot was performed with H1299 transfected with a Flag - tagged p53 or an empty vector ( Cont . ) for 48 h .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O

D, Western blot analysis was performed with H1299 transfected with a Flag-tagged p53 or an empty vector (Cont.) for 48 h.


E , 293T transfected with a control siRNA ( siCont . ) three p53 - specific siRNAs ( sip53 ) were subjected to Western blot .

O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

E, 293T cells transfected with a control siRNA (siCont.) and three p53-specific siRNAs (sip53) were subjected to Western blot analysis.


F , SMMC were treated with 100 μM etoposide ( + ) or untreated ( - ) for 24 h , Western blot was performed .

O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O

F, SMMC cells were treated with 100 μM etoposide (+) or untreated (-) for 24 h, and Western blot analysis was performed.


, Western blot analysis of A549 cells and H1299 cells.


H , H1299 transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) were analyzed by micrococcal nuclease ( MNase ) digestion .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O

H, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) were analyzed by micrococcal nuclease (MNase) digestion assay.


I , Western blot of H1299 transfected with a wild - type p53 ( p53 - WT ) or two " gain - of - function " mutants ( p53 - R273H p53 - R175H ) for 48 h .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

I, Western blot analysis of H1299 cells transfected with a wild-type p53 (p53-WT) or two "gain-of-function" mutants (p53-R273H and p53-R175H) for 48 h.


A , Coimmunoprecipitation ( Co - IP ) was performed in A549 with antibodies against p53 or USP7 .

O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O

A, Coimmunoprecipitation (Co-IP) analysis was performed in A549 cells with antibodies against p53 or USP7.


B , H1299 were transfected with an empty vector ( Cont . ) , a Flag - tagged p53 , a USP7 - specific shRNA , or last two plasmids together for 48 h . Western blot was then performed .

O O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

B, H1299 cells were transfected with an empty vector (Cont.), a Flag-tagged p53, a USP7-specific shRNA, or the last two plasmids together for 48 h. Western blot analysis was then performed.


C , extracts were differentially prepared from H1299 transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) . Western blot was then performed .

O O O O O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

C, Nuclear and cytosolic extracts were differentially prepared from H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.). Western blot analysis was then performed.


D , H1299 transfected with a Myc - tagged USP7 or a Myc - tagged USP7 together with a Flag - tagged p53 were subjected to IF ( upper ) . A549 transfected with a p53 - specific siRNA or a control siRNA ( siCont . ) were examined by IF , RNAi efficiency is shown ( lower ) . Representative are shown ( magnification , × 40 ) .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

D, H1299 cells transfected with a Myc-tagged USP7 or a Myc-tagged USP7 together with a Flag-tagged p53 were subjected to IF analysis (upper). A549 cells transfected with a p53-specific siRNA or a control siRNA (siCont.) were examined by IF analysis, and the RNAi efficiency is shown (lower). Representative images are shown (magnification, ×40).


F , IF of A549 transfected with a Myc - tagged wild - type USP7 ( Myc - USP7 - WT ) or three Myc - tagged truncated mutants . Representative are shown ( magnification , × 40 ) .

O O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O

F, IF analysis of A549 cells transfected with a Myc-tagged wild-type USP7 (Myc-USP7-WT) or three Myc-tagged truncated mutants. Representative images are shown (magnification, ×40).


G , H1299 were transfected with a Flag - tagged p53 or an empty vector together with a USP7 - specific siRNA or a control siRNA as indicated for 48 h . were then harvested subjected to a MNase digestion .

O O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O

G, H1299 cells were transfected with a Flag-tagged p53 or an empty vector together with a USP7-specific siRNA or a control siRNA as indicated for 48 h. Cells were then harvested and subjected to a MNase digestion assay.


A , Co - IP was performed in 293T with antibodies against p53 or H2Bub1 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O

A, Co-IP analysis was performed in 293T cells with antibodies against p53 or H2Bub1.


Peptide pull - down was performed with synthesized

Peptide pull-down assay was performed with synthesized peptides


D , Co - IP for H1299 transfected with a Flag - tagged p53 or an empty plasmid ( Cont . ) treated with a USP7 - specific inhibitor P5091 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O

D, Co-IP analysis for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid (Cont.) treated with a USP7-specific inhibitor P5091.


A , Western blot of 293T treated with 20 μM erastin for indicated times .

O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O

A, Western blot analysis of 293T cells treated with 20 μM erastin for the indicated times.


B , H1299 were transfected with Flag - tagged p53 for 24 h then treated with 12 μM erastin or untreated for another 24 h . Co - IP was performed with antibodies against Flag .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O

B, H1299 cells were transfected with Flag-tagged p53 for 24 h and then treated with 12 μM erastin or untreated for another 24 h. Co-IP analysis was performed with antibodies against Flag.


C , H1299 transfected with Flag - tagged p53 were treated with erastin or untreated for 24 h . extracts were differentially prepared subjected to Western blot .

O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O

C, H1299 cells transfected with Flag-tagged p53 were treated with erastin or untreated for 24 h. Nuclear and cytosolic extracts were differentially prepared and subjected to Western blot analysis.


D , 293T transfected with a p53 - specific siRNA , a USP7 - specific siRNA , or a control siRNA ( siCont . ) were treated with 20 μM erastin or untreated for 24 h as indicated . Western blot was then performed .

O O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O

D, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA, or a control siRNA (siCont.) were treated with 20 μM erastin or untreated for 24 h as indicated. Western blot analysis was then performed.


, qRT - PCR ( above ) Western blot ( below ) analyses for H1299 transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as indicated . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

, qRT-PCR (above) and Western blot (below) analyses for H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


F , ChIP of H1299 transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as indicated . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats .

O O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O 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, ChIP analysis of H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as indicated. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


G , H1299 transfected with Flag - tagged p53 were treated with 12 μM erastin or untreated for 24 h . ChIP with antibodies against Flag or USP7 was performed . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats .

O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

G, H1299 cells transfected with Flag-tagged p53 were treated with 12 μM erastin or untreated for 24 h. ChIP analysis with antibodies against Flag or USP7 was performed. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


H , 293T transfected with a p53 - specific siRNA , a USP7 - specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h . ChIP was performed with anti - H2Bub1 antibodies . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats .

O O B-CELL_LINE O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

H, 293T cells transfected with a p53-specific siRNA, a USP7-specific siRNA or a control were treated with 20 μM erastin or untreated for 24 h. ChIP analysis was performed with anti-H2Bub1 antibodies. Data information: Bars and error bars are mean±s.d., n=3 independent repeats.


