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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 abundance of liver biopsy , haptic artery portal vein as a function of rank , with total of top ten abundant number of 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 abundance of liver types as a function of rank , indicating total of top ten abundant number of that comprise 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 . bar plot shows contribution of each organelle to total mass , also accounting for unassigned . of cytosol , mitochondria , nucleoli , plasma 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 copy number of oxidative phosphorylation I - V in bulk liver tissue ( n = 6 independent biological replicates ) types ( isolated from n = 3 individuals for each type ) . Values are presented as mean ± s . d . B . copy number for members of oxidative phosphorylation I - V in hepatocytes Kupffer with Pearson correlation coefficient indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 per type with LFQ intensities [ Log10 ] copy numbers [ Log10 ] .

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


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

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

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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 . expression patterns over course of 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 , * * * 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

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 significantly differentially abundant between NASH ( n = 20 ) , cirrhosis ( n = 10 ) 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 were identified with Cluster 1 mainly upregulated in cirrhosis compared to NASH controls Cluster 2 downregulated . No additional replications of 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

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 ‐ ‐ whisker plot showing distribution of log2 ‐ values of statistical significantly regulated across three groups . Number of independent biological replicates is n = 15 , 20 10 for control , NASH cirrhosis group , respectively . black in middle of box is median , top bottom of box represent upper lower quartile values of data whiskers represent upper lower limits for considering outliers ( Q3 + 1 . 5 * IQR , Q1 ‐ 1 . 5 * IQR ) where IQR is 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 . of CYP 450 family members that are statistically significantly abundant between three groups . Data was presented as mean abundance followed by Z - score normalization across . Number of replicates is same as Panel ( C ) . F . Extracellular matrix ( ECM ) remodeling in liver cirrhosis . Upper lower panel shows abundance rank of involved in ECM organization in control ( upper ) cirrhosis ( lower ) groups , highlighting top shifted ECM components in cirrhosis group as compared with 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

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 of immunostained Eph4 epithelial sheets in apical plane . apical MTs were associated with both LUZP1 TJs . Scale bar , 10 μm ( low magnification ) 1 μm ( high magnification ) . Representative immunoelectron of Eph4 . Immunogold particles for LUZP1 preferentially accumulated at 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 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 B-MEASURED_VAR 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 of co - cultures of wild - type ( WT ) ZO - 1 / - 2 double knockout ( DKO ) Eph4 . ZO - 1 / - 2 DKO were marked by asterisks ( * ) . LUZP1 junctional localization was apparently disrupted in TJ - deficient ZO - 1 / - 2 DKO . Scale bar , 10 μm . Representative confocal of transient E - cadherin knockout ( KO ) in WT Eph4 . E - cadherin KO were marked by asterisks ( * ) . LUZP1 junctional localization seemed not to change between AJ - deficient E - cadherin KO WT . Scale bar , 10 μm .

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

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 within CRs were significantly reduced in LUZP1 KO compared to those in WT [ 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 ) . Bars error bars represent mean ± SD .

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 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

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 of co - cultures of wild - type ( WT ) ZO - 1 / - 2 double knockout ( DKO ) Eph4 . ppMLC within CRs were clearly reduced in ZO - 1 / - 2 DKO compared to those in WT . Scale bar , 10 μm . Bar plots with dot density plots showing that apical areas of WT were significantly smaller than those of ZO - 1 / - 2 DKO [ 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 ) . Bars error bars represent mean ± SD .

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 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 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 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

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

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

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 , Venus - LUZP1 - expressing LUZP1 knockout ( REV ) Eph4 treated with 100 nM calyculin A for 30 min . of ppMLC / MLC relative to WT control , confirming reversal of difference in ppMLC within CRs between WT LUZP1 KO 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 error bars represent mean ± SD .

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

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 using 1 μg GST - PP1c β / δ in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , 0 - 5 μg GST - LUZP1 . of di - phosphorylated MLC ( ppMLC ) / MLC relative to control showed that LUZP1 up - regulated ppMLC / MLC 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 followed by Steel ( compared with 1st lane ) ] . Bars error bars represent mean ± SD . In vitro Merlin phosphorylation using 1 μg GST - PP1c β / δ , 100 ng GST - Merlin , 2 pg p21 - activated kinase 1 ( PAK1 ) , 5 μg GST - LUZP1 . of phosphorylated Merlin ( pMerlin ) / Merlin relative to control showed that LUZP1 up - regulated pMerlin / Merlin [ 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 followed by Steel ( compared with 3rd lane ) ] . Bars error bars represent mean ± SD .

