IF analysis of P - IκBα in the intestine of 2 month - old WT ( C ) and quantification of P - IκBα positivity Scale bars in C , 25

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

IF analysis of P-IκBα in the intestine of 2 month-old WT (C) and quantification of P-IκBα positivity Scale bars in C, 25 μm


IF analysis of P - IκBα in the intestine of 2 month - old Lgr5 - GFP reporter mice ( D ) , and quantification of P - IκBα positivity inside the Lgr5 - GFP population . A minimum of 30 crypts was counted in 3 Lgr5 - GFP mice . Scale bars in D , 50 μm .

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

IF analysis of P-IκBα in the intestine of 2 month-old Lgr5-GFP reporter mice (D), and quantification of P-IκBα positivity inside the Lgr5-GFP population. A minimum of 30 crypts was counted in 3 Lgr5-GFP mice. Scale bars in D, 50 μm.


( E ) Distribution of P - IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted .

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

(E) Distribution of P-IκBα positive cells according to their position in the intestinal crypt from 200 crypts counted.


( F ) Double IF analysis of 2 month - old murine small intestine sections with the indicated antibodies and quantification of P - IκBα positivity inside the EphB2 + population . 70 crypts were counted . Bars represent mean values ± standard error of the mean ( s . e . m ) . Scale bars , 50 μm .

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

(F) Double IF analysis of 2 month-old murine small intestine sections with the indicated antibodies and quantification of P-IκBα positivity inside the EphB2+ population. 70 crypts were counted. Bars represent mean values ± standard error of the mean (s.e.m). Scale bars, 50 μm.


( G ) Double IF analysis of murine small intestine sections with the indicated antibodies . Scale bars , 50 μm .

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

(G) Double IF analysis of murine small intestine sections with the indicated antibodies. Scale bars, 50 μm.


( H ) Western blot analysis of cytoplasmic ( CYT ) and nuclear ( NUC ) extracts from isolated intestinal crypt cells .

(H) Western blot analysis of cytoplasmic (CYT) and nuclear (NUC) extracts from isolated intestinal crypt cells.


( I ) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti - SUMO2 / 3 antibody .

(I) Western blot analysis of crypt nuclear extracts immunoprecipitated with anti-SUMO2/3 antibody.


( J ) IF analysis of developing intestines from E12 . 5 to E17 . 5 with the indicated antibodies . Scale bars , 25 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

(J) IF analysis of developing intestines from E12.5 to E17.5 with the indicated antibodies. Scale bars, 25 μm.


( K ) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP - sequencing analysis ( n = 4 ) .

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

(K) Representation of IκBα distribution in the indicated genomic regions obtained from ChIP-sequencing analysis (n=4).


( A ) Representative images of P4 and P6 mice of the indicated IκBα genotypes .

(A) Representative images of P4 and P6 mice of the indicated IκBα genotypes.


Quantification of the animal weight ( B ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant .

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

Quantification of the animal weight (B) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.


Quantification of the intestinal length ( C ) from 50 WT / HET and 15 KO mice analyzed . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant .

Quantification of the intestinal length (C) from 50 WT/HET and 15 KO mice analyzed. Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant.


D - G ( D ) Immunohistochemical ( IHC ) , IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines ( D ) , 2 month - old IκBβ ; IκBε KO ( E ) and 2 month - old IκBαNES mice ( F and G ) . A minimum of 50 crypts / villus was counted per genotype ( 3 mice ) . Data information : bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * p - value < 0 . 05 , n . s . no significant . Scale bars in D , E , F and G , 50 μm .

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

D-G (D) Immunohistochemical (IHC), IF and Alcian Blue staining with quantification of P6 IκBα WT and KO intestines (D), 2 month-old IκBβ;IκBε KO (E) and 2 month-old IκBαNES mice (F and G). A minimum of 50 crypts/villus was counted per genotype (3 mice). Data information: bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, * p-value<0.05, n.s. no significant. Scale bars in D, E, F and G, 50 μm.


( A ) Representative image of the intestinal populations purified in the cell sorting experiments .

(A) Representative image of the intestinal populations purified in the cell sorting experiments.


( B ) IF analysis of EphB2 in P6 WT and IκBα KO intestine . Data information : Scale bars , 25 μm .

(B) IF analysis of EphB2 in P6 WT and IκBα KO intestine. Data information: Scale bars , 25 μm.


( C ) P - IκBα IF of EphB2 - high or negative sorted cells . Data information : Scale bars , 25 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

(C) P-IκBα IF of EphB2-high or negative sorted cells. Data information: Scale bars , 25 μm.


( D ) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA - seq . 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Clustering analysis of the indicated cell populations based on their transcriptional profiles obtained in RNA-seq. 3 mice per genotype were initially processed although 1 EphB2neg KO replicate was excluded from the analysis due to insufficient number of reads in the RNA seq.


( E ) Correlation plot of the indicated differentially expressed gene ( DEG ) sets . Values for DEG in fetal and adult ISC was obtained from Nusse et al , 2012 .

O O O O O O O O O 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) Correlation plot of the indicated differentially expressed gene (DEG) sets. Values for DEG in fetal and adult ISC was obtained from Nusse et al, 2012.


( F ) Table indicating the top 15 up - regulated genes in the IκBα KO EphB2 - high signature including 11 genes in the fetal ISC signature ( in orange ) .

(F) Table indicating the top 15 up-regulated genes in the IκBα KO EphB2-high signature including 11 genes in the fetal ISC signature (in orange).


( G ) GSEA of EphB2 - high cells indicating the enrichment of relevant ISC - related pathways .

(G) GSEA of EphB2-high cells indicating the enrichment of relevant ISC-related pathways.


( H ) Representative image of the Lgr5 + population purified in the cell sorting experiments .

(H) Representative image of the Lgr5+ population purified in the cell sorting experiments.


