( A ) Representative western blots showing the levels of SQR , TST , ETHE1 and SUOX protein from the brain of 5 wild - type ( WT ) and five mutant ( Mut ) mice ( B - E ) Relative levels of proteins normalized to Vinculin .

(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein from the brain of 5 wild-type (WT) and five mutant (Mut) mice (B-E) Relative levels of proteins normalized to Vinculin.


( A ) Total GSH amount normalized to protein from kidney and ( B ) brain of wild - type ( WT ) and mutant ( Mut ) mice . Error bars represent SDs of at least 3 animals per group .

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(A) Total GSH amount normalized to protein from kidney and (B) brain of wild-type (WT) and mutant (Mut) mice. Error bars represent SDs of at least 3 animals per group.


( C ) Thiosulfate levels in urine and ( D ) plasma normalized to protein amount . Error bars represent SDs of 9 animals per group ( urine ) and 6 animals per group ( plasma ) .

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(C) Thiosulfate levels in urine and (D) plasma normalized to protein amount. Error bars represent SDs of 9 animals per group (urine) and 6 animals per group (plasma).


( E ) H2S amount normalized to tissue from kidney and ( F ) brain . Error bars represent SDs of 7 / 8 animals per group .

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(E) H2S amount normalized to tissue from kidney and (F) brain. Error bars represent SDs of 7/8 animals per group.


( G ) Acylcarnitine C4 , C5 and C6 levels from plasma , normalized to volume . Error bars represent SDs of 5 wild - type and 3 mutants .

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(G) Acylcarnitine C4, C5 and C6 levels from plasma, normalized to volume. Error bars represent SDs of 5 wild-type and 3 mutants.


B . Table showing location , number of inserted or deleted residues and motility phenotype of all 18 stalk mutants . Quantification and classification are based on three technical repetitions . C . Example kymographs for ' Directional - WT like ' , ' Diffusive like ' , ' No movement ' , and ' Transient binding ' . Magnified area for ' Diffusive like ' motion shows run of a single molecule .

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B. Table showing location, number of inserted or deleted residues and motility phenotype of all 18 stalk mutants. Quantification and classification are based on three technical repetitions. C. Example kymographs for 'Directional - WT like', 'Diffusive like', 'No movement', and 'Transient binding'. Magnified area for 'Diffusive like' motion shows run of a single molecule.


D . Table showing the type of movement found for wild - type , mutant 2 , mutant 5 , and mutant 13 in a modified single - molecule assay with and without ATP . Classification of type of movement is based on two repetitions of different dynein preparations .

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D. Table showing the type of movement found for wild-type, mutant 2, mutant 5, and mutant 13 in a modified single-molecule assay with and without ATP. Classification of type of movement is based on two repetitions of different dynein preparations.


A - D Microtubule affinity measured by a cosedimentation assay in the apo state ( full line ) and in the presence of ATP ( dashed line ) , and AMPPNP ( dotted line ) for wild - type ( A ) , mutant 2 ( B ) , mutant 5 ( C ) , and mutant 13 ( D ) .

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A-D Microtubule affinity measured by a cosedimentation assay in the apo state (full line) and in the presence of ATP (dashed line), and AMPPNP (dotted line) for wild-type (A), mutant 2 (B), mutant 5 (C), and mutant 13 (D).


E . Microtubule stimulated ATPase activity of wild - type ( grey ) , mutant 2 ( red ) , mutant 5 ( orange ) , and mutant 13 ( purple ) . F . Bar plot of basal ATPase activity of wild - type ( grey ) , mutant 2 ( red ) , mutant 5 ( orange ) , and mutant 13 ( purple ) . Data information : Error bars show standard deviation of three repetitions of different dynein preparations .

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E. Microtubule stimulated ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). F. Bar plot of basal ATPase activity of wild-type (grey), mutant 2 (red), mutant 5 (orange), and mutant 13 (purple). Data information: Error bars show standard deviation of three repetitions of different dynein preparations.


A Proteins captured by BGL3 and RMRP antisense purifications . Proteins with an absolute log2 ( fold change ) > 1 and - log10 ( p - value ) > 1 . 3 ( compared with control purification ) were considered as proteins enriched by BGL3 specifically .

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A Proteins captured by BGL3 and RMRP antisense purifications. Proteins with an absolute log2 (fold change) > 1 and -log10 (p-value) > 1.3 (compared with control purification) were considered as proteins enriched by BGL3 specifically.


C & D RNA pull - down assay for BGL3 - BARD1 / PARP1 interactions before and after irradiation . Biotinylated in vitro - transcribed LncRNA - BGL3 sense or antisense transcripts were incubated with HEK - 293T cell lysates with or without ionizing radiation ( 10 Gy ) with 1 hr recovery for in vitro streptavidin RNA pull - down assays , followed by Western blots using the indicated antibodies .

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C&D RNA pull-down assay for BGL3-BARD1/PARP1 interactions before and after irradiation. Biotinylated in vitro-transcribed LncRNA-BGL3 sense or antisense transcripts were incubated with HEK-293T cell lysates with or without ionizing radiation (10 Gy) with 1 hr recovery for in vitro streptavidin RNA pull-down assays, followed by Western blots using the indicated antibodies.


HA - tagged full - length or deletion mutants of BARD1 were transfected into 293T cells . 48 hr later , BGL3 RNA pull - down assay were performed , and arrowheads indicate the bands for HA - tagged full - length or deletion mutants of

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HA-tagged full-length or deletion mutants of BARD1 were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed, and arrowheads indicate the bands for HA-tagged full-length or deletion mutants of BARD1


HA - tagged full - length or deletion mutants of PARP1 ( H ) were transfected into 293T cells . 48 hr later , BGL3 RNA pull - down assay were performed ,

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HA-tagged full-length or deletion mutants of PARP1(H) were transfected into 293T cells. 48 hr later, BGL3 RNA pull-down assay were performed,


A BGL3 is recruited to DNA damage sites . U2OS cells were subjected to laser micro - irradiation to generate DSBs in a line pattern . The relocation kinetics of BGL3 to DSBs was monitored by RNA fluorescence in situ hybridization ( FISH ) in a time course as indicated . BGL3 intensities at the laser line were normalized into a numerical value using Nikon NIS - Elements AR software ( version 4 . 40 . 00 ) . Data are presented as mean ± SD of four biological replicates .

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A BGL3 is recruited to DNA damage sites. U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. The relocation kinetics of BGL3 to DSBs was monitored by RNA fluorescence in situ hybridization (FISH) in a time course as indicated. BGL3 intensities at the laser line were normalized into a numerical value using Nikon NIS-Elements AR software (version 4.40.00). Data are presented as mean ±SD of four biological replicates.


B Chromosome aberrations induced by BGL3 depletion . Wild - type or BGL3 depleted BJ - 5ta cells were used in the metaphase spread analysis for spontaneous DNA breaks . Representative image ( left panel ) and the percentage of cells containing at least one DNA break ( right panel ) . Data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 .

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B Chromosome aberrations induced by BGL3 depletion. Wild-type or BGL3 depleted BJ-5ta cells were used in the metaphase spread analysis for spontaneous DNA breaks. Representative image (left panel) and the percentage of cells containing at least one DNA break (right panel). Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, ** P<0.01.


C MCF - 7 cells were treated as indicated , and cell response to ionizing radiation were analyzed by colony formation assays ( left panel ) . Data are presented as mean ± SEM of four independent experiments , analyzed by two - way analysis of variance ( ANOVA ) , * * P < 0 . 01 . Knock - down efficiency of BGL3 siRNA was examined by qRT - PCR and normalized to β - Actin ( middle panel ) , data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 . Knock - down efficiency of CtIP siRNA was examined by blotting ( right panel ) .

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C MCF-7 cells were treated as indicated, and cell response to ionizing radiation were analyzed by colony formation assays (left panel). Data are presented as mean ± SEM of four independent experiments, analyzed by two-way analysis of variance (ANOVA), **P < 0.01. Knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-Actin (middle panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01. Knock-down efficiency of CtIP siRNA was examined by blotting (right panel).


D MCF - 7 cells were transfected with the indicated siRNAs . Cell sensitivity to camptothecin ( CPT ) , hydroxyurea ( HU ) , etoposide ( ETO ) , or ionizing radiation was determined by MTS assays . Data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * P < 0 . 05 , * * P < 0 . 01 , NS : no significant difference .