A , H1299 transfected with a Flag - tagged p53 or an empty plasmid together with a USP7 - specific siRNA or a control siRNA ( siCont . ) as were treated with 12 μM erastin ( + ) or untreated ( - ) for 24 h as indicated . Representative phase - contrast were recorded ( magnification , × 20 ) , surviving were counted . Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 .

O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O 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, H1299 cells transfected with a Flag-tagged p53 or an empty plasmid together with a USP7-specific siRNA or a control siRNA (siCont.) as were treated with 12 μM erastin (+) or untreated (-) for 24 h as indicated. Representative phase-contrast images were recorded (magnification, ×20), and the surviving cells were counted. Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.


B , H1299 treated were subjected to transmission electron microscopy . Representative are shown . Arrow head indicates condensed marginated .

O O B-CELL_LINE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O

B, H1299 cells treated were subjected to transmission electron microscopy. Representative images are shown. Arrow head indicates condensed and marginated chromatin.


C , Lipid ROS were examined in treated Data information : Bars error bars are mean ± s . d . , n = 3 independent repeats . Two - unpaired Student ' s t - was performed . * : p < 0 . 05 .

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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, Lipid ROS levels were examined in cells treated Data information: Bars and error bars are mean±s.d., n=3 independent repeats. Two-tailed unpaired Student's t-test was performed. *: p<0.05.


Examples of CT scan of several metastases ( M032R1 , M032R2 , M032R5 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O O O O O

Examples of CT scan images of several metastases (M032R1, M032R2, and M032R5).


Volumetric measurements on PET CT scans could be generated from M032 , M032R1 , M032R5 . Graph illustrates that metastases R1 R5 initially expanded before start of treatment , but regressed upon vemurafenib treatment . M032R5 showed progressive disease after 4 months , whereas M032R1 still displayed stable disease . M032 was excised no recurrence was observed .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O

Volumetric measurements based on the PET and CT scans could be generated from M032, M032R1, and M032R5. Graph illustrates that the metastases R1 and R5 initially expanded before the start of the treatment, but regressed upon vemurafenib treatment. M032R5 showed progressive disease after 4 months, whereas M032R1 still displayed stable disease. M032 was excised and no recurrence was observed.


Hematoxylin - eosin ( H & amp ; amp ; E ) p - ERK stainings on FFPE material of metastases showed that vemurafenib - resistant tumors had reactivation of MAPK pathway . Scale bar represents 100

Hematoxylin-eosin (H&amp;E) and p-ERK stainings on FFPE material of all metastases showed that all vemurafenib-resistant tumors had reactivation of the MAPK pathway. Scale bar represents 100 μm


qPCR was performed on gDNA retrieved from each of metastases , using primers for BRAF CRAF normalized on . Bars represent mean of three replicates , error bars indicate standard deviation . results confirmed that BRAF was amplified in M032R1 , M032R2 , M032R5 .

B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O

qPCR was performed on gDNA retrieved from each of the metastases, using primers for BRAF and CRAF and normalized on LINE levels. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in M032R1, M032R2, and M032R5.


for BRAFV600E with a mutant epitope - specific antibody confirmed upregulation of BRAFV600E in R1 , R2 , R5 . Scale bar represents 100 μm .

O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

Staining for BRAFV600E with a mutant epitope-specific antibody confirmed the upregulation of BRAFV600E in R1, R2, and R5. Scale bar represents 100 μm.


Immunoblotting for MAPK pathway components confirmed reactivation of p - ERK p - MEK in resistant metastases .

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE O

Immunoblotting for MAPK pathway components confirmed reactivation of p-ERK and p-MEK in all resistant metastases.


Immunoblotting for M032 ' s samples using an antibody directed against either mutant BRAFV600E epitope or an N - terminal region of BRAF revealed expression of a shorter variant of BRAFV600E in M032R3 , of approximately 80 kDa . This 80 - kDa band could not be detected with N - terminus - recognizing antibody .

B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblotting for M032 patient's samples using an antibody directed against either the mutant BRAFV600E epitope or an N-terminal region of BRAF revealed the expression of a shorter variant of BRAFV600E in M032R3, of approximately 80 kDa. This 80-kDa band could not be detected with the N-terminus-recognizing antibody.


Validation of T55delinsRT was done by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used .

O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O

Validation of T55delinsRT was done by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used.


Transfection of pQXCIP - GFP , MEK1 - WT , or MEK1 - T55delinsRT into HEK293T showed that this mutation induces p - ERK p - RSK .

O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE O O O O O O O B-GENEPROD O O B-GENEPROD O

Transfection of pQXCIP-GFP, MEK1-WT, or MEK1-T55delinsRT into HEK293T cells showed that this mutation induces p-ERK and p-RSK.


Validation of expression of pQXCIP - GFP , MEK1WT , or MEK1T55delinsRT in A375 melanoma by immunoblotting .

O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O B-EXP_ASSAY O

Validation of expression of pQXCIP-GFP, MEK1WT, or MEK1T55delinsRT in A375 melanoma cells by immunoblotting.


Dose - response curves for A375 melanoma expressing GFP , MEK1WT , or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib , ERK inhibitor SCH772984 , or MEK inhibitor trametinib . Error bars indicate standard deviation .

O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O

Dose-response curves for A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT with indicated doses of BRAF inhibitor vemurafenib, ERK inhibitor SCH772984, or MEK inhibitor trametinib. Error bars indicate standard deviation.


Colony formation assays with A375 melanoma , infected with GFP , MEK1WT , or MEK1T55delinsRT - encoding lentivirus , treated with indicated doses inhibitors .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O

Colony formation assays with A375 melanoma cells, infected with GFP, MEK1WT, or MEK1T55delinsRT-encoding lentivirus, and treated with indicated doses and inhibitors.


Treatment of A375 MEK1T55delinsRT melanoma with DMSO ( − ) , 250 nM dabrafenib ( D ) , 10 nM trametinib ( T ) , or a combination ( D + T ) .

O O B-CELL_LINE B-GENEPROD O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

Treatment of A375 MEK1T55delinsRT melanoma cells with DMSO (−), 250 nM dabrafenib (D), 10 nM trametinib (T), or a combination (D+T).


A375 melanoma expressing GFP , MEK1WT , or MEK1T55delinsRT were injected into immune - deficient mice ( n = 8 per group ) , after of ˜ 100 mm3 was reached , mice were treated with 30 mg / kg dabrafenib or vehicle . Graphs represent fold change in volume normalized on volume on day of start of treatment . Error bars indicate standard error of mean .