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

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 of apical / basal in co - cultures of Venus - LUZP1 - expressing LUZP1 knockout ( REV ) LUZP1 knockout ( LUZP1 KO ) Eph4 ; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV [ 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 followed by Steel - Dwass ) . solid lines represent medians boxes represent inter - quartile ranges . error bars extending from box represents data within 1 . 5 times of interquartile range . Representative confocal of co - cultures of LUZP1 - expressing wild - type ( WT ) LUZP1 KO Eph4 treated with 2 μM nocodazole for 30 min . Nocodazole treatment partially reversed difference in di - phosphorylated MLC ( ppMLC ) within circumferential rings ( CRs ) between WT LUZP1 KO . Scale bar , 10 μm . Bar plots with dot density plots showing that ppMLC within CRs were significantly down - regulated in WT Eph4 after nocodazole treatment . Importantly , ppMLC in LUZP1 KO Eph4 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 followed by Steel - Dwass ) . Bars error bars represent mean ± standard deviation ( SD ) .

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

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 using 1 μg MTs in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , 1 μg GST - phosphatase 1c β / δ ( PP1c β / δ ) , 0 - 5 μg GST - LUZP1 . of relative ppMLC / MLC to 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 followed by Steel ( compared with 1st lane ) ] . Bars error bars represent 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 B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) CRY - deficient ( CRY Knockout ; CKO ) mice during constant ( yellow shading ) thereafter in constant darkness . Note 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 amplitude ( ± ) of mouse behavioural data ( n = 4 ) .

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


( C ) Longitudinal bioluminescence recordings of organotypic SCN slices from WT ( black ) CKO ( red ) PER2 : : LUC mice ( RLU ; relative 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 amplitude ( ± ) of rhythmic SCN bioluminescence traces .

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


( E ) Circadian PER2 : : LUC expression in immortalised WT 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 .

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

(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 , values ± shown ) . P - values were calculated by an unpaired t with Welch correction . Standard deviations differ significantly between WT CKO ( F : 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 CKO fibroblast at different constant conditions within physiological range ( n = 4 , solid lines : mean , dashed lines : ± ) . was changed from 37 ° C to 32 ° C halfway through experiment , as depicted by red / blue shading . Arrows represent medium changes . Note lack of rhythmicity in first three days in CKO appearance of rhythmicity after first medium change . ( B ) of period from recordings presented in ( A ) . Both WT CKO oscillations are compensated with respective Q10s of 1 . 05 0 . 95 ( n = 3 , mean ± ) . P - values were determined by two - t - . ( C ) Bioluminescence of WT CKO PER2 : : LUC during entrainment ( 12h 32 ° C ( blue ) - 12h 37 ° C ( red ) ) ( n = 3 , solid lines : mean , dashed lines : ± ) .

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

(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 at estimated peak of PER2 expression for each ( 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 CKO . were harvested at peak of PER2 expression , BMAL1 was immunoprecipitated , PER2 : : LUC binding was measured by bioluminescence measurements ( n = 3 , mean ± SD ) .

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

(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 in WT ( left ) CKO ( right ) 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 , ± ; PER2 : : LUC ( top ) presented as mean ( solid ) ± ( dashed ) , n = 3 . WT mRNA trace was preferentially fit by a circadian damped sine wave compared with straight ( p = 0 . 0412 , extra sum - of - square F - ) whereas CKO data was not ( ns ) .

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

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

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

(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 ( symbols ( 3 - hour moving average , n = 3 ± , 4 outliers removed ) ) as assayed in acute luciferase assays on lysates from harvested every hour over 48 hours , compared with parallel longitudinal co - recordings from in presence of 0 . 1 mM luciferin ( lines ( n = 6 , mean ± ) ) .

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

(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 CKO pulsed with proteasome inhibitor MG132 ( 10 µM , applied at arrow ) ( n = 3 , mean ± ) . ( C ) of relative PER2 : : LUC induction upon proteasome inhibition , n = 3 , mean ( solid ) ± ( 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 fitting resulting data with a one - phase exponential decay curve ( n = 3 , mean ± ) . Left image depicts timing of cycloheximide ( CHX , 10 µM ) pulses ( labelled I ( PER2 going up ) II ( PER2 going down ) ) , plotted on PER2 : : LUC bioluminescence traces of control ( colours ) . A representative trace of CHX - treated at time point I is shown in colours . Right image shows quantifications , p - values were calculated by unpaired t .