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D MCF-7 cells were transfected with the indicated siRNAs. Cell sensitivity to camptothecin (CPT), hydroxyurea (HU), etoposide (ETO), or ionizing radiation was determined by MTS assays. Data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.


E BGL3 deficiency inhibits DNA damage repair . Quantification of γ - H2AX foci at indicated times after irradiation ( 2Gy ) are presented . Data shown are results of three independent experiments ( 100 cells for each experiment ) , presented as mean ± SD , two - tailed Student ' s t test , * P < 0 . 05 , * * P < 0 . 01 , NS : no significant difference .

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E BGL3 deficiency inhibits DNA damage repair. Quantification of γ-H2AX foci at indicated times after irradiation (2Gy) are presented. Data shown are results of three independent experiments (100 cells for each experiment), presented as mean ± SD, two-tailed Student's t test, *P < 0.05, **P < 0.01, NS: no significant difference.


A & B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR ( A ) or NHEJ assay ( B ) as described in Methods . Data presented as mean ± SD of four biological replicates , and positive cell percentages as compared with control group . Two - tailed Student ' s t test , * * P < 0 . 01 . NS : no significant difference .

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A&B HEK293 cells integrated with HR or NHEJ reporter were transfected with the indicated siRNAs and subjected to the HR (A) or NHEJ assay (B) as described in Methods. Data presented as mean ±SD of four biological replicates, and positive cell percentages as compared with control group. Two-tailed Student's t test, **P < 0.01. NS: no significant difference.


C - F Wild - type ( CTRL ) or BGL3 knockdown U2OS cells were subjected to micro - irradiation ; 1 hr later , cells were fixed and processed for immunostaining with indicated antibodies . Representative images of BRCA1 ( C ) , BARD1 ( D ) , RAD51 ( E ) , and RPA70 ( F ) accumulation at sites of laser - induced DNA damage are shown .

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C-F Wild-type (CTRL) or BGL3 knockdown U2OS cells were subjected to micro-irradiation; 1 hr later, cells were fixed and processed for immunostaining with indicated antibodies. Representative images of BRCA1 (C), BARD1 (D), RAD51 (E), and RPA70 (F) accumulation at sites of laser-induced DNA damage are shown.


Parental or BGL3 - deficient U2OS cells were subjected to laser micro - irradiation to generate DSBs in a line pattern . DDR factor recruitment were examined 2 hr later . Data shown are the average of three independent experiments , and 100 cells were counted for each experiment . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , * * P < 0 . 01 .

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Parental or BGL3-deficient U2OS cells were subjected to laser micro-irradiation to generate DSBs in a line pattern. DDR factor recruitment were examined 2 hr later. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, **P < 0.01.


U2OS cells were transfected with the indicated siRNAs , and 48 hr later , HR efficiency were determined using the reporter assay , as described in the Methods . Data shown are the average of three independent experiments . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , NS : no significant difference .

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U2OS cells were transfected with the indicated siRNAs, and 48 hr later, HR efficiency were determined using the reporter assay, as described in the Methods. Data shown are the average of three independent experiments. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference.


E U2OS cells were transfected with the indicated siRNA , and RAD51 accumulation at sites of laser - induced DNA damage were examined . Data shown are the average of three independent experiments , and 100 cells were counted for each experiment . Data are presented as mean ± SD , analyzed by two - tailed Student ' s t test , NS : no significant difference . Knock - down efficiency of BARD1 siRNA was examined by blotting ( bottom left panel ) , and knock - down efficiency of BGL3 siRNA was examined by qRT - PCR and normalized to β - actin ( bottom right panel ) , data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 .

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E U2OS cells were transfected with the indicated siRNA, and RAD51 accumulation at sites of laser-induced DNA damage were examined. Data shown are the average of three independent experiments, and 100 cells were counted for each experiment. Data are presented as mean ± SD, analyzed by two-tailed Student's t test, NS: no significant difference. Knock-down efficiency of BARD1 siRNA was examined by blotting (bottom left panel), and knock-down efficiency of BGL3 siRNA was examined by qRT-PCR and normalized to β-actin (bottom right panel), data are presented as mean ± SD of three biological replicates. Two-tailed Student's t test, **P < 0.01.


A BGL3 promotes BARD1 - HP1γ interactions . 293T cells expressing the indicated siRNAs were irradiated ( 10 Gy ) , and BARD1 - HP1γ interactions examined at the indicated time points .

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A BGL3 promotes BARD1-HP1γ interactions. 293T cells expressing the indicated siRNAs were irradiated (10 Gy), and BARD1-HP1γ interactions examined at the indicated time points.


B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells . 293T cells expressing the indicated siRNAs and plasmids were irradiated ( 10 Gy ) . Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies .

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B BGL3 promotes RAP80 binding to K63 ubiquitin chain in cells. 293T cells expressing the indicated siRNAs and plasmids were irradiated (10 Gy). Cell lysates were immunoprecipitated with HA beads and subjected to immunoblot with the indicated antibodies.


C BGL3 promotes BARD1 - HP1γ ( up panel ) and BARD1 - RAD51 ( middle panel ) interactions in vitro . GST - HP1γ , GST - RAD51 , and FLAG - BARD1 were overexpressed and purified from cells as indicated in Methods . FLAG - BARD1 was divided into two equal parts , one part was used for BARD1 - HP1γ interaction analysis ( up panel ) , and another part was used for BARD1 - RAD51 interaction analysis ( middle panel ) . BARD1 - HP1γ ( up panel ) and BARD1 - RAD51 ( middle panel ) interactions with or without BGL3 were respectively detected by GST pull - down assays , here GST was used as a pull - down control .

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C BGL3 promotes BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions in vitro. GST-HP1γ, GST-RAD51, and FLAG-BARD1 were overexpressed and purified from cells as indicated in Methods. FLAG-BARD1 was divided into two equal parts, one part was used for BARD1-HP1γ interaction analysis (up panel), and another part was used for BARD1-RAD51 interaction analysis (middle panel). BARD1-HP1γ (up panel) and BARD1-RAD51 (middle panel) interactions with or without BGL3 were respectively detected by GST pull-down assays, here GST was used as a pull-down control.


A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs . U2OS cells were treated with DMSO , PARP inhibitor ( olaparib ) , ATM inhibitor ( KU - 55933 ) , ATR inhibitor ( VE - 821 ) or caffeine . BGL3 recruitment to DNA damage sites was assessed by RNA - FISH assays . B Quantification of the positive cells . For each group , 100 randomly selected cells were counted . Data are presented as mean ± SD of three biological replicates . Two - tailed Student ' s t test , * * P < 0 . 01 , N : no significant difference .

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A PARP1 inhibitor treatment abolished BGL3 recruitment to DSBs. U2OS cells were treated with DMSO, PARP inhibitor (olaparib), ATM inhibitor (KU-55933), ATR inhibitor (VE-821) or caffeine. BGL3 recruitment to DNA damage sites was assessed by RNA-FISH assays. B Quantification of the positive cells. For each group, 100 randomly selected cells were counted. Data are presented as mean ±SD of three biological replicates. Two-tailed Student's t test, **P < 0.01, N: no significant difference.


C PARP inhibitor mainly affected BGL3 recruitment at an early time point . U2OS cells were treated with DMSO or olaparib , and BGL3 recruitment was examined following laser micro - irradiation at indicated time point . Data presented are the average of three independent experiments , and 100 cells were counted for each experiment . Two - tailed Student ' s t test , * * P < 0 . 01 , N : no significant difference .

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C PARP inhibitor mainly affected BGL3 recruitment at an early time point. U2OS cells were treated with DMSO or olaparib, and BGL3 recruitment was examined following laser micro-irradiation at indicated time point. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.


D U2OS cells were transfected with the indicated siRNAs , and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH . Data presented are the average of three independent experiments , and 100 cells were counted for each experiment . Two - tailed Student ' s t test , * * P < 0 . 01 , N : no significant difference .

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D U2OS cells were transfected with the indicated siRNAs, and BGL3 recruitment to DSBs were monitored at the indicated time point by RNA FISH. Data presented are the average of three independent experiments, and 100 cells were counted for each experiment. Two-tailed Student's t test, **P < 0.01, N: no significant difference.


B . Total quantified proteome depth in tissues ( n = 18 ) , primary cells ( n = 12 ) , immortalized cell lines ( n = 4 ) and all samples ( n = 34 ) . In all cases , n means biological replicates unless otherwise indicated . The upper - and lower panel shows the number of quantified protein groups and peptides , respectively . C . Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank ( Liver : bulk liver biopsy , HepA : hepatic artery , PorV : portal vein , Cell lines : mixture of human liver - derived immortalized cell lines ) . Number of biological replicates is same as Panel ( A ) .