( I ) qPCR analysis of the indicated genes in purified Lgr5 + cells from the different IκBα backgrounds . 4 WT and 1 KO mice were analyzed with at least three technical replicates . Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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

(I) qPCR analysis of the indicated genes in purified Lgr5+ cells from the different IκBα backgrounds. 4 WT and 1 KO mice were analyzed with at least three technical replicates. Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, *p-value<0.05, n.s. no significant.


( A ) IF analysis of c - Rel and RelA / p65 in P6 WT and IκBα KO intestines . Scale bars in 25 μm .

(A) IF analysis of c-Rel and RelA/p65 in P6 WT and IκBα KO intestines. Scale bars in 25 μm.


( B ) Number of peaks from p65 ChIP - sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition ( n = 2 P6 WT and n = 2 P6 IκBα KO intestinal crypt cells ) . Data from individual experiments have been deposited at GSE131187 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Number of peaks from p65 ChIP-sequencing analysis associated with the different genomic localizations was obtained merging two biological replicates per condition (n=2 P6 WT and n=2 P6 IκBα KO intestinal crypt cells). Data from individual experiments have been deposited at GSE131187.


IF analysis of active Notch1 ( ICN1 ) ( C ) in the indicated IκBα backgrounds at P6 intestines . Scale bars in 25 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

IF analysis of active Notch1 (ICN1) (C) in the indicated IκBα backgrounds at P6 intestines. Scale bars in 25 μm.


IF analysis of β - catenin ( D ) in the indicated IκBα backgrounds at P6 intestines . Scale bars 25 μm .

IF analysis of β-catenin (D) in the indicated IκBα backgrounds at P6 intestines. Scale bars 25 μm.


( E ) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n = 3 P6 animals per genotype were analyzed ( a minimum of 15 crypts was counted per genotype ) . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 . Scale bars 25 μm .

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

(E) IF analysis of the PRC2 protein EZH2 and quantification of the EZH2 positive cells per crypt in n=3 P6 animals per genotype were analyzed (a minimum of 15 crypts was counted per genotype). Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001. Scale bars 25 μm.


( F ) Table indicating the methylation - related genes ( PRC2 members and KDM6 demethylase ) differentially express between IκBα WT and KO EphB2 - high sorted cells ( RNA - seq ) .

O O O O O O O O O O O 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) Table indicating the methylation-related genes (PRC2 members and KDM6 demethylase) differentially express between IκBα WT and KO EphB2-high sorted cells (RNA-seq).


( G ) Western blot analysis of IκBα IP from crypt nuclear extracts .

(G) Western blot analysis of IκBα IP from crypt nuclear extracts.


( I ) Representation of H3K27me3 distribution at embryonic E14 . 5 ( n = 2 WT and n = 2 KO mice ) and postnatal P6 ( n = 2 WT and n = 2 KO mice ) ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of the indicated fetal ISC genes .

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

(I) Representation of H3K27me3 distribution at embryonic E14.5 (n=2 WT and n=2 KO mice) and postnatal P6 (n=2 WT and n=2 KO mice) (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of the indicated fetal ISC genes.


( J ) Representation of IκBα ( n = 4 ) and H3K27me3 ( n = 2 ) distribution ( from ChIP - sequencing ) and expression levels ( from RNA - sequencing ) in the genomic region of Litaf .

O O O O O O O O O O O O O O O O O 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) Representation of IκBα (n=4) and H3K27me3 (n=2) distribution (from ChIP-sequencing) and expression levels (from RNA-sequencing) in the genomic region of Litaf.


( A ) Representative stereoscopic image of P6 WT and IκBα KO - derived intestinal organoids . Scale bars in A , 100

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

(A) Representative stereoscopic image of P6 WT and IκBα KO-derived intestinal organoids. Scale bars in A, 100 μm


( B ) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO - derived organoids . Scale bars in B 50 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

(B) IHC analysis with the indicated antibodies and Alcian Blue staining of WT and IκBα KO-derived organoids. Scale bars in B 50 μm.


( C ) qPCR analysis of the indicated ISC genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of the indicated ISC genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.


( D ) Correlation plots of the indicated differentially expressed gene ( DEG ) sets obtained in expression microarray ( 5 technical replicates of a minimum of two organoids per genotype were analyzed ) . Values for DEG in fetal and adult organoids were obtained from Mustata et al , 2013 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Correlation plots of the indicated differentially expressed gene (DEG) sets obtained in expression microarray (5 technical replicates of a minimum of two organoids per genotype were analyzed). Values for DEG in fetal and adult organoids were obtained from Mustata et al, 2013.


( E ) qPCR analysis of the indicated fetal genes in WT and IκBα KO - derived organoids . Data information : 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean ( s . e . m ) . p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 .

(E) qPCR analysis of the indicated fetal genes in WT and IκBα KO-derived organoids. Data information: 3 technical replicates of a minimum of two organoids per genotype were analyzed Bars represent mean values ± standard error of the mean (s.e.m). p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, **p-value<0.005, * p-value<0.05.


( F ) Western blot analysis of control and IκBα - deleted organoids using CRISPR - Cas9 technology .

(F) Western blot analysis of control and IκBα-deleted organoids using CRISPR-Cas9 technology.


( G ) Representative stereoscopic image of WT and IκBα - deleted organoids . Scale bars in G , 50 μm .

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

(G) Representative stereoscopic image of WT and IκBα-deleted organoids. Scale bars in G, 50 μm.


A ) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh - RNA for NF - κB elements . Data information Scale bars in A , 100 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

A) Representative stereoscopic images and Western blot analysis of IκBα KO organoids infected with the indicated sh-RNA for NF-κB elements. Data information Scale bars in A, 100 μm,


( B ) qPCR analysis of the indicated ISC and differentiation genes in the different sh - RNA treated organoids . The canonical NF - κB target gene Cxcl10 is shown as positive control of the experiment . Data information 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of the indicated ISC and differentiation genes in the different sh-RNA treated organoids. The canonical NF-κB target gene Cxcl10 is shown as positive control of the experiment. Data information 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


( C ) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information Scale bars in 50 μm .