B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O

A375 melanoma cells expressing GFP, MEK1WT, or MEK1T55delinsRT were injected into immune-deficient mice (n = 8 per group), and after the tumor size of ˜100 mm3 was reached, mice were treated with 30 mg/kg dabrafenib or vehicle. Graphs represent fold change in tumor volume normalized on the tumor volume on the day of the start of the treatment. Error bars indicate standard error of the mean.


Average of groups at end of experiment described in ( A ) . Error bars indicate standard deviation . Unpaired t - was used to calculate statistical significance . P - values are indicated in graph .

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

Average tumor weight of all groups at the end of the experiment described in (A). Error bars indicate standard deviation. Unpaired t-test was used to calculate statistical significance. P-values are indicated in the graph.


Immunoblotting for MAPK pathway components performed on tumors of groups .

Immunoblotting for MAPK pathway components performed on tumors of all groups.


Immunohistochemistry for Sirius Red ( for collagen ) H & amp ; amp ; E on tumors from groups , to identify grade of demarcation local invasion . Scale bar represents 500 ( upper row ) 100 ( lower row ) μm .

B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunohistochemistry for Sirius Red (staining for collagen) and H&amp;E on tumors from all groups, to identify grade of demarcation and local invasion. Scale bar represents 500 (upper row) and 100 (lower row) μm.


gDNA was isolated from another fragment of resection , PCR with insertion - specific primers was repeated . Primers amplifying exon 2 of MEK1WT were used as an input control . Source data are available online for this figure .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O

gDNA was isolated from another fragment of the tumor resection, and the PCR with the insertion-specific primers was repeated. Primers amplifying exon 2 of MEK1WT were used as an input control.Source data are available online for this figure.


for hematoxylin - eosin ( H & amp ; amp ; E ) p - ERK on PDX samples M032 . X1 , M032R1 . X1 , M032R2 . X1 , M032R4 . X1 , M032R5 . X1 . Stainings showed that p - ERK is higher in PDX derived from resistant metastases . Scale bar represents 100 μm .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O 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-GENEPROD O O O O O O O B-TISSUE O O O O O O O

Staining for hematoxylin-eosin (H&amp;E) and p-ERK on the PDX samples M032.X1, M032R1.X1, M032R2.X1, M032R4.X1, and M032R5.X1. Stainings showed that p-ERK is higher in the PDX derived from the resistant metastases. Scale bar represents 100 μm.


Immunoblotting for components of MAPK pathway confirmed reactivation of p - ERK in PDX derived from vemurafenib - resistant metastases , although p - ERK signal is heterogeneous in pre - treatment PDX .

B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O

Immunoblotting for components of the MAPK pathway confirmed reactivation of p-ERK in the PDX derived from the vemurafenib-resistant metastases, although the p-ERK signal is heterogeneous in the pre-treatment PDX.


Validation of BRAF amplification was performed by qPCR on gDNA derived from PDX , using primers for BRAF CRAF normalized on expression . Bars represent mean of three replicates , error bars indicate standard deviation . results confirmed that BRAF was amplified in PDX derived from M032R2 M032R5 , but not from M032R1 .

O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

Validation of the BRAF amplification was performed by qPCR on gDNA derived from the PDX, using primers for BRAF and CRAF and normalized on LINE expression. Bars represent the mean of three replicates, error bars indicate standard deviation. The results confirmed that BRAF was amplified in PDX derived from M032R2 and M032R5, but not from M032R1.


Presence of MEK1T55delinsRT was analyzed by PCR using insertion - specific primers . As a control , primers amplifying exon 2 of MEK1WT were used . insertion was found in M032R1 . X1 M032R4 . X1 .

O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

Presence of the MEK1T55delinsRT was analyzed by PCR using insertion-specific primers. As a control, primers amplifying exon 2 of MEK1WT were used. The insertion was found in M032R1.X1 and M032R4.X1.


A - C Calcium flux measured by FACScan for splenic B isolated from B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( see Figure EV1 , 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . addition of stimuli to is indicated by arrows .

O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

A-C Calcium flux measured by FACScan for splenic B cells isolated from B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (see Figure EV1, 80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). The addition of the stimuli to the cells is indicated by arrows.


D Representative microscopy showing Fab - PLA results measuring BCR proximity for IgM - BCR ( upper ) IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B . PLA signals are shown as red dots nuclei are visualized by DAPI . Scale bar : 5 μm . E Fab - PLA results are quantified by Blobfinder software presented as box plots . median values are highlighted as thick lines whiskers represent minimum maximum value . PLA signals ( dots / ) were counted from at least 100 for each sample . Data from treated samples were compared to data from resting , p - values were calculated by Kruskal - Wallis one - way of variance ( ANOVA ) .

O O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE B-CELL_TYPE O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-EXP_ASSAY 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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 Representative microscopy images showing Fab-PLA results measuring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. PLA signals are shown as red dots and the nuclei are visualized by DAPI staining. Scale bar: 5 μm.E The Fab-PLA results are quantified by Blobfinder software and presented as box plots. The median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. Data from treated samples were compared to data from resting cells, p-values were calculated by Kruskal-Wallis one-way analysis of variance (ANOVA).


A - C Calcium flux measured by FACScan for splenic B isolated from Lyn - deficient B1 - 8 transgenic mice after stimulation with ( A ) NIP15 - BSA ( 30 pM ) or 1NIP - pep ( 80 nM ) ; ( B ) Ac146 antibody ( 12 . 5 nM ) or Ac146Fab ( 25 nM ) ; ( C ) Ac38 antibody ( 12 . 5 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate addition of stimuli to .

O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-TISSUE B-CELL_TYPE O O B-GENEPROD O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

A-C Calcium flux measured by FACScan for splenic B cells isolated from Lyn-deficient B1-8 transgenic mice after stimulation with (A) NIP15-BSA (30 pM) or 1NIP-pep (80 nM); (B) Ac146 antibody (12.5 nM) or Ac146Fab (25 nM); (C) Ac38 antibody (12.5 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


D Representative microscopy showing Fab - PLA results monitoring BCR proximity for IgM - BCR ( upper ) IgD - BCR ( lower ) on untreated or treated B1 - 8 splenic B . Scale bar : 5 μm . E Quantified Fab - PLA results are presented as box plots , where median values are highlighted as thick lines whiskers represent minimum maximum value . PLA signals ( dots / ) were counted from at least 100 for each sample . p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O B-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE B-CELL_TYPE O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O 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 Representative microscopy images showing Fab-PLA results monitoring the BCR proximity for the IgM-BCR (upper) and the IgD-BCR (lower) on untreated or treated B1-8 splenic B cells. Scale bar: 5 μm.E Quantified Fab-PLA results are presented as box plots, where the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample. p-values were calculated by Kruskal-Wallis one-way ANOVA.