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B. Total quantified proteome depth in tissues (n=18), primary cells (n=12), immortalized cell lines (n=4) and all samples (n=34). In all cases, n means biological replicates unless otherwise indicated. The upper- and lower panel shows the number of quantified protein groups and peptides, respectively. C. Dynamic range of the different proteomes based on median LFQ intensity ordered by abundance rank (Liver: bulk liver biopsy, HepA: hepatic artery, PorV: portal vein, Cell lines: mixture of human liver-derived immortalized cell lines). Number of biological replicates is same as Panel (A).


B . Cumulative protein abundance of liver biopsy , haptic artery and portal vein as a function of protein rank , with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated .

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B. Cumulative protein abundance of liver biopsy, haptic artery and portal vein as a function of protein rank, with the total of the top ten abundant proteins and number of proteins that comprise four quartiles indicated.


D . Cumulative protein abundance of liver cell types as a function of protein rank , indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles .

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D. Cumulative protein abundance of liver cell types as a function of protein rank, indicating the total of the top ten abundant proteins and number of proteins that comprise the four quartiles.


F . The bar plot shows the contribution of each organelle to total cellular protein mass , also accounting for unassigned proteins . Percentage of cytosol , mitochondria , nucleoli , plasma membrane and actin filaments is indicated .

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F. The bar plot shows the contribution of each organelle to total cellular protein mass, also accounting for unassigned proteins. Percentage of cytosol, mitochondria, nucleoli, plasma membrane and actin filaments is indicated.


A . Total protein copy number of the oxidative phosphorylation complex I - V in bulk liver tissue ( n = 6 independent biological replicates ) and primary cell types ( isolated primary cells from n = 3 individuals for each cell type ) . Values are presented as mean ± s . d . B . Protein copy number for members of the oxidative phosphorylation complex I - V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated .

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A. Total protein copy number of the oxidative phosphorylation complex I-V in bulk liver tissue (n=6 independent biological replicates) and primary cell types (isolated primary cells from n=3 individuals for each cell type). Values are presented as mean ± s.d. B. Protein copy number for members of the oxidative phosphorylation complex I-V in hepatocytes and Kupffer cells with Pearson correlation coefficient indicated.


D . Top ten uniquely quantified proteins per cell type with LFQ intensities [ Log10 ] and copy numbers [ Log10 ] .

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D. Top ten uniquely quantified proteins per cell type with LFQ intensities [Log10] and copy numbers [Log10].


A . Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types , with columns showing three biological replicates ( n = 3 ) of four cell types and rows significant proteins . Significance was calculated by one - way ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Frames and numbers indicate four clusters of proteins highly enriched in each 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 O O O O O O O O O O O O

A. Unsupervised hierarchical clustering of proteins significantly differentially abundant across cell types, with columns showing three biological replicates (n=3) of four cell types and rows significant proteins. Significance was calculated by one-way ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Frames and numbers indicate four clusters of proteins highly enriched in each cell type.


A . Total quantified proteins at day one , three and seven upon primary cell culture in hepatocytes ( hHEP ) , hepatic stellate cells ( hHSC ) and liver sinusoidal endothelial cells ( LSEC ) . In all cases , number of biological replicates is 3 . Values are presented as mean ± s . d . No additional replications of the experiment was done in laboratory .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Total quantified proteins at day one, three and seven upon primary cell culture in hepatocytes (hHEP), hepatic stellate cells (hHSC) and liver sinusoidal endothelial cells (LSEC). In all cases, number of biological replicates is 3. Values are presented as mean ± s.d. No additional replications of the experiment was done in laboratory.


F . Protein expression patterns over the course of primary hHSC culture ( n = 3 ) . Values are presented as mean ± s . d . Significance was calculated by ANOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * P < 0 . 01 , and * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Protein expression patterns over the course of primary hHSC culture (n=3). Values are presented as mean ± s.d. Significance was calculated by ANOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of **P < 0.01, and ***P < 0.001.


A . Hierarchical clustering of proteins significantly differentially abundant between NASH ( n = 20 ) , cirrhosis ( n = 10 ) and control groups ( n = 15 ) . Significance was calculated by ANCOVA , followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) . Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated . No additional replications of the experiment was done in laboratory .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Hierarchical clustering of proteins significantly differentially abundant between NASH (n=20), cirrhosis (n=10) and control groups (n=15). Significance was calculated by ANCOVA, followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05). Two major clusters of proteins were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH and controls and Cluster 2 downregulated. No additional replications of the experiment was done in laboratory.


D . Box ‐ and ‐ whisker plot showing the distribution of log2 ‐ intensity values of statistical significantly regulated proteins across three groups . Number of independent biological replicates is n = 15 , 20 and 10 for the control , NASH and cirrhosis group , respectively . The black line in the middle of the box is the median , the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers ( Q3 + 1 . 5 * IQR , Q1 ‐ 1 . 5 * IQR ) where IQR is the interquartile range ( Q3 - Q1 ) . Significance was defined by ANCOVA followed by Benjamini - Hochberg correction for multiple hypothesis testing ( FDR < 0 . 05 ) with a significance level of * * * P < 0 . 001 . E . Heatmap of CYP 450 family members that are statistically significantly abundant between three groups . Data was presented as mean protein abundance followed by Z - score normalization across . Number of replicates is same as Panel ( C ) . F . Extracellular matrix ( ECM ) remodeling in liver cirrhosis . Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control ( upper ) and cirrhosis ( lower ) groups , highlighting top shifted ECM components in the cirrhosis group as compared with the control group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Box‐and‐whisker plot showing the distribution of log2‐intensity values of statistical significantly regulated proteins across three groups. Number of independent biological replicates is n=15, 20 and 10 for the control, NASH and cirrhosis group, respectively. The black line in the middle of the box is the median, the top and the bottom of the box represent the upper and lower quartile values of the data and the whiskers represent the upper and lower limits for considering outliers (Q3+1.5*IQR, Q1‐1.5*IQR) where IQR is the interquartile range (Q3-Q1). Significance was defined by ANCOVA followed by Benjamini-Hochberg correction for multiple hypothesis testing (FDR < 0.05) with a significance level of ***P < 0.001. E. Heatmap of CYP 450 family members that are statistically significantly abundant between three groups. Data was presented as mean protein abundance followed by Z-score normalization across. Number of replicates is same as Panel (C). F. Extracellular matrix (ECM) remodeling in liver cirrhosis. Upper and lower panel shows the abundance rank of proteins involved in ECM organization in the control (upper) and cirrhosis (lower) groups, highlighting top shifted ECM components in the cirrhosis group as compared with the control group.


Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane . The apical MTs were associated with both LUZP1 and TJs . Scale bar , 10 μm ( low magnification ) and 1 μm ( high magnification ) . Representative immunoelectron micrographs of Eph4 cells . Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin . MV , microvilli ; AJ , adherens junction , DS , desmosome . Scale bars , 200 nm .

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

Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane. The apical MTs were associated with both LUZP1 and TJs. Scale bar, 10 μm (low magnification) and 1 μm (high magnification).   Representative immunoelectron micrographs of Eph4 cells. Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin. MV, microvilli; AJ, adherens junction, DS, desmosome. Scale bars, 200 nm.


Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ZO - 1 / - 2 DKO cells were marked by asterisks ( * ) . The LUZP1 junctional localization was apparently disrupted in TJ - deficient ZO - 1 / - 2 DKO cells . Scale bar , 10 μm . Representative confocal micrographs of transient E - cadherin knockout ( KO ) in WT Eph4 cells . E - cadherin KO cells were marked by asterisks ( * ) . The LUZP1 junctional localization seemed not to change between AJ - deficient E - cadherin KO and WT cells . Scale bar , 10 μm .

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

Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ZO-1/-2 DKO cells were marked by asterisks (*). The LUZP1 junctional localization was apparently disrupted in TJ-deficient ZO-1/-2 DKO cells. Scale bar, 10 μm.   Representative confocal micrographs of transient E-cadherin knockout (KO) in WT Eph4 cells. E-cadherin KO cells were marked by asterisks (*). The LUZP1 junctional localization seemed not to change between AJ-deficient E-cadherin KO and WT cells. Scale bar, 10 μm.


Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [ 19 . 19 ± 10 . 02 arbitrary units ( a . u . ) ( WT ) vs . 2 . 92 ± 1 . 81 a . u . ( LUZP1 KO ) ] . n = 3 . * * p < 0 . 01 ( Mann - Whitney U test ) . Bars and error bars represent the mean ± SD .

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

Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [19.19 ± 10.02 arbitrary units (a.u.) (WT) vs. 2.92 ± 1.81 a.u. (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD.


Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ppMLC levels within CRs were clearly reduced in ZO - 1 / - 2 DKO cells compared to those in WT cells . Scale bar , 10 μm . Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO - 1 / - 2 DKO cells [ 119 . 62 ± 31 . 62 μm2 ( WT ) vs . 199 . 96 ± 94 . 22 μm2 ( LUZP1 KO ) ] ( See also Fig 1I ) . n = 3 . * * p < 0 . 01 ( unpaired t test ) . Bars and error bars represent the mean ± SD .

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

Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ppMLC levels within CRs were clearly reduced in ZO-1/-2 DKO cells compared to those in WT cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO-1/-2 DKO cells [119.62 ± 31.62 μm2 (WT) vs. 199.96 ± 94.22 μm2 (LUZP1 KO)] (See also Fig 1I). n = 3. **p < 0.01 (unpaired t test). Bars and error bars represent the mean ± SD.


In vitro direct binding assay between FLAG - LUZP1 and GST - MLC . Representative immunoblots are shown . S , supernatant ; P , pellet . A representative plot in which the amount of bound GST - WT - , AA - , or DD - MLC / FLAG - LUZP1 was plotted with the Michaelis - Menten fitted curve . Bar plots with dot density plots showing that the dissociation constant ( Kd ) of LUZP1 from DD - MLC was significantly lower than that from WT - MLC and AA - MLC [ 4 . 04 ± 1 . 05 μM ( WT - MLC ) vs . 4 . 88 ± 1 . 79 μM ( AA - MLC ) vs . 0 . 85 ± 0 . 39 μM ( DD - MLC ) ] . n = 5 ( WT - MLC and AA - MLC ) and n = 4 ( DD - MLC ) . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel - Dwass test ] . Bars and error bars represent the mean ± SD .

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

In vitro direct binding assay between FLAG-LUZP1 and GST-MLC. Representative immunoblots are shown. S, supernatant; P, pellet.   A representative plot in which the amount of bound GST-WT-, AA-, or DD-MLC/FLAG-LUZP1 was plotted with the Michaelis-Menten fitted curve.   Bar plots with dot density plots showing that the dissociation constant (Kd) of LUZP1 from DD-MLC was significantly lower than that from WT-MLC and AA-MLC [4.04 ± 1.05 μM (WT-MLC) vs. 4.88 ± 1.79 μM (AA-MLC) vs. 0.85 ± 0.39 μM (DD-MLC)]. n = 5 (WT-MLC and AA-MLC) and n = 4 (DD-MLC). *p < 0.05 [Kruskal-Wallis test followed by Steel-Dwass test]. Bars and error bars represent the mean ± SD.


Representative immunoblot of WT , LUZP1 KO , and Venus - LUZP1 - expressing LUZP1 knockout ( REV ) Eph4 cells treated with 100 nM calyculin A for 30 min . Quantification of the ppMLC / MLC ratio relative to WT control , confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [ WT , 1 . 00 ( control ) vs . 1 . 40 ± 0 . 06 ( calyculin A ) vs . 1 . 14 ± 0 . 33 ( washout ) ; KO , 0 . 09 ± 0 . 04 ( control ) vs . 1 . 49 ± 0 . 06 ( calyculin A ) vs . 0 . 81 ± 0 . 99 ( washout ) ; REV , 2 . 06 ± 1 . 78 ( control ) vs . 1 . 82 ± 1 . 50 ( calyculin A ) vs . 1 . 80 ± 1 . 14 ( washout ) ] . n = 3 . Bars and error bars represent the mean ± SD .

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

Representative immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.


In vitro MLC phosphorylation assay using 1 μg GST - PP1c β / δ in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , and 0 - 5 μg GST - LUZP1 . Quantification of the di - phosphorylated MLC ( ppMLC ) / MLC ratio relative to the control showed that LUZP1 up - regulated ppMLC / MLC levels in a dose - dependent manner [ 1 . 00 ( 1st lane , control ) vs . 1 . 27 ± 0 . 33 ( 2nd lane ) vs . 1 . 76 ± 0 . 68 ( 3rd lane ) vs . 2 . 53 ± 1 . 65 ( 4th lane ) vs . 2 . 93 ± 2 . 45 ( 5th lane ) ] . n = 3 or 6 . * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD . In vitro Merlin phosphorylation assay using 1 μg GST - PP1c β / δ , 100 ng GST - Merlin , 2 pg p21 - activated kinase 1 ( PAK1 ) , and 5 μg GST - LUZP1 . Quantification of the phosphorylated Merlin ( pMerlin ) / Merlin ratio relative to the control showed that LUZP1 up - regulated pMerlin / Merlin levels [ 0 . 23 ± 0 . 15 ( 1st lane ) vs . 1 . 00 ( 2nd lane , control ) vs . 0 . 32 ± 0 . 17 ( 3rd lane ) vs . 0 . 97 ± 0 . 42 ( 4th lane ) vs . 1 . 25 ± 0 . 39 ( 5th lane ) ] . n = 4 or 9 . * p < 0 . 05 , * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 3rd lane ) ] . Bars and error bars represent the mean ± SD .

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

In vitro MLC phosphorylation assay using 1 μg GST-PP1c β/δ in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 up-regulated ppMLC/MLC levels in a dose-dependent manner [1.00 (1st lane, control) vs. 1.27 ± 0.33 (2nd lane) vs. 1.76 ± 0.68 (3rd lane) vs. 2.53 ± 1.65 (4th lane) vs. 2.93 ± 2.45 (5th lane)]. n = 3 or 6. **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.   In vitro Merlin phosphorylation assay using 1 μg GST-PP1c β/δ, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 μg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 up-regulated pMerlin/Merlin levels [0.23 ± 0.15 (1st lane) vs. 1.00 (2nd lane, control) vs. 0.32 ± 0.17 (3rd lane) vs. 0.97 ± 0.42 (4th lane) vs. 1.25 ± 0.39 (5th lane)]. n = 4 or 9. *p < 0.05, **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 3rd lane)]. Bars and error bars represent the mean ± SD.


Box plots with dot density plots showing the ratio of the apical area / basal area in co - cultures of Venus - LUZP1 - expressing LUZP1 knockout ( REV ) and LUZP1 knockout ( LUZP1 KO ) Eph4 cells ; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [ REV , 0 . 65 ± 0 . 16 ( control ) vs . 0 . 90 ± 0 . 18 ( nocodazole ) vs . 0 . 64 ± 0 . 16 ( washout ) ; KO , 1 . 30 ± 0 . 17 ( control ) vs . 1 . 07 ± 0 . 13 ( nocodazole ) vs . 1 . 32 ± 0 . 19 ( washout ) ] . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . The solid lines represent the medians and the boxes represent the inter - quartile ranges . The error bars extending from the box represents the data within 1 . 5 times of the interquartile range . Representative confocal micrographs of co - cultures of LUZP1 - expressing wild - type ( WT ) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min . Nocodazole treatment partially reversed the difference in di - phosphorylated MLC ( ppMLC ) levels within circumferential rings ( CRs ) between WT and LUZP1 KO cells . Scale bar , 10 μm . Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down - regulated in WT Eph4 cells after nocodazole treatment . Importantly , ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [ WT , 21 . 43 ± 6 . 96 arbitrary units ( a . u . ) ( control ) vs . 17 . 67 ± 5 . 40 a . u . ( nocodazole ) vs . 20 . 84 ± 7 . 19 a . u . ( washout ) ; KO , 8 . 74 ± 1 . 71 a . u . ( control ) vs . 8 . 67 ± 1 . 89 a . u . ( nocodazole ) vs . 7 . 96 ± 2 . 35 a . u . ( washout ) ] . n = 3 . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . Bars and error bars represent the mean ± standard deviation ( SD ) .

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

Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).