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

(C) Representative stereoscopic images of IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information Scale bars in 50 μm.


( D - E ) qPCR analysis of the indicated adult and fetal ISC genes ( D ) and elements of the Notch and IFN pathway ( E ) ( differentially expressed in IκBα KO organoids ) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ - 6438 . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of the indicated adult and fetal ISC genes (D) and elements of the Notch and IFN pathway (E) (differentially expressed in IκBα KO organoids) in IκBα KO organoids untreated or treated with the PRC2 inhibitor EPZ-6438. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


( F ) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor . Data information Scale bars in F , 50 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

(F) Representative stereoscopic images of IκBα KO organoids treated with dexamethasone as IFN inhibitor and DAPT as Notch inhibitor. Data information Scale bars in F, 50 μm.


( G ) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT . Data information : 3 technical replicates of a minimum of two organoids per condition were analyzed . Bars represent mean values ± standard error of the mean ( s . e . m ) ; p values were derived from an unpaired t - test , two - tailed , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR analysis of IκBα KO organoids untreated or treated with dexamethasone and DAPT. Data information: 3 technical replicates of a minimum of two organoids per condition were analyzed. Bars represent mean values ± standard error of the mean (s.e.m); p values were derived from an unpaired t-test, two-tailed, ****p-value<0.0001, ***p-value<0.0005, **p-value<0.005, * p-value<0.05, n.s. no significant.


( A ) IF analysis of colonic tissue from control and DSS - treated animals including acute damaged ( AD ) areas and recovery ( R ) areas . Data information : Scale bars 100

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

(A) IF analysis of colonic tissue from control and DSS-treated animals including acute damaged (AD) areas and recovery (R) areas. Data information: Scale bars 100 μm


IF analysis of P - IκBα and the indicated markers in the areas of acute damage and recovery ( B ) Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 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

IF analysis of P-IκBα and the indicated markers in the areas of acute damage and recovery (B) Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.


IF analysis of P - IκBα and colocalization of P - IκBα with the ISC marker Olfm4 in acute damaged areas ( C ) . Asterisks indicate the presence of rare cells with nuclear P - IκBα . Data information : Scale bars 50 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

IF analysis of P-IκBα and colocalization of P-IκBα with the ISC marker Olfm4 in acute damaged areas (C). Asterisks indicate the presence of rare cells with nuclear P-IκBα. Data information: Scale bars 50 μm.


( D ) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time - points . 3 technical replicates of organoids from each genotype were analyzed .

O O O O O O O O O O O O O O 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) qPCR analysis of the indicated genes in WT and IκBα KO organoids treated with TNFα and collected at the indicated time-points. 3 technical replicates of organoids from each genotype were analyzed.


( E - F ) Representative images of the colonic tissue from 2 months - old DSS - treated WT and IκBα KO mice are shown . Alcian Blue staining was used to identify the mucus - secreting goblet cells in the colonic glands and Ki67 as proliferation marker . Nuclear counterstain is Fast Red . The table shows number of ulcerations present in the intestines of mice analysed ( 3 WT , 2 IκBα KO ) . Data information : Scale bars in E and F , 100

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Representative images of the colonic tissue from 2 months-old DSS-treated WT and IκBα KO mice are shown. Alcian Blue staining was used to identify the mucus-secreting goblet cells in the colonic glands and Ki67 as proliferation marker. Nuclear counterstain is Fast Red. The table shows number of ulcerations present in the intestines of mice analysed (3 WT, 2 IκBα KO). Data information: Scale bars in E and F, 100 μm


A and B ) Confocal microscopy images showing mitochondrial fragmentation in HBV - and HBx - expressing cells , respectively . Huh7 cells were transiently transfected with HBV ( A ) or the HBx - flag construct ( B ) . At 2 days post - transfection , cells prestained with MitoTracker ( Mito , white ) were immunostained with anti - HBsAg ( A , green ) and anti - flag ( B , green ) antibodies , respectively . In the zoomed images , typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown .

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

A and B) Confocal microscopy images showing mitochondrial fragmentation in HBV- and HBx-expressing cells, respectively. Huh7 cells were transiently transfected with HBV (A) or the HBx-flag construct (B). At 2 days post-transfection, cells prestained with MitoTracker (Mito, white) were immunostained with anti-HBsAg (A, green) and anti-flag (B, green) antibodies, respectively. In the zoomed images, typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown.


C ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins . β - actin was used as an internal loading control . Mitochondrial fraction ( bottom panel ) isolated from HepAD38 cells ( see the purity in Figure 2B ) was analyzed by Western blotting with phospho - Drp1 ( Ser616 ) antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody.


( D - F ) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV - and HBx - expressing cells , respectively . Huh7 cells transfected with HBV ( D and E ) or HBx - flag construct ( F ) were immunostained with antibodies specific to TOM20 ( red ) , Drp1 ( D , green ) , and phospho - Drp1 ( Ser616 ) ( E and F , green ) , respectively . HBV and HBx gene expression ( white ) was verified using anti - HBsAg ( D and E ) and anti - flag ( F ) antibodies , respectively . In the zoomed images , the yellow color indicates the merge of Drp1 or phospho - Drp1 ( Ser616 ) with mitochondria . ( A , B , D , E , and F ) Transfected ( + ) and untransfected ( − ) cells are marked . Nuclei were stained with DAPI ( blue ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV- and HBx-expressing cells, respectively. Huh7 cells transfected with HBV (D and E) or HBx-flag construct (F) were immunostained with antibodies specific to TOM20 (red), Drp1 (D, green), and phospho-Drp1 (Ser616) (E and F, green), respectively. HBV and HBx gene expression (white) was verified using anti-HBsAg (D and E) and anti-flag (F) antibodies, respectively. In the zoomed images, the yellow color indicates the merge of Drp1 or phospho-Drp1 (Ser616) with mitochondria. (A, B, D, E, and F) Transfected (+) and untransfected (−) cells are marked. Nuclei were stained with DAPI (blue).