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

(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 CKO PER2 : : LUC in presence or absence of CK1δ / ε inhibitor PF670462 ( 0 . 3 µM ; PF ) .

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

(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 CKO PER2 : : LUC in presence or absence of GSK3 inhibitor CHIR99021 ( 5 µM ; CHIR ) .

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

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 CKO PER2 : : LUC in presence or absence of CRY inhibitor KL001 ( 1 µM ) .

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

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 lysate of MutuDC + pRBC ; S0 : supernatant following incubation with - G beads ; S1 : 1st wash of - G beads ; IP MHC II : eluates from Y - 3P - immunoprecipitated MHC II , boiled or not . Representative of 2 independent experiments .

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

(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 ) Mus musculus ( right ) identified in experiments performed with PbK pRBC ( blue ) 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 proportion of CD11a + CD49d + activated CD4 T that produce IFN 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 ) of IFN / TNF - double producing among activated CD4 T for each peptide . Basal level with MutuDC alone was subtracted . C , D : Data show mean + / - . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon 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 other peptides , pooled from 3 replicates .

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 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

(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 of each peptide - specific response with respect to total - specific response obtained with PbA pRBC - loaded MutuDC . C , D : Data show mean + / - . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon 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 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 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

(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 among activated CD4 T , after subtracting basal level with MutuDC alone ( mean + / - ) . Asterisks show statistical significance assessed by paired non - parametric Wilcoxon 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 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 + / - ) 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 7 pi , in order to avoid ECM without blunting infection . Day 6 , P = 0 . 29 ; Day 7 , P = 0 . 0013 by multiple unpaired t - .

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

(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 among activated CD4 T , at day 14 pi following CQ treatment ( mean + / - ) . 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 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 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

(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 for MHC II presentation of PbA non - PbA antigens ( A ) Gating strategy for FACS - sorting pDC ( Lin - CD11clo B220 + Ly6C + ) , cDC1 ( Lin - CD11c + CD8α + ) , cDC2 ( Lin - CD11c + CD8α - ) 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 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 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 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 .

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

(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 + / - of 5 experiments ) . Difference is not significant on unpaired t - 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

(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 indicated concentrations , assessed with BTg01Z hybridomas . Dotted in D , E , F , H , I shows basal response of hybridomas in 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

(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 indicated , assessed with BTg01Z hybridomas . Dotted in D , E , F , H , I shows basal response of hybridomas in 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

(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 indicated , assessed with 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 in D , E , F , H , I shows basal response of hybridomas in 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

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

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

(G) 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 of 5 : 1 , assessed with BEZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted in D , E , F , H , I shows basal response of hybridomas in 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

(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 of 20 : 1 , assessed with OTIIZ hybridomas . H , I : representative of 2 independent experiments with 20 naïve mice pooled per experiment . Dotted in D , E , F , H , I shows basal response of hybridomas in 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

(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 + / - of 4 experiments ) . Difference is not significant on unpaired t - 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

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

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

(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 proportion of CD11a + CD49d + CD4 T that produce IFN TNF in response to MutuDC loaded with RBC , PbA pRBC or ETRAMP10 . 2 peptide .

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

(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 that co - produce IFN TNF ( mean + / - ) in response to MutuDC loaded with indicated antigen , isolated from Karma mice treated ( open circles ) or not ( black circles ) with DT . Asterisks show significant differences between no DT + DT group by multiple unpaired t - 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 B-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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 responding to indicated stimuli ( mean + / - ) . GAPDH . 1 , P = 0 . 0047 ; EF1a , 0 . 00039 ; ETRAMP , P = 0 . 00022 ; pRBC , P = 0 . 00019 by multiple unpaired t - 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 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

(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 + / - ) at day 4 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 - without assuming consistent SD . C , E , F , G : No DT group , N = 5 mice ; + DT group , N = 6 mice . Representative of 3 independent experiments .

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

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

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

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

(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 , in response to MutuDC loaded with indicated antigen ( mean + / - ) . 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 - without assuming consistent SD . H , I , J : N = 3 mice per group .

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

(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 . of oocytes from with MOS variants . day of oocyte retrieval was defined as day 0 . Polar bodies were indicated by black arrows . Scale bar = 20 µm .