A - D Proximity between IgM - BCR on surface of 3046SM ( A , C ) or 3046M ( B , D ) before after a 1 - min stimulation with indicated reagents , assayed by Fab - PLA . Results are presented as representative microscopy ( A , B ) box plots after ( C , D ) . Scale bar : 5 μm . In box plots , median values are highlighted as thick lines whiskers represent minimum maximum value . PLA signals ( dots / ) were counted from at least 100 for each sample p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O 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-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O 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-D Proximity between IgM-BCR on the surface of 3046SM (A, C) or 3046M (B, D) cells before and after a 1-min stimulation with the indicated reagents, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). Scale bar: 5 μm. In the box plots, the median values are highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SM ( E , F ) 3046M ( G , H ) after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate addition of stimuli to .

O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O

E-H Calcium flux measured by FACScan for 3046SM (E, F) and 3046M (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


A - D Proximity between IgD - BCRs on surface of 3046SD ( A , C ) or 3046D ( B , D ) before after a 1 - min stimulation , assayed by Fab - PLA . Results are presented as representative microscopy ( A , B ) box plots after ( C , D ) . PLA signals are shown as red dots nuclei were visualized by DAPI . Scale bar : 5 μm . In box plots , median values were highlighted as thick lines whiskers represent minimum maximum value . PLA signals ( dots / ) were counted from at least 100 for each sample p - values were calculated by Kruskal - Wallis one - way ANOVA .

O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O 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-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O B-EXP_ASSAY 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-EXP_ASSAY O O O O 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-D Proximity between IgD-BCRs on the surface of 3046SD (A, C) or 3046D (B, D) cells before and after a 1-min stimulation, assayed by Fab-PLA. Results are presented as representative microscopy images (A, B) and box plots after quantification (C, D). PLA signals are shown as red dots and nuclei were visualized by DAPI staining. Scale bar: 5 μm. In the box plots, the median values were highlighted as thick lines and the whiskers represent the minimum and maximum value. PLA signals (dots/cells) were counted from at least 100 cells for each sample and p-values were calculated by Kruskal-Wallis one-way ANOVA.


E - H Calcium flux measured by FACScan for 3046SD ( E , F ) 3046D ( G , H ) after stimulation with NIP15 - BSA ( 30 pM ) , 1NIP - pep ( 80 nM ) , Ac146Fab ( 25 nM ) or Ac38Fab ( 25 nM ) . Arrows indicate addition of stimuli to .

E-H Calcium flux measured by FACScan for 3046SD (E, F) and 3046D (G, H) cells after stimulation with NIP15-BSA (30 pM), 1NIP-pep (80 nM), Ac146Fab (25 nM) or Ac38Fab (25 nM). Arrows indicate the addition of the stimuli to the cells.


Calcium flux measured by FACScan for 3046M ( A , D ) , 3046SD ( B , E ) 3046D ( C , F ) stimulated with anti - HC ( 2 μl / ml ) ( A , B , C ) , or anti - λ ( 2 μl / ml ) ( D , E , F ) antibodies with or without 1 min prestimulation with 1NIP - pep ( 80 nM ) . addition of antibodies is indicated by black arrows . 1NIP - pep was added immediately before recording . Data are representative of three independent experiments .

B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O

Calcium flux measured by FACScan for 3046M (A, D), 3046SD (B, E) and 3046D (C, F) cells stimulated with anti-HC (2 μl/ml) (A, B, C), or anti-λ (2 μl/ml) (D, E, F) antibodies with or without 1 min prestimulation with 1NIP-pep (80 nM). The addition of antibodies is indicated by black arrows. The 1NIP-pep was added immediately before recording. Data are representative of three independent experiments.


A - E B1 - 8 B , 3046SM , 3046M , 3046SD , or 3046D were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD19 antibody ( 62 nM ) , or costimulated with 1NIP - pep anti - CD19 antibody . calcium fluxes were measured by FACScan .

O O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O

A-E B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD19 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD19 antibody. The calcium fluxes were measured by FACScan.


F - J B1 - 8 B , 3046SM , 3046M , 3046SD , or 3046D were stimulated with 1NIP - pep ( 80 nM ) , or anti - CD20 antibody ( 62 nM ) , or costimulated with 1NIP - pep anti - CD20 antibody . calcium fluxes were measured by FACScan .

O O O B-ORGANISM I-ORGANISM I-ORGANISM B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O

F-J B1-8 B cells, 3046SM cells, 3046M cells, 3046SD cells, or 3046D cells were stimulated with 1NIP-pep (80 nM), or anti-CD20 antibody (62 nM), or costimulated with 1NIP-pep and anti-CD20 antibody. The calcium fluxes were measured by FACScan.


API delivery is blocked at low temperatures . SEY6210 were labeled for 5 min chased for times indicated . Identical experiments were performed at 10 ° , 14 ° , 30 ° C . API was recovered by immunoprecipitation followed by SDS - PAGE detected by a STORM phosphorimager ( Molecular Dynamics , Sunnyvale , CA ) . positions of prAPI mAPI are indicated .

B-GENEPROD O O O O O O O B-ORGANISM O 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-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O

API delivery is blocked at low temperatures. SEY6210 cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and 30°C. API was recovered by immunoprecipitation followed by SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and mAPI are indicated.


Precursor API is trapped in a prevacuolar compartment in vps18 . ( A ) Yeast JSR18Δ1 with plasmid pJSR9 ( vps18 ts ) was grown at permissive of 26 ° C . Before labeling , were incubated at either 26 ° or 38 ° C for 10 min . labeling was for 5 min , followed by indicated chase times .

O B-GENEPROD O O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times.


( B ) vps18 spheroplasts were shifted to 38 ° C for 5 min , labeled for 5 min , then chased for either 0 or 60 min . At each time point spheroplasts were collected by centrifugation resuspended in PS0 buffer . Total ( T ) fraction either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in presence of 0 . 2 % Triton X - 100 . supernatant ( S0 ) pellet ( P0 ) fractions were collected after centrifugation at 10 , 000 g for 5 min .

O O O B-GENEPROD B-CELL_TYPE O O O O 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-CELL_TYPE O O 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-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.


A mutant in API propeptide causes accumulation of prAPI on a nonvacuolar compartment . ( A ) Spheroplasts from an ape1Δ containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer . Total ( T ) fraction ( after differential lysis ) either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in presence of 0 . 2 % Triton X - 100 .