In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , 1 μg GST - protein phosphatase 1c β / δ ( PP1c β / δ ) , and 0 - 5 μg GST - LUZP1 . Quantification of the relative ppMLC / MLC ratio to the control showed that MTs promote LUZP1 - mediated inhibition of PP1c β / δ [ 1 . 00 ( 1st - lane , control ) vs . 1 . 42 ± 0 . 59 ( 2nd - lane ) vs . 1 . 72 ± 0 . 76 ( 3rd - lane ) vs . 1 . 99 ± 0 . 56 ( 4th - lane ) vs . 1 . 14 ± 0 . 37 ( 5th - lane ) vs . 2 . 87 ± 1 . 51 ( 6th - lane ) vs . 2 . 74 ± 1 . 19 ( 7th - lane ) vs . 2 . 50 ± 0 . 88 ( 8th - lane ) ] . n = 6 . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD .

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

In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.


( A ) Representative double - plotted actograms showing wheel - running activity of wild type ( WT ) and CRY - deficient ( CRY Knockout ; CKO ) mice during constant light ( yellow shading ) and thereafter in constant darkness . Note the 48 hour x - axis for WT versus 32 hour for CKO .

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(A) Representative double-plotted actograms showing wheel-running activity of wild type (WT) and CRY-deficient (CRY Knockout; CKO) mice during constant light (yellow shading) and thereafter in constant darkness. Note the 48 hour x-axis for WT versus 32 hour for CKO.


( B ) Mean period and amplitude ( ± SEM ) of mouse behavioural data ( n = 4 ) .

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(B) Mean period and amplitude (±SEM) of mouse behavioural data (n=4).


( C ) Longitudinal bioluminescence recordings of organotypic SCN slices from WT ( black ) and CKO ( red ) PER2 : : LUC mice ( RLU ; relative light units ) .

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(C) Longitudinal bioluminescence recordings of organotypic SCN slices from WT (black) and CKO (red) PER2::LUC mice (RLU; relative light units).


( D ) Mean period and amplitude ( ± SEM ) of rhythmic SCN bioluminescence traces .

(D) Mean period and amplitude (±SEM) of rhythmic SCN bioluminescence traces.


( E ) Circadian PER2 : : LUC expression in immortalised WT and CKO adult lung fibroblasts . Left panel shows two raw traces of a representative longitudinal bioluminescence recording , right panel shows same data detrended with a 24 - hour moving average to remove differences in baseline expression .

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(E) Circadian PER2::LUC expression in immortalised WT and CKO adult lung fibroblasts. Left panel shows two raw traces of a representative longitudinal bioluminescence recording, right panel shows same data detrended with a 24-hour moving average to remove differences in baseline expression.


( F ) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments ( n ≥ 3 per experiment , individual values ± SEM shown ) . P - values were calculated by an unpaired t test with Welch correction . Standard deviations differ significantly between WT and CKO ( F test : p < 0 . 0001 ) .

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(F) Period of rhythmic fibroblast bioluminescence traces from at least 31 experiments (n≥3 per experiment, individual values ±SEM shown). P-values were calculated by an unpaired t test with Welch correction. Standard deviations differ significantly between WT and CKO (F test: p <0.0001).


( A ) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range ( n = 4 , solid lines : mean , dashed lines : ± SEM ) . Temperature was changed from 37 ° C to 32 ° C halfway through the experiment , as depicted by red / blue shading . Arrows represent medium changes . Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change . ( B ) Quantification of period from recordings presented in ( A ) . Both WT and CKO oscillations are temperature compensated with respective Q10s of 1 . 05 and 0 . 95 ( n = 3 , mean ± SEM ) . P - values were determined by two - tailed t - test . ( C ) Bioluminescence of WT and CKO PER2 : : LUC cells during temperature entrainment ( 12h 32 ° C ( blue ) - 12h 37 ° C ( red ) ) ( n = 3 , solid lines : mean , dashed lines : ± SEM ) .

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

(A) Detrended traces of bioluminescence recordings of WT and CKO fibroblast at different constant temperature conditions within the physiological range (n=4, solid lines: mean, dashed lines: ±SEM). Temperature was changed from 37°C to 32°C halfway through the experiment, as depicted by red/blue shading. Arrows represent medium changes. Note the lack of rhythmicity in the first three days in CKO and the appearance of rhythmicity after the first medium change. (B) Quantification of period from recordings presented in (A). Both WT and CKO oscillations are temperature compensated with respective Q10s of 1.05 and 0.95 (n=3, mean ±SEM). P-values were determined by two-tailed t-test. (C) Bioluminescence of WT and CKO PER2::LUC cells during temperature entrainment (12h 32°C (blue) - 12h 37°C (red)) (n=3, solid lines: mean, dashed lines: ±SEM).


( A ) Mean number of PER2 : : LUC molecules per cell at the estimated peak of PER2 expression for each cell line ( mean of three experiments , n = 3 each ) .

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(A) Mean number of PER2::LUC molecules per cell at the estimated peak of PER2 expression for each cell line (mean of three experiments, n=3 each).


( B ) PER2 : : LUC binding to BMAL1 in WT and CKO cells . Cells were harvested at the peak of PER2 expression , BMAL1 was immunoprecipitated , and PER2 : : LUC binding was measured by bioluminescence measurements ( n = 3 , mean ± SD ) .

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(B) PER2::LUC binding to BMAL1 in WT and CKO cells. Cells were harvested at the peak of PER2 expression, BMAL1 was immunoprecipitated, and PER2::LUC binding was measured by bioluminescence measurements (n=3, mean ±SD).


( C ) Per2 mRNA levels in WT ( left ) and CKO ( right ) cells were determined by qPCR over one circadian cycle ( bottom ) , while PER2 : : LUC bioluminescence ( top , min - max normalised ) was recorded from parallel cultures . Per2 mRNA reported relative to Rns18s ( bottom ) , n = 3 , ± SEM ; PER2 : : LUC ( top ) presented as mean ( solid ) ± SEM ( dashed ) , n = 3 . The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line ( p = 0 . 0412 , extra sum - of - square F - test ) whereas CKO data was not ( ns ) .

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(C) Per2 mRNA levels in WT (left) and CKO (right) cells were determined by qPCR over one circadian cycle (bottom), while PER2::LUC bioluminescence (top, min-max normalised) was recorded from parallel cultures. Per2 mRNA reported relative to Rns18s (bottom), n=3, ±SEM; PER2::LUC (top) presented as mean (solid) ±SEM (dashed), n=3. The WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight line (p=0.0412, extra sum-of-square F-test) whereas CKO data was not (ns).


( D ) Detrended Per2 and Nr1d1 promoter activity in WT , CKO and quadruple Cry1 / 2 - Per1 / 2 knockout ( CPKO ) mouse embryonic fibroblasts ( MEFs ) recorded at 37 ° C , n = 3 , mean ( solid ) ± SEM ( dashed ) . Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line ( p < 0 . 0001 , extra sum - of - squares F - test ) ( right hand graphs , solid lines ; error bars , SEM ) .

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(D) Detrended Per2 and Nr1d1 promoter activity in WT, CKO and quadruple Cry1/2-Per1/2 knockout (CPKO) mouse embryonic fibroblasts (MEFs) recorded at 37°C, n=3, mean (solid) ±SEM (dashed). Nr1d1 data were preferentially fit with a circadian damped sine wave over straight line (p<0.0001, extra sum-of-squares F-test) (right hand graphs, solid lines; error bars, SEM).


( A ) Actual PER2 : : LUC levels ( dark symbols ( 3 - hour moving average , n = 3 ± SEM , 4 outliers removed ) ) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours , compared with parallel longitudinal co - recordings from cells in the presence of 0 . 1 mM luciferin ( light lines ( n = 6 , mean ± SEM ) ) .

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(A) Actual PER2::LUC levels (dark symbols (3-hour moving average, n=3 ±SEM, 4 outliers removed)) as assayed in acute luciferase assays on cell lysates from cells harvested every hour over 48 hours, compared with parallel longitudinal co-recordings from cells in the presence of 0.1 mM luciferin (light lines (n=6, mean ±SEM)).


( B ) PER2 : : LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 ( 10 µM , applied at the arrow ) ( n = 3 , mean ± SEM ) . ( C ) Quantification of relative PER2 : : LUC induction upon proteasome inhibition , n = 3 , mean ( solid ) ± SEM ( dashed ) .

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(B) PER2::LUC recording of asynchronous WT and CKO cells pulsed with proteasome inhibitor MG132 (10 µM, applied at the arrow) (n=3, mean ±SEM). (C) Quantification of relative PER2::LUC induction upon proteasome inhibition, n=3, mean (solid) ±SEM (dashed).