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

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

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

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 of MEK1 / 2 ERK1 / 2 activation after transfection of FLAG - tagged wild - type MOS identified MOS variants in HEK 293 .

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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 ) pERK1 / 2 ( grey ) in oocytes after injection of different MOS variant mRNAs combined with mCherry mRNAs . signal of mCherry was directly captured after fixation , with DAPI for 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 ) 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 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 . immunofluorescence of F - actin ( red ) TPX2 ( green ) in WT Erk1 / 2oo - / - oocytes , using DAPI ( blue ) co - ( n = 15 - 20 oocytes for each group ) . zoomed of F - actin are displayed in gray . Scale bar = 10 µm . D . box plot summarizing relative intensities of F - actin between control ERK1 / 2oo - / - oocytes ( n = 7 - 10 oocytes for each group ) .

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

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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 . bar graphs show expression 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 . showing expression level of selected maternal genes , mitochondrial translation genes mitochondrial respiratory chain genes regulated by MOS mutation or ERK1 / 2 inactivation .

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

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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


C . representative showing JC - 1 monomers ( green ) ( red ) of mouse oocytes after treatment with DMSO ( control , Ctrl , n = 34 ) or U0126 ( n = 40 ) in in vitro culture medium for 16 h . Scale bar = 100 µm . D . bar graph of of intensities of JC - 1 to those of monomers in ( C ) . intensities of JC - 1 were quantified by Image J software . oocytes number used was labeled .

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C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E


E . Representative displaying JC - 1 monomers ( green ) , ( red ) bright filed of GV MII oocytes from wild - type ( WT ) Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big multiple polar bodies . Scale bar = 100 µm .

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E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.


F . Confocal of JC - 1 monomers ( green ) ( red ) of GV MII oocytes from wild - type ( WT ) Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big multiple polar bodies , indicated by red arrows . Hoechst 33342 ( blue ) was co - stained to represent nucleus or chromosome . Scale bar = 10 µm .

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F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.


( A Representative confocal of HeLa TRIM16KO treated with ( A ) MG132 ( 20 µM , 2h ) , scale bar : 7 . 5 µm . samples were processed for IF with Ub p62 antibody . ( B Representative confocal of control siRNA TRIM16 siRNA transfected treated with ( B ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub p62 antibodies . ( C graph shows of with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(A Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


D ) Representative confocal of HeLa TRIM16KO treated with ( D ) H2O2 ( 200 µM , 2h ) samples were processed for IF with Ub p62 antibody . E ) Representative confocal of control siRNA TRIM16 siRNA transfected treated with ( E ) H2O2 , ( 200 µM , 2h ) , where IF was conducted with Ub p62 antibodies . F ) graph shows of with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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D) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


( G ) Western blot ( WB ) of detergent - soluble - insoluble fractions of HeLa TRIM16KO treated with MG132 ( 50 µM , 1h ) probed with indicated antibodies .

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(G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies.


( H , I ) Densitometric ( using ImageJ ) of WB normalized with α - Tubulin or Lamin - B1 . Data information : Unless otherwise stated , scale bar : 10 µm .

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(H, I) Densitometric analysis (using ImageJ) of WB normalized with α-Tubulin or Lamin-B1. Data information: Unless otherwise stated, scale bar: 10 µm.


( J ) Representative confocal of HeLa TRIM16KO treated with MG132 ( 20 µM , 2h ) processed for IF with Ub LC3B antibody .

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(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.


( K , L ) WB of LC3B in lysates of HeLa TRIM16KO treated with ( K ) Bafilomycin A1 ( 300 nM , 3h ) alone or ( L ) with MG132 ( 10 µM , 4h ) as indicated . L . E , Low exposure ; H . E , high exposure .

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(K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure.


( M ) Representative confocal HeLa TRIM16KO treated with H2O2 ( 200 µM , 2h ) processed for IF with Proteostat dye , Ub , p62 antibody . To show details , brightness of TRIM16KO was increased more than control

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(M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells


( A ) Western blot of HeLa TRIM16KO lysates probed with indicated antibodies .

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(A) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.


( B ) Western blot of lysates from HeLa TRIM16KO treated with or without MG132 probed with indicated antibodies .

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(B) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.


( C ) Left panel , western blotting with HeLa TRIM16KO lysates probed with indicated antibodies . Right panel , densitometric ( mean ± SD ) of band relative to actin , n = 3 , * * * p < 0 . 0005 ( Student ' s unpaired t - ) .