( A ) Huh7 cells transfected with HBV construct were prestained with MitoTracker ( Mito , red ) and subsequently , immunostained with anti - Parkin ( green ) and anti - HBsAg ( white ) antibodies . Nuclei are demarcated with white dots . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria . Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV - expressing cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Huh7 cells transfected with HBV construct were prestained with MitoTracker (Mito, red) and subsequently, immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria. Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV-expressing cells.


( B ) The cytosolic ( Cyto ) and mitochondrial ( Mito ) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h . Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins . Fractions : WCL , whole cell lysates ; Cyto , purified cytoplasm ; Mito , purified mitochondria . Organelle markers : VDAC1 , mitochondria ; GAPDH , cytoplasm .

O O 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) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm.


( C ) Parkin protein in whole cell lysates of HBV - expressing cells was immunoprecipitated by anti - Parkin antibody , followed by immunoblotting ( IB ) with anti - ubiquitin ( Ub ) antibody . Normal rabbit IgG was used as a control for immunoprecipitation ( IP ) . Western blot analysis for β - actin indicates equivalent amount of cell lysates for IP ( C and D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Parkin protein in whole cell lysates of HBV-expressing cells was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a control for immunoprecipitation (IP). Western blot analysis for β-actin indicates equivalent amount of cell lysates for IP (C and D).


( D ) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non - targeting ( NT ) and Parkin ( P ) siRNA , respectively , for 48 h was immunoprecipitated by anti - Mfn2 antibody , followed by immunoblotting ( IB ) with anti - ubiquitin ( Ub ) antibody . Normal mouse IgG was used as a control for immunoprecipitation ( IP ) . The protein expression was analyzed by Western blotting with antibodies specific to Mfn2 , Parkin , and β - actin proteins . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non-targeting (NT) and Parkin (P) siRNA, respectively, for 48 h was immunoprecipitated by anti-Mfn2 antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal mouse IgG was used as a control for immunoprecipitation (IP). The protein expression was analyzed by Western blotting with antibodies specific to Mfn2, Parkin, and β-actin proteins. (


( E ) Intracellular mRNA levels of Parkin , PINK1 , and LC3B were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in Parkin , PINK1 , and LC3B gene expression .

O O O O O O O O O O O O O O O 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) Intracellular mRNA levels of Parkin, PINK1, and LC3B were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin, PINK1, and LC3B gene expression.


( F ) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins . β - actin was used as a loading cont ( F ) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins . β - actin was used as a loading control ( F and G ) . The relative intensity of ATF4 , Parkin , PINK1 , and LC3B protein expression normalized to β - actin was analyzed by ImageJ software .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading cont(F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading control (F and G). The relative intensity of ATF4, Parkin, PINK1, and LC3B protein expression normalized to β-actin was analyzed by ImageJ software.


. ( G ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins .

. (G) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins.


( A ) Huh7 cells transiently expressing GFP - LC3 protein were transfected with HBV construct in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 8 h before fixation . At 2 days post - transfection , cells were immunostained with antibodies against HBsAg ( white ) , TOM20 ( blue ) , and Parkin ( red ) . Nuclei are demarcated with white dots . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , the arrows ( white puncta ) indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and Parkin .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies against HBsAg (white), TOM20 (blue), and Parkin (red). Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin.


( B and C ) Quantification of the number of total GFP - LC3 puncta ( B ) and GFP - LC3 puncta colocalized with TOM20 ( C ) in the panel ( A ) . ( D ) Quantitative analysis of the area of GFP - LC3 puncta ( white ) representing merge of GFP - LC3 puncta , TOM20 , and Parkin in the 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

(B and C) Quantification of the number of total GFP-LC3 puncta (B) and GFP-LC3 puncta colocalized with TOM20 (C) in the panel (A). (D) Quantitative analysis of the area of GFP-LC3 puncta (white) representing merge of GFP-LC3 puncta, TOM20, and Parkin in the panel (A).


( B ) Huh7 cells transiently expressing Mito - mRFP - EGFP were transfected with HBV , HBx - flag , and HBV - ΔX constructs , respectively , for 48 h . Cells were immunostained with antibodies specific to HBsAg and flag ( white ) , respectively . Nuclei were stained with DAPI ( blue ) . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , fluorescence signals indicate the expression of Mito - mRFP - EGFP targeting mitochondria : yellow color , no mitophagy ; red color , mitophagy .

(B) Huh7 cells transiently expressing Mito-mRFP-EGFP were transfected with HBV, HBx-flag, and HBV-ΔX constructs, respectively, for 48 h. Cells were immunostained with antibodies specific to HBsAg and flag (white), respectively. Nuclei were stained with DAPI (blue). Transfected (+) and untransfected (−) cells are marked. In the zoomed images, fluorescence signals indicate the expression of Mito-mRFP-EGFP targeting mitochondria: yellow color, no mitophagy; red color, mitophagy.


( C ) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel ( B ) .

(C) Quantitative analysis of the fluorescence signal targeted to mitochondria in the panel (B).


( D ) GFP - LC3 - expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 8 h before fixation . At 2 days post - transfection , cells prestained with LysoTracker ( Lyso , red ) were immunostained with antibodies against HBsAg ( white ) and TOM20 ( blue ) . Nuclei are demarcated with white dot circles . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoom images , the arrows denoting white puncta indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and lysosome . ( E ) Quantification of the colocalization of GFP - LC3 puncta containing lysosome with mitochondria in the panel ( D ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3-expressing Huh7 cells were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells prestained with LysoTracker (Lyso, red) were immunostained with antibodies against HBsAg (white) and TOM20 (blue). Nuclei are demarcated with white dot circles. Transfected (+) and untransfected (−) cells are marked. In the zoom images, the arrows denoting white puncta indicate GFP-LC3 puncta (green) colocalized with TOM20 and lysosome. (E) Quantification of the colocalization of GFP-LC3 puncta containing lysosome with mitochondria in the panel (D).