O O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O 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-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O

A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.


( B ) supernatant ( S200 ) pellet ( P200 ) fractions were collected after centrifugation at 5 , 000 g for 5 min . P200 fraction was resuspended in 10 % Ficoll , vacuoles were isolated by flotation through 4 % Ficoll . V , vacuole ; I , 4 / 10 % Ficoll interface ; P , gradient pellet . were detected by Western blotting . positions of prAPI , mAPI , mPrA are indicated .

O O O B-SUBCELLULAR O O O B-SUBCELLULAR 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-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(B) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


( C ) ape1Δ containing P22L API on a plasmid were labeled for 5 min , harvested by centrifugation , subjected to a nonradioactive chase in either nitrogen - containing ( SMD ) or nitrogen starvation ( SD - N ) medium . Samples were collected at times indicated immunoprecipitated with API antiserum . resulting SDS - PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager .

O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(C) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.


Precursor API is trapped within a vesicle in cvt17 mutants . ( A ) Precursor API is in a protease - protected compartment in cvt17 mutants . THY32 ( cvt17 ) spheroplasts were subjected to differential lysis protease treatment in PS200 buffer as in Fig

O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O

Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3


B ) Precursor API is not free in vacuolar lumen in cvt17 mutants . Differential fractionation was continued by resuspending P200 fraction in PS0 buffer separating supernatant ( S0 ) pellet ( P0 ) fractions by centrifugation at 10 , 000 g . S0 P0 fractions were treated with proteinase K in presence absence of Triton X - 100 as indicated . fractions were precipitated with 10 % TCA resolved by SDS PAGE ; of interest were detected by Western blotting . positions of prAPI , mAPI , mPrA are indicated .

O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


Precursor API is contained within subvacuolar vesicles in cvt17 mutants . ( A ) Isolated vacuoles ( V ) from cvt17 were lysed in PS0 buffer , loaded on top of an Optiprep step gradient , centrifuged at 170 , 000 g for 60 min as described in Materials Methods . Fractions VV , 0 / 1 . 06 interface ; LD , 1 . 06 region ; SV , 1 . 06 / 1 . 12 interface ; HD , 1 . 12 region .

O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Precursor API is contained within subvacuolar vesicles in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were lysed in PS0 buffer, loaded on top of an Optiprep step gradient, and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV, 1.06/1.12 interface; HD, 1.12 region.


( B ) An aliquot of fractions VV SV was treated with proteinase K either in presence or absence of Triton X - 100 as indicated . Because of presence of protease inhibitors in vesicle isolation procedure , an intermediate - sized API degradation product results from proteinase K treatment . V fraction represents one - tenth of load of Optiprep gradient . were detected by Western blotting . positions of prAPI mALP are indicated .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.


Immuno - EM of cvt17 . Yeast strains SEY6210 ( parental wild type ) THY32 ( cvt17 ) were grown in YPD prepared for microscopy as described in Materials Methods . ( A ) Wild - type probed with antibody against mature API . ( B ) A cvt17 section showing a vesicle containing prAPI probed with antibody against API proregion . ( C ) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API . N , nucleus ; V , vacuole ; ( arrows ) API containing vesicles . Bars , 0 . 5 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-ORGANISM O O O O O B-ORGANISM O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O O

Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and prepared for microscopy as described in Materials and Methods. (A) Wild-type cells probed with antibody against mature API. (B) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API. N, nucleus; V, vacuole; (arrows) API containing vesicles. Bars, 0.5 μm.


Characterization of API - containing vesicles . ( A ) Fractionations were performed as in Fig . 5 . total ( T ) , vacuole ( V ) , vacuolar vesicle ( VV ) , subvacuolar vesicle ( SV ) fractions are shown . were detected by immunoblotting as indicated in figure .

O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O

Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V), vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are shown. Proteins were detected by immunoblotting as indicated in the figure.


( B ) SYPRO orange detected by scanning mode on a Molecular Dynamics STORM phosphorimager . relative mobility of molecular mass standards are indicated on left . Polypeptides enriched in subvacuolar vesicles are indicated on right .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O

(B) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are indicated on the left. Polypeptides enriched in the subvacuolar vesicles are indicated on the right.


C Maximal cytochrome c release for DX / REL pairs with ≥ 3 biological replicates ) ; box - whisker plots summarize data ( box 25 % - 75 % ; belt = median ; dot = mean ; whiskers = minimum maximum values ) . Statistical analyses were performed using an unpaired two - sided Student ' s t - , with significance p < 0 . 05 ( trend p < 0 . 10 ) . Cyto c , cytochrome

O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE

C Maximal cytochrome c release for all DX/REL pairs with ≥3 biological replicates); box-whisker plots summarize data (box 25%-75%; belt=median; dot=mean; whiskers=minimum and maximum values). Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend p<0.10). Cyto c, cytochrome c


D Bak oligomerization in response to escalating tBid for DX / REL pairs . Relative mitochondrial loading per lane is assessed by densitometry , showing ration of loading control in REL lane with - matched DX lane at same tBid exposure ( no REL lane is underloaded compared to DX lane ) .

O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

D Bak oligomerization in response to escalating tBid concentration for DX/REL pairs. Relative mitochondrial protein loading per lane is assessed by densitometry, showing ration of loading control in REL lane with patient-matched DX lane at same tBid exposure (no REL lane is underloaded compared to DX lane).


A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin . Results are shown for three DX / REL neuroblastoma pairs , two with attenuated cytochrome c release ( CHLA15 / CHLA20 SKNBE1 / SKNBE2C ) one without ( CHLA122 / CHLA136 ) . Data information : data points are mean SD from triplicate wells , experiments are representative of at least three biological replicates dotted - represents 50 % viability .

O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-DISEASE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O B-CELL_LINE O B-CELL_LINE O O O O 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-EXP_ASSAY O

A In vitro viability curves after 72hr exposure to mafosfamide or doxorubicin. Results are shown for three DX/REL neuroblastoma cell line pairs, two with attenuated cytochrome c release (CHLA15/CHLA20 and SKNBE1/SKNBE2C) and one without (CHLA122/CHLA136). Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates dotted-line represents 50% viability.


C , In vitro viability for CHLA15 ( DX ) CHLA20 ( REL ) following ionizing radiation at 7 days Data information : data points are mean SD from triplicate wells , experiments are representative of at least three biological replicates , dotted - represents 50 % viability .

O O O O B-EXP_ASSAY O B-CELL_LINE O O O B-CELL_LINE O O O O O O O O O O O O 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-EXP_ASSAY O

C, In vitro viability for CHLA15 (DX) and CHLA20 (REL) following ionizing radiation at 7 days Data information: data points are mean and SD from triplicate wells, experiments are representative of at least three biological replicates, dotted-line represents 50% viability.


F Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 SY5Y , in comparison to cultured in escalating concentrations of crizotinib until resistant ( NB1643 - ALKR SY5Y - ALKR ) . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at points ) in a representative experiment from at least two biological replicates .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_LINE O B-GENEPROD B-CELL_LINE O B-GENEPROD O O O O O O O O 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 Cytochrome c release from mitochondria after exposure to tBid or BimBH3 peptide for parental NB1643 and SY5Y cells, in comparison to cells cultured in escalating concentrations of crizotinib until resistant (NB1643-ALKR and SY5Y-ALKR). Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C Transmission electron microscopy image was used to quantify mitochondrial , circularity roundness in DX / REL neuroblastoma pairs ( mean + / - SD shown ) . Data information : , n = 137 - 241 mitochondria per statistical analyses were performed using a two - Mann - Whitney U , with significance p < 0 . 05 ( trend p < 0 . 10 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O 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, C Transmission electron microscopy image analysis was used to quantify mitochondrial size, circularity and roundness in DX/REL neuroblastoma pairs (mean +/- SD shown). Data information: , n=137-241 mitochondria per cell line statistical analyses were performed using a two-tailed Mann-Whitney U test, with significance p<0.05 (trend p<0.10).


H In vitro viability of CHLA15 - ctrl , CHLA15 - shMFN2 , CHLA15 - shPACS2 , CHLA20 following 72hr exposure to ABT - 737 ; dotted - represents 50 % viability . Data information : data points are mean SD from triplicate wells , experiments are representative of at least two biological replicates .

O O O B-EXP_ASSAY O B-CELL_LINE O O O B-CELL_LINE O O O B-CELL_LINE O O O B-CELL_LINE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O

H In vitro viability of CHLA15-ctrl, CHLA15-shMFN2, CHLA15-shPACS2, and CHLA20 cells following 72hr exposure to ABT-737; dotted-line represents 50% viability. Data information: data points are mean and SD from triplicate wells, experiments are representative of at least two biological replicates.


I Mitochondrial cytochrome c release in response to tBid BimBH3 peptide in CHLA15 - Ctrl , CHLA15 - shMFN2 CHLA15 - shPACS2 . Data information : , data points are mean of duplicate wells ( SD < 0 . 05 at points ) in a representative experiment from at least two biological

O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD B-SMALL_MOLECULE O O B-CELL_LINE O O O B-CELL_LINE O O B-CELL_LINE O O O O O O O O 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 Mitochondrial cytochrome c release in response to tBid and BimBH3 peptide in CHLA15-Ctrl, CHLA15-shMFN2 and CHLA15-shPACS2 cells. Data information: , data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates


A , B ( black - tracing ) mitochondrial ( colored - tracing ) Ca2 + measured using reporters in CHLA15 ( n = 29 ) / CHLA20 ( n = 65 ) SKNBE1 ( n = 37 ) / SKNBE2C ( n = 52 ) pairs . ER Ca2 + release was induced by 100μM carbachol , an IP3R - agonist ; time added indicated by arrow ; tracings are mean + / - SD error bars . Coupling time ( time between achieving 50 % of maximal mitochondrial concentrations ) is an index of ER - mitochondrial proximity transfer efficiency ; plotted below . Data information : data is derived from at least 3 separate transfections ( biological replicates ) . statistical analyses were performed using an unpaired two - sided Student ' s t - , with significance p < 0 . 05 .

O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O 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-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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 Cytosolic (black-tracing) and mitochondrial (colored-tracing) Ca2+ concentration measured using fluorescent reporters in the CHLA15 (n=29 cells)/CHLA20 (n=65 cells) and SKNBE1 (n=37 cells)/SKNBE2C (n=52 cells) pairs. ER Ca2+ release was induced by 100μM carbachol, an IP3R-agonist; time added indicated by arrow; tracings are mean +/- SD error bars. Coupling time (time between achieving 50% of maximal cytosolic and mitochondrial concentrations) is an index of ER-mitochondrial proximity and transfer efficiency; plotted below. Data information: data is derived from at least 3 separate cell transfections (biological replicates). statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05.


F Cytochrome c release in response to tBid BimBH3 assessed . Data information : data points are mean of duplicate wells ( SD < 0 . 05 at points ) in a representative experiment from at least two biological replicates .

O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O 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 Cytochrome c release in response to tBid and BimBH3 assessed. Data information: data points are mean of duplicate wells (SD<0.05 at all points) in a representative experiment from at least two biological replicates.


A , B , C of ceramide species as measured by LC / MS from DX REL pair whole pellets . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates

O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-EXP_ASSAY 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, C Concentration of ceramide species as measured by LC/MS from DX and REL pair whole cell pellets. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each


D , E Cumulative ceramides sphingomyelins ( of C32 - C36 chain ) for DX / REL pairs . Data information : show mean of 3 - 4 biological replicates plotted ; 3 technical replicates each error bars are + / - SD .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O 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 Cumulative ceramides and sphingomyelins (of C32-C36 chain length) for DX/REL pairs. Data information: show mean of 3-4 biological replicates plotted; 3 technical replicates each error bars are +/- SD.


H Summary data for CHLA15 / CHLA20 SKNBE1 / SKNBE2C showing relative cytochrome c release when pre - incubated with GW4869 to inhibit ceramide generation , compared with untreated . Data information : data points from two ( CHLA15 ) or three ( SKNBE1 ) biological replicates are depicted in H . Statistical analyses were performed using an unpaired two - sided Student ' s t - , with significance p < 0 . 05 ( trend , p < 0 . 10 ) .

O O O O B-CELL_LINE O B-CELL_LINE I-CELL_LINE O B-CELL_LINE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O 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 Summary data for CHLA15/CHLA20 and SKNBE1/SKNBE2C showing relative cytochrome c release when pre-incubated with GW4869 to inhibit ceramide generation, compared with untreated cells. Data information: All data points from two (CHLA15) or three (SKNBE1) biological replicates are depicted in H. Statistical analyses were performed using an unpaired two-sided Student's t-test, with significance p<0.05 (trend, p<0.10).


A - D Mesenteric blood ( PECAM1 , red ) lymphatic vessels ( LYVE1 , green ; PROX1 , gray ) . Asterisks indicate lymphatic valves , arrow indicates a lymphatic vessel stub , arrowhead indicates an isolated lymphatic vessel fragmentE - H Blood vessels ( PECAM1 , green ) lymphatic vessels ( VEGFR - 3 , red ) in small intestinal wall .