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(C) Left panel, western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, ***p < 0.0005 (Student's unpaired t-test).


D - G ( D , F ) HEK293T were either transiently transfected with an empty vector or a Flag - tagged - TRIM16 expressing vector immunoblotted with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . ( E , G ) Densitometric ( mean ± SD ) of band relative to actin , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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D-G (D, F) HEK293T cells were either transiently transfected with an empty vector or a Flag-tagged-TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. (E, G) Densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, *p < 0.05 (Student's unpaired t-test).


( H ) WB of HeLa TRIM16KO lysates of treated with cycloheximide ( 100 µM ) for indicated period probed with different antibodies as indicated . blots of control TRIM16KO are exposed for equal duration developed together on same X - ray film . ( I ) of NRF2 , p62 , KEAP1 band intensities relative to actin . n = 3 , * * p < 0 . 005 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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(H) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 µM) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X-ray film. (I) Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n=3, **p < 0.005, *p < 0.05 (Student's unpaired t-test).


( J ) Immunoprecipitation ( IP ) of interaction between endogenous TRIM16 endogenous p62 in HeLa lysates in absence presence of MG132 ( 10 µM , 4h ) .

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(J) Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 µM, 4h).


IP to map interaction between p62 TRIM16 domains in HEK293T lysates in ( L ) absence of MG132 ( 10 µM , 4h ) .

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IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence of MG132 (10 µM, 4h).


IP to map interaction between p62 TRIM16 domains in HEK293T lysates in ( M ) presence of MG132 ( 10 µM , 4h ) .

IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (M) presence of MG132 (10 µM, 4h).


( N ) IP of interaction between endogenous TRIM16 endogenous NRF2 in HeLa lysates . Mr , Molecular Marker .

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(N) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, Molecular Weight Marker.


( O ) Co - IP to map interaction between NRF2 TRIM16 domains in HEK293T lysates .

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(O) Co-IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.


( P ) IP of interaction between endogenous TRIM16 endogenous KEAP1 in HeLa lysates .

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(P) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates.


( A , B ) Co - IP of interaction between NRF2 KEAP1 in absence presence of TRIM16 .

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(A, B) Co-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16.


( C ) Co - IP of interaction between NRF2 p62 in absence presence of TRIM16 .

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(C) Co-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16.


( D ) Co - IP of interaction between KEAP1 p62 in absence presence of TRIM16 .

(D) Co-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16.


( A - C ) of NRF2 ubiquitination in absence presence of TRIM16 by Co - IP assays using transiently transfected plasmid constructs as indicated . Two different variants of ubiquitin are used , one that can only be ubiquitinated at Lysine 48 residue ( HA - K48 - UB ) other that can be only ubiquitinated at Lysine 63 residue ( HA - K63 - UB ) . other lysine residues are mutated . L . E , Low exposure ; H . E , High Exposure .

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(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure.


of NRF2 ubiquitination in absence presence of TRIM16 by Ni - NTA pull - down assays using transiently transfected plasmid constructs as indicated . His - tagged ubiquitin which is mutated at lysines except 63 position is used in these assays .

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Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.


( F ) of NRF2 ubiquitination in absence presence of TRIM16 deletion variants by Co - IP assays using transiently transfected plasmid constructs as indicated .

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(F) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co-IP assays using transiently transfected plasmid constructs as indicated.


( G ) Western blot of lysates from HeLa , TRIM16KO TRIM16KO complemented with TRIM16 deletion constructs where were treated with MG132 ( 20 µM , 2h ) blot is probed with indicated antibodies .

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(G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( H ) Western blot of lysates from HeLa , TRIM16KO TRIM16KO complemented with TRIM16 deletion constructs where were treated with MG132 ( 20 µM , 2h ) blot is probed with indicated antibodies .

(H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( A ) Western blot of HeLa TRIM16KO lysates of treated with increasing concentrations of H2O2 as specified probed with antibodies as indicated .

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(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( B ) WB of HeLa TRIM16KO lysates of treated with 400 µM of H2O2 for different durations as indicated probed with antibodies as shown . red arrows indicate time point where were retreated with 400 µM of H2O2 . L . E , Low exposure ; H . E , High Exposure .

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(B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure.


( C ) Densitometric of NRF2 band normalized with actin .

(C) Densitometric analysis of NRF2 band intensity normalized with actin.