( A - C ) Parkin silencing accelerates HBV - induced mitochondrial apoptotic signaling . HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non - targeting ( NT ) and Parkin siRNA , respectively , for 72 h . Cells were used for Western blot analysis using antibodies specific to the indicated proteins ( A ) , caspase - 3 / 7 activity assay ( B ) , and TUNEL assay ( C )

O O 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

(A-C) Parkin silencing accelerates HBV-induced mitochondrial apoptotic signaling. HepG2 and HepAD38 cells grown in the absence or presence of tetracycline were transfected with non-targeting (NT) and Parkin siRNA, respectively, for 72 h. Cells were used for Western blot analysis using antibodies specific to the indicated proteins (A), caspase-3/7 activity assay (B), and TUNEL assay (C)


( a - c ) HeLa cells were either left untreated , amino - acid ( aa ) / FBS starved for 5 h , or starved and then recovered in amino - acid / FBS - containing medium , then immunostained , or immunoblotted using antibodies as shown . Co - localization panels show an overlap between mTOR and LAMP1 signals . Note that changes in the positioning of lysosomal mTOR ( quantified as the percentage of cells with predominantly peripheral localization of LAMP1 - positive vesicles ; a , c ) correlate with mTORC1 activity ( levels of phosphorylated S6K relative to the total S6K ; b ) .

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 O O O O O O O O O O O O O O O O O O O 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-c) HeLa cells were either left untreated, amino-acid (aa)/FBS starved for 5 h, or starved and then recovered in amino-acid/FBS-containing medium, then immunostained, or immunoblotted using antibodies as shown. Co-localization panels show an overlap between mTOR and LAMP1 signals. Note that changes in the positioning of lysosomal mTOR (quantified as the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles; a,c) correlate with mTORC1 activity (levels of phosphorylated S6K relative to the total S6K; b).


( d ) Visualization of Akt activated in response to recovery after serum starvation . After nutrient recovery , LAMP1 - positive vesicles localize to peripheral regions with higher concentrations of phospho - Akt . DAPI , 4 , 6 − diamidino − 2 − phenylindole . For all panels , values are means ± s . e . m . of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; other comparisons are not significant ( n . s . ) . Representative maximum - intensity projections of serial confocal optical sections are shown . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

(d) Visualization of Akt activated in response to recovery after serum starvation. After nutrient recovery, LAMP1-positive vesicles localize to peripheral regions with higher concentrations of phospho-Akt. DAPI, 4,6−diamidino−2−phenylindole. For all panels, values are means ± s.e.m. of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons are not significant (n.s.). Representative maximum-intensity projections of serial confocal optical sections are shown. Uncropped images of blots are shown in Supplementary Fig. S7.


( a , b ) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability . Cells were treated as in Fig . 1a followed by incubation with dimethylsulphoxide ( vehicle ) or with nocodazole during the last 2 h before fixation / lysis . Samples were analysed by immunofluorescence ( a ) or immunoblotting ( b ) . Quantification of phopho - S6K levels is shown in ( b ) .

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

(a,b) Nocodazole flattens the differences in lysosomal mTOR localization and dampens mTORC1 signalling in response to changes in nutrient availability. Cells were treated as in Fig. 1a followed by incubation with dimethylsulphoxide (vehicle) or with nocodazole during the last 2 h before fixation/lysis. Samples were analysed by immunofluorescence (a) or immunoblotting (b). Quantification of phopho-S6K levels is shown in (b).


( c - e ) Changes in lysosomal positioning induced by kinesin - or small GTPase - family members ( c , d ) correlate with changes in mTORC1 activity ( e ) . HeLa cells were transfected with overexpression constructs ( OE ) or with siRNA as shown , followed by immunofluorescence ( c , d ) or by immunoblotting ( e ) analyses . Values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-e) Changes in lysosomal positioning induced by kinesin- or small GTPase-family members (c,d) correlate with changes in mTORC1 activity (e). HeLa cells were transfected with overexpression constructs (OE) or with siRNA as shown, followed by immunofluorescence (c,d) or by immunoblotting (e) analyses. Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


( a - c ) HeLa cells transfected with ARL8B or KIF2 siRNA , or with ARL8B overexpression construct ( non - targeting siRNA and empty pcDNA vector used as transfection controls ) , were either left untreated , serum / amino - acid starved for 5 h , or starved and then recovered in amino - acid - and FBS - containing medium for 30 min . Cells were immunostained using LAMP1 antibody ( a ) and the percentage of cells with predominantly peripheral localization of LAMP1 - positive vesicles was quantified ( b ) or subjected to immunoblotting ( c ) using antibodies as shown . Quantification of phospho - S6K levels relative to the total S6K is shown in ( c ) . Values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-c) HeLa cells transfected with ARL8B or KIF2 siRNA, or with ARL8B overexpression construct (non-targeting siRNA and empty pcDNA vector used as transfection controls), were either left untreated, serum/amino-acid starved for 5 h, or starved and then recovered in amino-acid- and FBS-containing medium for 30 min. Cells were immunostained using LAMP1 antibody (a) and the percentage of cells with predominantly peripheral localization of LAMP1-positive vesicles was quantified (b) or subjected to immunoblotting (c) using antibodies as shown. Quantification of phospho-S6K levels relative to the total S6K is shown in (c). Values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Starvation increases pHi in HeLa cells allowed to starve using three different protocols ( see Methods ) with or without subsequent recovery .

O O 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) Starvation increases pHi in HeLa cells allowed to starve using three different protocols (see Methods) with or without subsequent recovery.


( b - d ) Changing pHi from 7 . 1 to 7 . 7 is sufficient to affect localization of lysosomes and mTORC1 activity . pHi was titrated in full tissue - culture medium containing nigericin , which enables changes in pHi to be forced by altering pH in the medium , followed by immunostaining ( b , c ) or western blotting ( d ) using antibodies as shown .