O O O O B-TISSUE O B-GENEPROD O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE O

A-D Mesenteric blood (PECAM1, red) and lymphatic vessels (LYVE1, green; PROX1, gray). Asterisks indicate lymphatic valves, arrow indicates a lymphatic vessel stub, and arrowhead indicates an isolated lymphatic vessel fragmentE-H Blood vessels (PECAM1, green) and lymphatic vessels (VEGFR-3, red) in the small intestinal wall.


I , J Detection of chylous ascites ( arrows ) in VCiΔR26 mice at P6 .

O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O

I, J Detection of chylous ascites (arrows) in the VCiΔR26 mice at P6.


K , L LYVE1 of intestinal lymphatic vessels at P6 .

O O O B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O O O

K, L LYVE1 staining of intestinal lymphatic vessels at P6.


M - T LYVE1 of lymphatic vessels in intestinal wall in adult mice . Genotypes deletion lengths are indicated in ( U ) . U of LYVE1 areas in ( M - T ) . of i ∆ Vegfc gene deletion is indicated in months ( mo ) , Vegfd indicates VEGF - D genotype . Data are represented as mean ± . Significant differences were determined using one - way ANOVA Bonferroni post hoc compared to WT intestine represented in ( M ) . * P = 0 . 003 , * * P = 0 . 001 , * * * P = 0 . 0008 , ♯ P = 0 . 0002 , § P = 0 . 0001 .

O O O B-GENEPROD O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M-T LYVE1 staining of lymphatic vessels in the intestinal wall in adult mice. Genotypes and deletion lengths are indicated in (U).U Quantification of LYVE1 areas in (M-T). Length of the i∆Vegfc gene deletion is indicated in months (mo), and Vegfd indicates the VEGF-D genotype. Data are represented as mean ± SEM. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT intestine represented in (M). *P = 0.003, **P = 0.001, ***P = 0.0008, ♯P = 0.0002, §P = 0.0001.


Overview of small intestine cross section stained with red highlighting location of higher magnification in ( B - F ) ; ( I ) for entire villus ( II ) for villus base . β - Gal pattern of villus in wild - type ( Ctrl ) , Vegfc / LacZ ( VC ) , Vegfr3 / LacZ ( VR - 3 ) , Vegfr2 / LacZ ( VR - 2 ) mice . Higher magnification representing β - Gal of villus base in Vegfc / LacZmice . Data information : Arrows indicate VEGF - C expression in arterialSMC , arrowheads indicate VEGF - C expression in SMCfibers in villus , asterisks highlight VEGF - C expression in circular smooth muscle layer of intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

O O B-TISSUE I-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-ORGANISM O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

Overview of the small intestine cross section stained with nuclear red and highlighting the location of higher magnification images in (B-F); (I) for the entire villus and (II) for the villus base. β-Gal staining pattern of the villus in wild-type (Ctrl), Vegfc/LacZ (VC), Vegfr3/LacZ (VR-3), and Vegfr2/LacZ (VR-2) mice.Higher magnification images representing β-Gal staining of the villus base in Vegfc/LacZmice.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal reaction with smooth muscle actin ( SMA ) peroxidase . Data information : Arrows indicate VEGF - C expression in arterialSMC , arrowheads indicate VEGF - C expression in SMCfibers in villus , asterisks highlight VEGF - C expression in circular smooth muscle layer of intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

Vegfc/LacZβ-Gal staining reaction with smooth muscle actin (SMA) peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Vegfc / LacZβ - Gal LYVE1 peroxidase . Data information : Arrows indicate VEGF - C expression in arterialSMC , arrowheads indicate VEGF - C expression in SMCfibers in villus , asterisks highlight VEGF - C expression in circular smooth muscle layer of intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

Vegfc/LacZβ-Gal staining and LYVE1 peroxidase staining.Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Surface image of Vegfc / LacZβ - Gal - stained intestine ( left ) cross section counterstaining with PECAM1 ( right ) . Data information : Arrows indicate VEGF - C expression in arterialSMC , arrowheads indicate VEGF - C expression in SMCfibers in villus , asterisks highlight VEGF - C expression in circular smooth muscle layer of intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

Surface image of Vegfc/LacZβ-Gal-stained intestine (left) and cross section counterstaining with PECAM1 (right).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Immunofluorescence of lacteal lymphatic vessel ( LYVE1 ) , blood capillaries ( PECAM1 ) , smooth muscle ( SMA ) . Data information : Arrows indicate VEGF - C expression in arterialSMC , arrowheads indicate VEGF - C expression in SMCfibers in villus , asterisks highlight VEGF - C expression in circular smooth muscle layer of intestinal wall . Scale bars : 50 μm , except ( C ) inset 25 μm .

B-EXP_ASSAY O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O O B-TISSUE I-TISSUE O B-GENEPROD O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O

Immunofluorescence staining of lacteal lymphatic vessel (LYVE1), blood capillaries (PECAM1), and smooth muscle cells (SMA).Data information: Arrows indicate the VEGF-C expression in arterialSMC, arrowheads indicate the VEGF-C expression in SMCfibers in the villus, and asterisks highlight the VEGF-C expression in circular smooth muscle cell layer of the intestinal wall. Scale bars: 50 μm, except (C) inset 25 μm.


Two - month - old mice received tamoxifen were fed on high - fat diet ( HFD ) for seven before . change during seven of HFD , expressed as average fold change in comparison with starting . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 . comparisons at seven of HFD . Significant differences were determined using one - way ANOVA Bonferroni post hoc compared to WT . * P = 0 . 004 ; * * P = 0 . 003 . n = 16 , WT ; n = 6 , Vegfd − / − ; n = 16 , VCiΔR26 ; n = 5 , Vegfd − / − ; VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O B-ORGANISM O O O O O B-GENEPROD O O O O B-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Body weight change during seven weeks of HFD, expressed as average fold change in comparison with the starting weight. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.Body weight comparisons at seven weeks of HFD. Significant differences were determined using one-way ANOVA and Bonferroni post hoc analysis compared to WT. *P = 0.004; **P = 0.003. n = 16, WT; n = 6, Vegfd−/−; n = 16, VCiΔR26; n = 5, Vegfd−/−; VCiΔR26.