( D ) WB of HeLa TRIM16KO lysates of treated with 2 . 5 µM ( 2 h ) of As2O3 .

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(D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3.


( E - G ) isolated from HeLa TRIM16KO , untreated or treated with MG132 ( 20 µM , 2h ) were subjected to qRT - PCR with primers of genes as indicated . fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 , * * p < 0 . 005 ( Student ' s unpaired t - ) .

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(E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05, **p < 0.005 (Student's unpaired t-test).


isolated from control NRF2 knockdown HeLa transfected with control vector or Myc - TRIM16 subjected to qRT - PCR with primers of genes as indicated . Mean ± SD , n = 3 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Student ' s unpaired t - ) .

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RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).


( K ) Western blot of HeLa TRIM16KO lysates of treated with h increasing concentrations of H2O2 as specified probed with antibodies as indicated .

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(K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( L ) Densitometric of Ub smear normalized with actin . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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(L) Densitometric analysis of Ub smear intensity normalized with actin. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).


( M ) Representative confocal of control , Ubb Ube2n siRNA transfected treated with H2O2 ( 200 µM , 2h ) IF was performed with Ub p62 antibodies . Scale bar : 10 µm .

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(M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm.


( N ) graph shows of with . Data from ≥ 10 microscopic fields for each condition ( 40X ) , n = 2 , Mean ± SD .

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(N) The graph shows the percentage of cells with protein aggregates. Data from ≥ 10 microscopic fields for each condition (40X), n=2, Mean ±SD.


( O ) Western blot of Ubb Ube2n siRNA transfected lysates with antibodies as indicated .

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(O) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated.


( A ) Control or NRF2 siRNA transfected HeLa lysates were subjected to western blotting with indicated antibodies .

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(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.


( B , C ) isolated from untreated or MG132 - treated ( 20 µM , 2h ) control or NRF2 siRNA transfected were subjected to qRT - PCR with primers of genes as indicated . fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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

(B, C) RNA isolated from untreated or MG132-treated (20 µM, 2h) control or NRF2 siRNA transfected cells were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


WB of control siRNA or NRF2 siRNA transfected lysates of HeLa , untreated or treated with MG132 ( 20 µM , 2h ) as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

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WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with MG132 (20 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


WB of control siRNA or NRF2 siRNA transfected lysates of HeLa , untreated or treated with H2O2 ( 200 µM , 2h ) as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


Left panels : Representative confocal of control siRNA NRF2 siRNA transfected treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub p62 antibodies . Right panels : tracing corresponding to a in zoom panel .

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Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (F) MG132 (20 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


Left panels : Representative confocal of control siRNA NRF2 siRNA transfected treated with ( G ) H2O2 ( 200 µM , 2h ) , where IF was conducted with Ub p62 antibodies . Right panels : tracing corresponding to a in zoom panel .

Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


( H ) Hela , TRIM16KO , TRIM16KO complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) subjected to WB with Myc , GFP indicated antibodies .

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(H) Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 or GFP-NRF2 were treated with MG132 (20 µM, 2h) and subjected to WB analysis with Myc, GFP and indicated antibodies.


( I ) Representative confocal of Hela , TRIM16KO , TRIM16KO complemented with Myc - NRF2 which were treated with MG132 ( 20 µM , 2h ) subjected to IF with Ub p62 antibodies .

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(I) Representative confocal images of Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 which were treated with MG132 (20 µM, 2h) and subjected to IF with Ub and p62 antibodies.


( J ) Graph represent % of with Ub - p62 dots , data from ≥ 10 microscopic fields ( 40X ) , n = 3 , mean ± SD , * p < 0 . 005 ( one - way ANOVA ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(J) Graph represent % of cells with Ub-p62 dots, data from ≥ 10 microscopic fields (40X), n=3, mean ±SD, *p < 0.005 (one-way ANOVA test). Data information: Unless otherwise stated, scale bar: 10 µm.


WB of detergent - soluble - insoluble fractions of HeLa TRIM16KO expressing either ( A ) Flag - p62 probed with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric of WB ' s normalized with α - Tubulin or Lamin - B1 .

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WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (A) Flag-p62 probed with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.


WB of detergent - soluble - insoluble fractions of HeLa TRIM16KO expressing either ( C ) GFP - polyQ74 probed with or GFP or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric of WB ' s normalized with α - Tubulin or Lamin - B1 .

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

WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (C) GFP-polyQ74 probed with or GFP or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.