( D ) Phase - dependent PER2 : : LUC half - life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one - phase exponential decay curve ( n = 3 , mean ± SEM ) . Left image depicts the timing of cycloheximide ( CHX , 10 µM ) pulses ( labelled I ( PER2 levels going up ) and II ( PER2 levels going down ) ) , plotted on PER2 : : LUC bioluminescence traces of control cells ( dark colours ) . A representative trace of CHX - treated cells at time point I is shown in light colours . Right image shows quantifications , p - values were calculated by unpaired t test .

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(D) Phase-dependent PER2::LUC half-life was determined by inhibiting translation at different circadian phases and fitting the resulting data with a one-phase exponential decay curve (n=3, mean ±SEM). Left image depicts the timing of cycloheximide (CHX, 10 µM) pulses (labelled I (PER2 levels going up) and II (PER2 levels going down)), plotted on PER2::LUC bioluminescence traces of control cells (dark colours). A representative trace of CHX-treated cells at time point I is shown in light colours. Right image shows quantifications, p-values were calculated by unpaired t test.


( A ) Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of CK1δ / ε inhibitor PF670462 ( 0 . 3 µM ; PF ) .

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(A) Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CK1δ/ε inhibitor PF670462 (0.3 µM; PF).


Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 ( 5 µM ; CHIR ) .

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Period analyses of WT and CKO PER2::LUC cells in the presence or absence of GSK3 inhibitor CHIR99021 (5 µM; CHIR).


Period analyses of WT and CKO PER2 : : LUC cells in the presence or absence of CRY inhibitor KL001 ( 1 µM ) .

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Period analyses of WT and CKO PER2::LUC cells in the presence or absence of CRY inhibitor KL001 (1 µM).


( B ) Western blot revealed with anti - I - Abβ JV2 antibody . WCL : whole cell lysate of MutuDC + pRBC ; S0 : supernatant following incubation with protein - G beads ; S1 : 1st wash of protein - G beads ; IP MHC II : eluates from Y - 3P - immunoprecipitated MHC II , boiled or not . Representative of 2 independent experiments .

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(B) Western blot revealed with anti-I-Abβ JV2 antibody. WCL: whole cell lysate of MutuDC + pRBC ; S0: supernatant following incubation with protein-G beads ; S1: 1st wash of protein-G beads ; IP MHC II: eluates from Y-3P-immunoprecipitated MHC II, boiled or not. Representative of 2 independent experiments.


( C ) Venn diagram of unique peptides from P . berghei ( left ) and Mus musculus ( right ) identified in the experiments performed with PbK pRBC ( blue ) and PbA pRBC ( orange ) .

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(C) Venn diagram of unique peptides from P. berghei (left) and Mus musculus (right) identified in the experiments performed with PbK pRBC (blue) and PbA pRBC (orange).


( B ) Gating strategy to calculate the proportion of CD11a + CD49d + activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC , PbA pRBC , OVA peptide ( IR17 ) or ETRAMP10 . 2 peptide ( NL17 ) .

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(B) Gating strategy to calculate the proportion of CD11a+ CD49d+ activated CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC, OVA peptide (IR17) or ETRAMP10.2 peptide (NL17).


( C ) Percentage of IFN / TNF - double producing cells among activated CD4 T cells for each peptide . Basal level with MutuDC alone was subtracted . C , D : Data show the mean + / - SEM . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 0098 ; TIM14 , P = 0 . 078 ; ENO , P = 0 . 016 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 0039 ; EF1α , P = 0 . 0039 ; M1 , P = 0 . 016 ; MSP1 , P = 0 . 031 ; LDH , P = 0 . 062 ; ETRAMP , P = 0 . 001 ; ATPSYN , P = 0 . 25 ; END70 , P = 0 . 25 ; PDI , P = 0 . 16 ; GAPDH . 2 , P = 0 . 99 ; pRBC , P = 0 . 001 . N = 6 mice for ATPSYN , END70 , PDI , GAPDH . 2 , N = 11 mice for all other peptides , pooled from 3 replicates .

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

(C) Percentage of IFN/TNF-double producing cells among activated CD4 T cells for each peptide. Basal level with MutuDC alone was subtracted. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.


( D ) Hierarchy of immunodominance , depicted as the percentage of each peptide - specific response with respect to the total parasite - specific response obtained with PbA pRBC - loaded MutuDC . C , D : Data show the mean + / - SEM . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 0098 ; TIM14 , P = 0 . 078 ; ENO , P = 0 . 016 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 0039 ; EF1α , P = 0 . 0039 ; M1 , P = 0 . 016 ; MSP1 , P = 0 . 031 ; LDH , P = 0 . 062 ; ETRAMP , P = 0 . 001 ; ATPSYN , P = 0 . 25 ; END70 , P = 0 . 25 ; PDI , P = 0 . 16 ; GAPDH . 2 , P = 0 . 99 ; pRBC , P = 0 . 001 . N = 6 mice for ATPSYN , END70 , PDI , GAPDH . 2 , N = 11 mice for all other peptides , pooled from 3 replicates .

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

(D) Hierarchy of immunodominance, depicted as the percentage of each peptide-specific response with respect to the total parasite-specific response obtained with PbA pRBC-loaded MutuDC. C,D: Data show the mean +/- SEM. Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.0098 ; TIM14, P = 0.078 ; ENO, P = 0.016 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.0039 ; EF1α, P = 0.0039 ; M1, P = 0.016 ; MSP1, P = 0.031 ; LDH, P = 0.062 ; ETRAMP, P = 0.001 ; ATPSYN, P = 0.25 ; END70, P = 0.25 ; PDI, P = 0.16 ; GAPDH.2, P = 0.99 ; pRBC, P = 0.001. N = 6 mice for ATPSYN, END70, PDI, GAPDH.2, N = 11 mice for all other peptides, pooled from 3 replicates.


( B ) IFN / TNF - double producing cells among activated CD4 T cells , after subtracting the basal level with MutuDC alone ( mean + / - SEM ) . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon tests in comparison with OVA peptide . AMA1 , P = 0 . 027 ; TIM14 , P = 0 . 84 ; ENO , P = 0 . 84 ; GAPDH . 1 , P = 0 . 002 ; MAHRP , P = 0 . 71 ; EF1α , P = 0 . 001 ; M1 , P = 0 . 032 ; MSP1 , P = 0 . 23 ; LDH , P = 0 . 016 ; ETRAMP , P = 0 . 064 ; ATPSYN , P = 0 . 77 ; END70 , P = 0 . 097 ; PDI , P = 0 . 0052 ; GAPDH . 2 , P = 0 . 19 ; pRBC , P = 0 . 001 . N = 15 mice pooled from 3 replicates .

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

(B) IFN/TNF-double producing cells among activated CD4 T cells, after subtracting the basal level with MutuDC alone (mean +/- SEM). Asterisks show statistical significance assessed by paired non-parametric Wilcoxon tests in comparison with OVA peptide. AMA1, P = 0.027 ; TIM14, P = 0.84 ; ENO, P = 0.84 ; GAPDH.1, P = 0.002 ; MAHRP, P = 0.71; EF1α, P = 0.001 ; M1, P = 0.032 ; MSP1, P = 0.23 ; LDH, P = 0.016 ; ETRAMP, P = 0.064 ; ATPSYN, P = 0.77 ; END70, P = 0.097 ; PDI, P = 0.0052 ; GAPDH.2, P = 0.19 ; pRBC, P = 0.001. N = 15 mice pooled from 3 replicates.


( C ) Blood parasitemia ( mean + / - SEM ) monitored by flow cytometry after inoculation of 5x104 PbA . GFP spz in B6 mice , treated ( open squares ) or not ( black squares ) with CQ at day 6 and 7 pi , in order to avoid ECM without blunting infection . Day 6 , P = 0 . 29 ; Day 7 , P = 0 . 0013 by multiple unpaired t - tests .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Blood parasitemia (mean +/- SEM) monitored by flow cytometry after inoculation of 5x104 PbA.GFP spz in B6 mice, treated (open squares) or not (black squares) with CQ at day 6 and 7 pi, in order to avoid ECM without blunting infection. Day 6, P = 0.29 ; Day 7, P = 0.0013 by multiple unpaired t-tests.


( D ) IFN / TNF - double producing cells among activated CD4 T cells , at day 14 pi following CQ treatment ( mean + / - SEM ) . Basal level with MutuDC alone was subtracted . GAPDH . 1 , P = 0 . 99 ; EF1α , P = 0 . 062 ; ETRAMP , P = 0 . 031 ; pRBC , P = 0 . 031 by paired non - parametric Wilcoxon tests in comparison with OVA peptide .