(b-d) Changing pHi from 7.1 to 7.7 is sufficient to affect localization of lysosomes and mTORC1 activity. pHi was titrated in full tissue-culture medium containing nigericin, which enables changes in pHi to be forced by altering pH in the medium, followed by immunostaining (b,c) or western blotting (d) using antibodies as shown.


( e ) Changes in lysosomal localization have no effect on pHi . ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement .

O O O O O 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) Changes in lysosomal localization have no effect on pHi. ARL8B and KIF2 were overexpressed or knocked down in HeLa cells followed by pHi measurement.


( f , g ) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions . Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation - recovery ( f ) or to 1 h changes of pHi in full tissue - culture medium containing nigericin ( g ) are shown .

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

(f,g) Nutrients and pHi affect levels of ARL8 and KIF2 in lysosomal fractions. Protein levels and their quantification in total cellular lysates or in isolated lysosomal fractions from HeLa cells subjected to 1 h nutrient deprivation-recovery (f) or to 1 h changes of pHi in full tissue-culture medium containing nigericin (g) are shown.


( h , i ) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules . HeLa cells treated as in ( f ) and ( g ) , followed by isolation of polymerized microtubule fraction and western blotting . The asterisk indicates a nonspecific band . For all panels values are means ± s . e . m . of three independent experiments carried out in triplicate . All comparisons are with the control within each treatment condition , * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; n . s . not significant . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

(h,i) Effect of nutrients and pHi on binding of ARL8 and KIF2 to polymerized microtubules. HeLa cells treated as in (f) and (g), followed by isolation of polymerized microtubule fraction and western blotting. The asterisk indicates a nonspecific band. For all panels values are means ± s.e.m. of three independent experiments carried out in triplicate. All comparisons are with the control within each treatment condition, *P0.05, **P0.01, ***P0.005 Student's t-test; n.s. not significant. Uncropped images of blots are shown in Supplementary Fig. S7.


( b ) Serum and amino - acid starvation of HeLa cells for 5 h reduces LC3 - II levels ( above ) , but increases autolysosome numbers detected using tfLC3 ( below ) . With tfLC3 , GFP - and RFP - positive puncta represent autophagosomes before lysosomal fusion , whereas RFP - positive / GFP - negative puncta represent autolysosomes - GFP is more rapidly quenched by low lysosomal pH ( see Methods ) . Increased autolysosomes indicate enhanced starvation - induced flux of LC3 to lysosomes .

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

(b) Serum and amino-acid starvation of HeLa cells for 5 h reduces LC3-II levels (above), but increases autolysosome numbers detected using tfLC3 (below). With tfLC3, GFP- and RFP-positive puncta represent autophagosomes before lysosomal fusion, whereas RFP-positive/GFP-negative puncta represent autolysosomes-GFP is more rapidly quenched by low lysosomal pH (see Methods). Increased autolysosomes indicate enhanced starvation-induced flux of LC3 to lysosomes.


( c ) ARL8B knockdown increases autophagosomal synthesis . siRNA - transfected HeLa cells were incubated for 48 h , then left untreated or incubated with bafilomycin A1 . LC3 - II levels versus actin were quantified ( bottom graphs ) . Asterisk : nonspecific band .

O O O 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

(c) ARL8B knockdown increases autophagosomal synthesis. siRNA-transfected HeLa cells were incubated for 48 h, then left untreated or incubated with bafilomycin A1. LC3-II levels versus actin were quantified (bottom graphs). Asterisk: nonspecific band.


( d ) ARL8B overexpression inhibits autophagosome synthesis and degradation . HeLa cells overexpressing ARL8B or empty vector were analysed as in ( c ) .

(d) ARL8B overexpression inhibits autophagosome synthesis and degradation. HeLa cells overexpressing ARL8B or empty vector were analysed as in (c).


( e ) HeLa cells were co - transfected with either ARL8B overexpression construct or siRNA ( non - targeting siRNA and empty peGFP vector were transfection controls ) together with mCherry - LC3 for 48 h . After fixation , cells were stained for endogenous LAMP1 and DNA ( DAPI ) . Representative maximum - intensity projections of serial confocal optical sections are shown . Co - localization panels show overlapping mCherry - LC3 and LAMP1 signals . ( f - h ) Quantification of autophagosome - lysosome fusion in HeLa cells . Percentages of autolysosomes ( positive for both mCherry - LC3 and LAMP1 ) to autophagosomes ( positive for mCherry - LC3 and negative for LAMP1 ) were quantified . In ( h ) , we analysed cells treated for 2 h with nocodazole before fixation , which dispersed the perinuclear lysosomal cluster ( see Fig . 2a ) . ( i ) Autophagosomal and autolysosomal numbers in tfLC3 - expressing cells after ARL8B overexpression and knockdown ( Supplementary Fig . S5o shows representative cells . ) . In ( f - i ) we analysed 20 cells per group in three independent experiments . Values are means ± s . e . m of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - test ; other comparisons not significant ( n . s . ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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

(e) HeLa cells were co-transfected with either ARL8B overexpression construct or siRNA (non-targeting siRNA and empty peGFP vector were transfection controls) together with mCherry-LC3 for 48 h. After fixation, cells were stained for endogenous LAMP1 and DNA (DAPI). Representative maximum-intensity projections of serial confocal optical sections are shown. Co-localization panels show overlapping mCherry-LC3 and LAMP1 signals. (f-h) Quantification of autophagosome-lysosome fusion in HeLa cells. Percentages of autolysosomes (positive for both mCherry-LC3 and LAMP1) to autophagosomes (positive for mCherry-LC3 and negative for LAMP1) were quantified. In (h), we analysed cells treated for 2 h with nocodazole before fixation, which dispersed the perinuclear lysosomal cluster (see Fig. 2a). (i) Autophagosomal and autolysosomal numbers in tfLC3-expressing cells after ARL8B overexpression and knockdown (Supplementary Fig. S5o shows representative cells.). In (f-i) we analysed 20 cells per group in three independent experiments. Values are means ± s.e.m of three independent experiments carried out in triplicate. *P0.05, **P0.01, ***P0.005 Student's t-test; other comparisons not significant (n.s.). Uncropped images of blots are shown in Supplementary Fig. S7.