Two - month - old mice received tamoxifen were fed on high - fat diet ( HFD ) for seven before . Glucose tolerance ( GTT ) after six of HFD . Significant differences were determined using unpaired two - t - . * P = 0 . 014 ; * * P = 0 . 041 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Glucose tolerance test (GTT) after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.014; **P = 0.041. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen were fed on high - fat diet ( HFD ) for seven before . Total fatweight , fat from composition measurements after six of HFD , weights of visceral fat ( VF ) subcutaneous fat ( SF ) at time of necropsy . Significant differences were determined using unpaired two - t - . * P = 0 . 006 ; * * P = 0 . 001 ; # P = 0 . 008 ; § P = 0 . 006 . n = 4 in each group .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Total fatweight, fat percentage from body composition measurements after six weeks of HFD, and weights of visceral fat (VF) and subcutaneous fat (SF) at the time of necropsy. Significant differences were determined using unpaired two-tailed t-test. *P = 0.006; **P = 0.001; #P = 0.008; §P = 0.006. n = 4 in each group.


Two - month - old mice received tamoxifen were fed on high - fat diet ( HFD ) for seven before . Food consumption during fifth week of HFD . n = 9 , WT ; n = 10 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Food consumption during the fifth week of HFD. n = 9, WT; n = 10, VCiΔR26.


Two - month - old mice received tamoxifen were fed on high - fat diet ( HFD ) for seven before . Whole - mount immunofluorescence of blood ( PECAM1 , red ) lymphatic vessels ( LYVE1 , green ) in intestinal villi intestinal wall . of lacteal villus ( solid striped color bars , respectively ) intestinal wallLYVE1 + from represented in ( F ) . Significant differences were determined using unpaired two - t - . * P = 0 . 0002 ; * * P = 0 . 00007 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O B-GENEPROD O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis.Whole-mount immunofluorescence staining of blood (PECAM1, red) and lymphatic vessels (LYVE1, green) in intestinal villi and intestinal wall.Quantification of the lacteal and villus length (solid and striped color bars, respectively) and the intestinal wallLYVE1+ area percentage from images represented in (F). Significant differences were determined using unpaired two-tailed t-test. *P = 0.0002; **P = 0.00007. n = 5, WT; n = 6, VCiΔR26.


Two - month - old mice received tamoxifen were fed on high - fat diet ( HFD ) for seven before . Free fatty acid ( FFA ) cholesterol measurements from feces after six of HFD . Significant differences were determined using unpaired two - t - . * P = 0 . 001 ; * * P = 0 . 007 . n = 5 , WT ; n = 6 , VCiΔR26 .

O O O O O B-ORGANISM O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O O O O O O O O O O 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-ORGANISM O

Two-month-old mice received tamoxifen and were fed on high-fat diet (HFD) for seven weeks before analysis. Free fatty acid (FFA) and cholesterol measurements from the feces after six weeks of HFD. Significant differences were determined using unpaired two-tailed t-test. *P = 0.001; **P = 0.007. n = 5, WT; n = 6, VCiΔR26.


D , RT - qPCR time courses . Promoters of indicated oscillating genes were used to drive expression of a destabilized , GFP from a single copy integrated transgene ; gfp mRNA endogenous transcript driven by same promoter were quantified from same samples . Relative expression was calculated as - dCT = - ( target CT values - actin CT values ) then mean normalized for each trace individually . Peak phases ( ϕPeak ) amplitudes ( A ) for endogenous transcripts are from ( Meeuse et al . , 2020 ) . qPCR was performed in technical replicate , shown are averages .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O 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-GENEPROD O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O

D, RT-qPCR time courses. Promoters of the indicated oscillating genes were used to drive expression of a destabilized, nuclear GFP protein from a single copy integrated transgene; gfp mRNA and the endogenous transcript driven by the same promoter were quantified from the same RNA samples. Relative expression was calculated as -dCT = - (target CT values - actin CT values) and then mean normalized for each trace individually. Peak phases (ϕPeak) and amplitudes (A) for the endogenous transcripts are from (Meeuse et al., 2020). qPCR was performed in technical replicate, shown are averages.


B , C showing trend - corrected luminescence as in ( A ) , for indicated RNAi conditions causing altered numbers ( B ) or durations ( C ) of molts , respectively .

O O O O O O O B-EXP_ASSAY O O 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, C Heatmap showing trend-corrected luminescence as in (A), for indicated RNAi conditions causing altered numbers (B) or durations (C) of molts, respectively.


C , Image of an L1 synchronized grh - 1 : : aid ( HW2418 ) grown at 20 ° C on a 250 μM auxin - containing plates . A lethargic was transferred to an agar - pad containing microscopy slide were collected every 1 sec , using DIC , 100x magnification . Selected of Movie EV1 are shown . Time stamp ( min : sec ) is indicated . Arrows indicate phenotypic features : loosening of cuticle ( 0 : 00 ) ; back - - forth movements ( 0 : 05 ) ; inflation of cuticle ( 2 : 46 ) ; vesicles underneath loosened cuticle ( 6 : 11 ) ; rupturing of cuticle ( 6 : 20 , 6 : 40 ) .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O 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-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O O O

C, Image sequence of an L1 synchronized grh-1::aid animal (HW2418) grown at 20°C on a 250 μM auxin-containing plates. A lethargic animal was transferred to an agar-pad containing microscopy slide and images were collected every 1 sec, using DIC, 100x magnification. Selected images of Movie EV1 are shown. Time stamp (min:sec) is indicated. Arrows indicate phenotypic features: loosening of the cuticle (0:00); back-and-forth movements (0:05); inflation of the cuticle (2:46); vesicles underneath loosened cuticle (6:11); rupturing of the cuticle (6:20, 6:40).


A , of of grh - 1 : : aid constitutively expressing luciferase ( HW2434 ) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O

A, Quantification of the percentage of grh-1::aid animals constitutively expressing luciferase (HW2434) that enter each of four molts molt upon hatching into increasing concentrations of auxin as indicated.


B , Boxplot showing duration of M1 , M2 M3 of treated with indicated concentrations of auxin . that failed to develop beyond M3 in ( A ) were excluded . horizontal represents median , hinges extend to first third quartiles , whiskers extend up to most extreme value within 1 . 5 * IQR ( interquartile range ) of median . Significant differences relative to 0 nM auxin are indicated . P - values were determined by a non - parametric Wilcoxon rank sum that does not assume normal distribution .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O

B, Boxplot showing the duration of M1, M2 and M3 of animals treated with indicated concentrations of auxin. Animals that failed to develop beyond M3 in (A) were excluded. The horizontal line represents the median, hinges extend to first and third quartiles, and the whiskers extend up to the most extreme value within 1.5*IQR (interquartile range) of the median. Significant differences relative to 0 nM auxin are indicated. P-values were determined by a non-parametric Wilcoxon rank sum test that does not assume normal distribution.