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

(D) IFN/TNF-double producing cells among activated CD4 T cells, at day 14 pi following CQ treatment (mean +/- SEM). Basal level with MutuDC alone was subtracted. GAPDH.1, P = 0.99 ; EF1α, P = 0.062 ; ETRAMP, P = 0.031 ; pRBC, P = 0.031 by paired non-parametric Wilcoxon tests in comparison with OVA peptide.


Figure 4 . cDC1 are superior to other spleen antigen - presenting cells for MHC II presentation of PbA and non - PbA antigens ( A ) Gating strategy for FACS - sorting pDC ( Lin - CD11clo B220 + Ly6C + ) , cDC1 ( Lin - CD11c + CD8α + ) , cDC2 ( Lin - CD11c + CD8α - ) and a mix of macrophages moDC ( Lin - CD11c - CD11b + Ly6C + ) from spleens of mice infected for 6 days with PbA pRBC .

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

Figure 4. cDC1 are superior to other spleen antigen-presenting cells for MHC II presentation of PbA and non-PbA antigens (A) Gating strategy for FACS-sorting pDC (Lin- CD11clo B220+ Ly6C+), cDC1 (Lin- CD11c+ CD8α+), cDC2 (Lin- CD11c+ CD8α-) and a mix of macrophages moDC (Lin- CD11c- CD11b+ Ly6C+) from spleens of mice infected for 6 days with PbA pRBC.


( B ) I - Ab presentation of ETRAMP10 . 2 peptide by the indicated APC , measured with BEZ hybridomas . PbA pRBC - loaded MutuDC were used as controls . Representative of 2 experiments with 4 infected mice pooled per replicate .

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(B) I-Ab presentation of ETRAMP10.2 peptide by the indicated APC, measured with BEZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. Representative of 2 experiments with 4 infected mice pooled per replicate.


( C ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in day 6 - infected mice ( mean + / - SEM of 5 experiments ) . Difference is not significant based on unpaired t - test with Welch ' s correction .

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

(C) Fold-change surface expression of I-Ab in cDC2 over cDC1 in day 6-infected mice (mean +/- SEM of 5 experiments). Difference is not significant based on unpaired t-test with Welch's correction.


( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( D ) I - Ab - AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations , assessed with the BTg01Z hybridomas . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (D) I-Ab-AS15 presentation after exogenous addition of AS15 peptide at the indicated concentrations, assessed with the BTg01Z hybridomas.Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( E ) I - Ab - AS15 presentation by DC incubated with heat - killed T . gondii at the indicated ratio , assessed with the BTg01Z hybridomas . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (E) I-Ab-AS15 presentation by DC incubated with heat-killed T. gondii at the indicated ratio, assessed with the BTg01Z hybridomas. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( D , E , F ) I - Ab presentation of non - PbA antigens by cDC isolated from day 6 PbA - infected mice . ( F ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at the indicated ratio , assessed with the OTIIZ hybridomas . PbA pRBC - loaded MutuDC were used as controls . D , E , F : representative of 2 experiments with 4 infected mice pooled per replicate . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(D,E,F) I-Ab presentation of non-PbA antigens by cDC isolated from day 6 PbA-infected mice. (F) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at the indicated ratio, assessed with the OTIIZ hybridomas. PbA pRBC-loaded MutuDC were used as controls. D,E,F: representative of 2 experiments with 4 infected mice pooled per replicate. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( G ) In vivo pRBC uptake by cDC1 ( XCR1 + CD172a - ) and cDC2 ( XCR1 - CD172a + ) analyzed at 6 days post - infection with PbA . GFP . Gates were positioned based on the auto - fluorescence of the cDC subset in day 6 PbA - infected mice . As GFP + events are rare , 107 events were acquired for each mouse . Symbols show the mean percentage + / - SEM ( N = 4 mice per group ) of GFP + cells in each DC subset . * , P = 0 . 0005 by unpaired t - test without assuming consistent SD .

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(G) In vivo pRBC uptake by cDC1 (XCR1+ CD172a-) and cDC2 (XCR1- CD172a+) analyzed at 6 days post-infection with PbA.GFP. Gates were positioned based on the auto-fluorescence of the cDC subset in day 6 PbA-infected mice. As GFP+ events are rare, 107 events were acquired for each mouse. Symbols show the mean percentage +/- SEM (N = 4 mice per group) of GFP+ cells in each DC subset. *, P = 0.0005 by unpaired t-test without assuming consistent SD.


( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( H ) I - Ab - NL17 presentation by DC incubated with PbA pRBC at a ratio of 5 : 1 , assessed with the BEZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

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

(H,I) I-Ab presentation by cDC isolated from naïve mice. (H) I-Ab-NL17 presentation by DC incubated with PbA pRBC at a ratio of 5:1, assessed with the BEZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( H , I ) I - Ab presentation by cDC isolated from naïve mice . ( I ) I - Ab - IR17 presentation by DC incubated with OVA - expressing E . coli at a ratio of 20 : 1 , assessed with the OTIIZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted line in D , E , F , H , I shows the basal response of the hybridomas in the absence of antigen .

O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,I) I-Ab presentation by cDC isolated from naïve mice. (I) I-Ab-IR17 presentation by DC incubated with OVA-expressing E. coli at a ratio of 20:1, assessed with the OTIIZ hybridomas. H,I: representative of 2 independent experiments with 20 naïve mice pooled per experiment. Dotted line in D,E,F,H,I shows the basal response of the hybridomas in the absence of antigen.


( J ) Fold - change surface expression of I - Ab in cDC2 over cDC1 in naïve mice ( mean + / - SEM of 4 experiments ) . Difference is not significant based on unpaired t - test with Welch ' s correction .

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(J) Fold-change surface expression of I-Ab in cDC2 over cDC1 in naïve mice (mean +/- SEM of 4 experiments). Difference is not significant based on unpaired t-test with Welch's correction.


( B ) Gating strategy to assess efficiency of cDC1 depletion .

(B) Gating strategy to assess efficiency of cDC1 depletion.


( C ) Percentage ( mean + / - SEM ) of CD8α + out of Lin - B220 - CD11c + MHC IIhi cells . * , P = 0 . 0043 by Mann - Whitney test .

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(C) Percentage (mean +/- SEM) of CD8α+ out of Lin- B220- CD11c+ MHC IIhi cells. *, P = 0.0043 by Mann-Whitney test.


( D ) FACS plots showing the proportion of CD11a + CD49d + CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC , PbA pRBC or ETRAMP10 . 2 peptide .

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(D) FACS plots showing the proportion of CD11a+ CD49d+ CD4 T cells that produce IFN and TNF in response to MutuDC loaded with RBC, PbA pRBC or ETRAMP10.2 peptide.


( E ) Proportion of CD11a + CD49d + CD4 T cells that co - produce IFN and TNF ( mean + / - SEM ) in response to MutuDC loaded with the indicated antigen , isolated from Karma mice treated ( open circles ) or not ( black circles ) with DT . Asterisks show significant differences between no DT and + DT group by multiple unpaired t - tests without assuming consistent SD . RBC , P = 0 . 18 ; GAPDH . 1 , P = 0 . 019 ; EF1a , 0 . 039 ; ETRAMP , P = 0 . 00054 ; OVA , P = 0 . 25 ; pRBC , P = 0 . 015 .

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

(E) Proportion of CD11a+ CD49d+ CD4 T cells that co-produce IFN and TNF (mean +/- SEM) in response to MutuDC loaded with the indicated antigen, isolated from Karma mice treated (open circles) or not (black circles) with DT. Asterisks show significant differences between no DT and + DT group by multiple unpaired t-tests without assuming consistent SD. RBC, P = 0.18 ; GAPDH.1, P = 0.019 ; EF1a, 0.039 ; ETRAMP, P = 0.00054 ; OVA, P = 0.25 ; pRBC, P = 0.015.


( F ) Absolute numbers of IFN + TNF + CD11a + CD49d + CD4 + T cells responding to the indicated stimuli ( mean + / - SEM ) . GAPDH . 1 , P = 0 . 0047 ; EF1a , 0 . 00039 ; ETRAMP , P = 0 . 00022 ; pRBC , P = 0 . 00019 by multiple unpaired t - tests without assuming consistent SD .