( A ) ELISA of supernatant IL - 1β for unprimed THP - 1 cells ( UT ) or LPS - primed ( 500 ng / ml , 3 hrs ) THP - 1 cells then infected with ZIKV ( MOI = 1 ) for 36 hrs . Uninfected cells serves as mock control .

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

(A) ELISA of supernatant IL-1β for unprimed THP-1 cells (UT) or LPS-primed (500 ng/ml, 3 hrs) THP-1 cells then infected with ZIKV (MOI=1) for 36 hrs. Uninfected cells serves as mock control.


( B ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells , left untreated or primed by LPS ( 500 ng / ml , 3 hrs ) , after ZIKV infection ( MOI = 1 ) for 36 hrs .

O O O 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

(B) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells, left untreated or primed by LPS (500 ng/ml, 3 hrs), after ZIKV infection (MOI=1) for 36 hrs.


( C ) ELISA of IL - 1β in the supernatants of wild type ( WT ) , NLRP3 knockout ( KO ) # 1 or AIM2 KO # 1 THP - 1 cells left untreated ( UT ) , or pretreated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) stimulation , or stimulated with poly ( dA : dT ) ( 1mg / ml , 6 hrs ) , or infected with ZIKV ( MOI = 1 , 36 hrs ) .

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

(C) ELISA of IL-1β in the supernatants of wild type (WT), NLRP3 knockout (KO) #1 or AIM2 KO#1 THP-1 cells left untreated (UT), or pretreated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) stimulation, or stimulated with poly (dA:dT) (1mg/ml, 6 hrs), or infected with ZIKV (MOI=1, 36 hrs).


( D ) ELISA of IL - 1β in the supernatants of two clones of NLRP3 KO THP - 1 cells with or without ZIKV infection ( MOI = 1 ) at the indicated time points .

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

(D) ELISA of IL-1β in the supernatants of two clones of NLRP3 KO THP-1 cells with or without ZIKV infection (MOI=1) at the indicated time points.


( E ) ELISA of IL - 1β in the supernatants of PBMCs from two donors transfected with control ( ctrl ) siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

O O O O O O O O O O O O O O O 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) ELISA of IL-1β in the supernatants of PBMCs from two donors transfected with control (ctrl) siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for 36 hrs.


( F ) ELISA of IL - 1β in the supernatant of WT , NLRP3 KO or AIM2 KO THP - 1 cells with ZIKV ( MR766 or GZ01 ) ( MOI = 1 ) or DENV - 2 ( MOI = 1 ) infection for 36 hrs .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA of IL-1β in the supernatant of WT, NLRP3 KO or AIM2 KO THP-1 cells with ZIKV (MR766 or GZ01) (MOI=1) or DENV-2 (MOI=1) infection for 36 hrs.


( G ) ELISA of IL - 1β in the supernatants of THP - 1 cells pre - infected with ZIKV for 24 hrs or mock control . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) treatment for 6 hrs .

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

(G) ELISA of IL-1β in the supernatants of THP-1 cells pre-infected with ZIKV for 24 hrs or mock control. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) treatment for 6 hrs.


( H ) Immunoblot analysis of supernatant ( Sup ) and cell extracts ( Lys ) of THP - 1 cells pre - infected with ZIKV ( MOI = 1 ) or mock control for 24 hrs . The cells were then treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM ) stimulation for 6 hrs .

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

(H) Immunoblot analysis of supernatant (Sup) and cell extracts (Lys) of THP-1 cells pre-infected with ZIKV (MOI=1) or mock control for 24 hrs. The cells were then treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM) stimulation for 6 hrs.


( I ) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3 , ASC , pro - caspase - 1 and pro - IL - 1β together with NS1 .

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

(I) Immunoblot analysis of supernatants and cell extracts of 293T cells transfected with plasmids expressing NLRP3, ASC, pro-caspase-1 and pro-IL-1β together with NS1.


( J ) Immunoblot analysis of protein extracts of Flag - tagged NS1 - inducible THP - 1 cells treated with increasing doses of doxycycline ( Dox ) for 24 hrs .

O O O O O O O O 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) Immunoblot analysis of protein extracts of Flag-tagged NS1-inducible THP-1 cells treated with increasing doses of doxycycline (Dox) for 24 hrs.


( K ) ELISA of IL - 1β in the supernatants of Flag - NS1 - inducible THP - 1 cells left unprimed then treated with ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) , or pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation .

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

(K) ELISA of IL-1β in the supernatants of Flag-NS1-inducible THP-1 cells left unprimed then treated with ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs), or pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.


( L ) Immunoblot analysis of supernatants ( Sup ) and cell extracts ( Lys ) of Flag - NS1 - inducible THP - 1 cells pre - treated with LPS ( 500 ng / ml , 3 hrs ) followed by ATP ( 5 mM , 6 hrs ) or poly ( I : C ) ( 2 mg / ml , 6 hrs ) stimulation .

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

(L) Immunoblot analysis of supernatants (Sup) and cell extracts (Lys) of Flag-NS1-inducible THP-1 cells pre-treated with LPS (500 ng/ml, 3 hrs) followed by ATP (5 mM, 6 hrs) or poly (I:C) (2 mg/ml, 6 hrs) stimulation.


( A ) Relative qRT - PCR analysis of ZIKV RNA in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(A) Relative qRT-PCR analysis of ZIKV RNA in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.


( B ) Relative qRT - PCR analysis of ZIKV RNA in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(B) Relative qRT-PCR analysis of ZIKV RNA in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( C ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs by the indicated antibodies .

(C) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs by the indicated antibodies.