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

(F) Absolute numbers of IFN+ TNF+ CD11a+ CD49d+CD4+ T cells responding to the indicated stimuli (mean +/- SEM). GAPDH.1, P = 0.0047 ; EF1a, 0.00039 ; ETRAMP, P = 0.00022 ; pRBC, P = 0.00019 by multiple unpaired t-tests without assuming consistent SD.


( G ) Blood parasitemia ( mean + / - SEM ) at day 4 and 6 pi in Karma mice treated ( open circles ) or not ( black circles ) with DT . Day 4 , P = 0 . 022 ; Day 6 , P = 0 . 77 by multiple unpaired t - tests without assuming consistent SD . C , E , F , G : No DT group , N = 5 mice ; + DT group , N = 6 mice . Representative of 3 independent experiments .

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(G) Blood parasitemia (mean +/- SEM) at day 4 and 6 pi in Karma mice treated (open circles) or not (black circles) with DT. Day 4, P = 0.022 ; Day 6, P = 0.77 by multiple unpaired t-tests without assuming consistent SD. C,E,F,G: No DT group, N = 5 mice ; + DT group, N = 6 mice. Representative of 3 independent experiments.


( H ) Absolute numbers of CD4 + T cells producing IFN , TNF or IL - 10 , either individually or in combination , following restimulation with MutuDC + PbA pRBC ( mean + / - SEM ) . These cells are called cytokine + in the next panels . * , P = 0 . 0023 by unpaired t - test without assuming consistent SD .

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(H) Absolute numbers of CD4+ T cells producing IFN, TNF or IL-10, either individually or in combination, following restimulation with MutuDC+PbA pRBC (mean +/- SEM). These cells are called cytokine+ in the next panels. *, P = 0.0023 by unpaired t-test without assuming consistent SD.


( I ) Multifunctionality analysis ( IFN , TNF , IL - 10 ) of cytokine + T cells responding to MutuDC loaded with PbA pRBC .

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

(I) Multifunctionality analysis (IFN, TNF, IL-10) of cytokine+ T cells responding to MutuDC loaded with PbA pRBC.


( J ) Proportion of IL - 10 + IFN - TNF - among cytokine + T cells , in response to MutuDC loaded with the indicated antigen ( mean + / - SEM ) . GAPDH . 1 , P = 0 . 32 ; EF1a , 0 . 16 ; ETRAMP , P = 8 . 5x10 - 5 ; OVA , P = 0 . 11 ; pRBC , P = 0 . 0015 by multiple unpaired t - tests without assuming consistent SD . H , I , J : N = 3 mice per group .

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(J) Proportion of IL-10+ IFN- TNF- among cytokine+ T cells, in response to MutuDC loaded with the indicated antigen (mean +/- SEM). GAPDH.1, P = 0.32 ; EF1a, 0.16 ; ETRAMP, P = 8.5x10-5 ; OVA, P = 0.11 ; pRBC, P = 0.0015 by multiple unpaired t-tests without assuming consistent SD. H,I,J: N = 3 mice per group.


D . The morphology of oocytes and embryos from female patients with MOS variants . The day of oocyte retrieval was defined as day 0 . Polar bodies were indicated by black arrows . Scale bar = 20 µm .

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D. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar=20 µm.


B . The immunofluorescence images show the pERK1 / 2 ( red ) dynamics in immature ( GV , n = 5 ) , mature ( MII , n = 10 ) , and fertilized oocytes from unidentified control patients ( n = 6 ) , as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants . pERK1 / 2 activation was inhibited after U0126 treatment ( n = 6 ) . FITC - α - tubulin ( Green ) and DAPI ( blue ) were used for co - staining . Scale bar = 10 µm .

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B. The immunofluorescence images show the pERK1/2 (red) dynamics in immature (GV, n=5), mature (MII, n=10), and fertilized oocytes from unidentified control patients (n=6), as well as in mature oocytes from patient 1 carrying homozygous MOSAsn95Lys variants. pERK1/2 activation was inhibited after U0126 treatment (n=6). FITC-α-tubulin (Green) and DAPI (blue) were used for co-staining. Scale bar=10 µm.


C . Western blot analysis of the MEK1 / 2 and ERK1 / 2 activation after transfection of FLAG - tagged wild - type MOS and identified MOS variants in HEK 293 cells .

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C. Western blot analysis of the MEK1/2 and ERK1/2 activation after transfection of FLAG-tagged wild-type MOS and identified MOS variants in HEK 293 cells.


F . Immunofluorescence of FLAG ( green ) and pERK1 / 2 ( grey ) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs . The signal of mCherry was directly captured after fixation , with DAPI staining for DNA visualization ( blue ) . ( n = over 30 oocytes in each group ) . Scale bar = 10 µm .

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F. Immunofluorescence of FLAG (green) and pERK1/2 (grey) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs. The signal of mCherry was directly captured after fixation, with DAPI staining for DNA visualization (blue). (n=over 30 oocytes in each group). Scale bar=10 µm.


A . Immunofluorescence of FLAG ( red ) and F - actin ( gray ) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild - type human MOS or MOS variants mRNAs and culture in medium with 2 . 5 µM milrinone for 24 h , followed by release to maturation ( n = 15 - 20 oocytes each group ) . Scale bar = 10 µm .

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A. Immunofluorescence of FLAG (red) and F-actin (gray) in oocytes after microinjection of negative control or mouse Mos siRNAs combined with or without wild-type human MOS or MOS variants mRNAs and culture in medium with 2.5 µM milrinone for 24 h, followed by release to maturation (n=15-20 oocytes each group). Scale bar= 10 µm.


C . The immunofluorescence of F - actin ( red ) and TPX2 ( green ) in WT and Erk1 / 2oo - / - oocytes , using DAPI ( blue ) co - staining ( n = 15 - 20 oocytes for each group ) . The zoomed images of F - actin are displayed in gray . Scale bar = 10 µm . D . The box plot summarizing the relative intensities of F - actin between control and ERK1 / 2oo - / - oocytes ( n = 7 - 10 oocytes for each group ) .

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

C. The immunofluorescence of F-actin (red) and TPX2 (green) in WT and Erk1/2oo-/- oocytes, using DAPI (blue) co-staining (n=15-20 oocytes for each group). The zoomed images of F-actin are displayed in gray. Scale bar=10 µm. D. The box plot summarizing the relative intensities of F-actin between control and ERK1/2oo-/- oocytes (n=7-10 oocytes for each group).


E . Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1 / 2oo - / - mice . The MII oocyte , zygote , 2 - cell and blastocyst embryos were harvested in vivo at 14 , 24 , 48 , and 96 h post - hCG , respectively ( n = 3 mice for each timepoints in two groups ) . Scale bar = 100 µm . F , G . Bar graphs shows the blastocyst percentage ( F ) and fragment percentage ( G ) in wild - type ( n = 51 ) and maternal ERK1 / 2 deletion embryos ( n = 63 ) .

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

E. Representative bright field images showing the morphology of oocytes and embryos from wildtype and ERK1/2oo-/- mice. The MII oocyte, zygote, 2-cell and blastocyst embryos were harvested in vivo at 14, 24, 48, and 96 h post-hCG, respectively (n=3 mice for each timepoints in two groups). Scale bar=100 µm. F, G. Bar graphs shows the blastocyst percentage (F) and fragment percentage (G) in wild-type (n=51) and maternal ERK1/2 deletion embryos (n=63).


H . The bar graphs show the expression levels of selected MOS - regulated decay genes ( n = 2 or 3 biological replicates ) .

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H. The bar graphs show the expression levels of selected MOS-regulated decay genes (n=2 or 3 biological replicates).


H . Heatmap showing the expression level of selected maternal genes , mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1 / 2 inactivation .

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

H. Heatmap showing the expression level of selected maternal genes, mitochondrial translation genes and mitochondrial respiratory chain genes regulated by MOS mutation or ERK1/2 inactivation.


A . The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO ( control , Ctrl , n = 11 ) or 20 uM U0126 treatment ( n = 12 ) . B . The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO ( control , Ctrl , n = 19 ) or 20 uM U0126 ( n = 17 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The scatter plots showing the ATP levels of human MII oocyte matured in medium with DMSO (control, Ctrl, n=11) or 20 uM U0126 treatment (n=12). B. The scatter plots showing the ATP levels of mouse MII oocytes after in vitro maturation with DMSO (control, Ctrl, n=19) or 20 uM U0126 (n=17). C