( D ) Relative qRT - PCR analysis of ZIKV RNA in WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(D) Relative qRT-PCR analysis of ZIKV RNA in WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.


( E ) Plaque titration of ZIKV in supernatants of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(E) Plaque titration of ZIKV in supernatants of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.


( F ) Immunoblot analysis of extracts of WT or caspase - 1 KO THP - 1 cells by the indicated antibodies .

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

(F) Immunoblot analysis of extracts of WT or caspase-1 KO THP-1 cells by the indicated antibodies.


( G ) Relative qRT - PCR analysis of ZIKV RNA in WT or caspase - 1 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(G) Relative qRT-PCR analysis of ZIKV RNA in WT or caspase-1 KO THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( H ) Relative qRT - PCR analysis of ZIKV RNA in NS - 1 - inducible THP - 1 cells treated with or without doxycycline ( Dox ) , then left untreated or treated with Ac - YVAD - cmk ( 20 μΜ ) for 3 hrs followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-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

(H) Relative qRT-PCR analysis of ZIKV RNA in NS-1-inducible THP-1 cells treated with or without doxycycline (Dox), then left untreated or treated with Ac-YVAD-cmk (20 μΜ) for 3 hrs followed by ZIKV infection (MOI=1) for the indicated time points.


( I ) Relative qRT - PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors .

(I) Relative qRT-PCR analysis of NLRP3 knockdown efficiency in PBMCs from two donors.


( J ) Relative qRT - PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs .

(J) Relative qRT-PCR analysis of ZIKV RNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.


( K ) qRT - PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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

(K) qRT-PCR analysis of ZIKV RNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


( A ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - NS1 together with Flag - NLRP3 , Flag - ASC , Flag - caspase - 1 and Flag - IL - 1β . WCL , whole cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-NS1 together with Flag-NLRP3, Flag-ASC, Flag-caspase-1 and Flag-IL-1β. WCL, whole cell lysates.


( B ) Co - immunoprecipitation and immunoblot analysis of extracts of NS1 - inducible THP - 1 cells , which expressed Flag - tagged NS1 , treated with or without doxycycline ( Dox ) ( 100 ng / ml ) .

O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of NS1-inducible THP-1 cells, which expressed Flag-tagged NS1, treated with or without doxycycline (Dox) (100 ng/ml).


( C ) Immunoblot analysis of extracts of THP - 1 cells treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) followed by increasing doses of Dox treatment .

O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of extracts of THP-1 cells treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs) followed by increasing doses of Dox treatment.


( D ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - NS1 . Below , caspase - 1 mRNA RT - PCR ; RPL13A mRNA RT - PCR serves as a loading control .

(D) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-NS1. Below, caspase-1 mRNA RT-PCR; RPL13A mRNA RT-PCR serves as a loading control.


( E ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with increasing amount of HA - DENV - 2 - NS1 .

O O O O O O 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) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with increasing amount of HA-DENV-2-NS1.


( F ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 followed by cycloheximide ( CHX ) treatment ( 100 μg / ml ) for the indicated time points .

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

(F) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 followed by cycloheximide (CHX) treatment (100 μg/ml) for the indicated time points.


( G ) Immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 together with empty vector or HA - NS1 then treated with MG132 ( 10 mM ) , Lactacystin ( 5 μM ) , Carfilzomib ( 100 mM ) or CQ ( 50 mM ) for 6 hrs .

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

(G) Immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 together with empty vector or HA-NS1 then treated with MG132 (10 mM), Lactacystin (5 μM), Carfilzomib (100 mM) or CQ (50 mM) for 6 hrs.


( H ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) .

O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml).


( I ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - NS1 , followed by MG132 ( 10 mM ) treatment for 6 hrs .

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

(I) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-NS1, followed by MG132 (10 mM) treatment for 6 hrs.


( A ) Immunoblot analysis of extracts of wild type ( WT ) and USP8 knockout ( KO ) 293T cells transfected with Flag - caspase - 1 together with empty vector or Myc - NS1 .

O O O O O O O O O O O O O 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) Immunoblot analysis of extracts of wild type (WT) and USP8 knockout (KO) 293T cells transfected with Flag-caspase-1 together with empty vector or Myc-NS1.


( B ) Immunoblot analysis of extracts of transfected with Flag - caspase - 1 together with empty vector or Myc - USP8 then treated with or without MG132 ( 10 mM ) for 6 hrs .

O O O O 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

(B) Immunoblot analysis of extracts of transfected with Flag-caspase-1 together with empty vector or Myc-USP8 then treated with or without MG132 (10 mM) for 6 hrs.


( C ) Co - immunoprecipitation and immunoblot analysis of extracts of Flag - NS1 - inducible THP - 1 cells treated with or without Dox ( 100 ng / ml ) then transfected with or without poly ( I : C ) ( 2 mg / ml , 6 hrs ) .

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

(C) Co-immunoprecipitation and immunoblot analysis of extracts of Flag-NS1-inducible THP-1 cells treated with or without Dox (100 ng/ml) then transfected with or without poly (I:C) (2 mg/ml, 6 hrs).


( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 and Myc - USP8 together with HA - NS1 followed by MG132 treatment ( 10 mM ) for 6 hrs .

(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 and Myc-USP8 together with HA-NS1 followed by MG132 treatment (10 mM) for 6 hrs.


( E ) Co - immunoprecipitation and immunoblot analysis of extracts of THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs .

O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.


( F ) Co - immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP - 1 cells infected with ZIKV ( MOI = 1 ) for 24 hrs .

O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of WT and USP8 KO THP-1 cells infected with ZIKV (MOI=1) for 24 hrs.


( G ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag - caspase - 1 and indicated HA - tagged WT ubiquitin ( Ub ) or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - and K63 - Only ) , together with empty vector or Myc - USP8 , followed by MG132 ( 10 mM ) treatment for 6 hrs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with plasmids expressing Flag-caspase-1 and indicated HA-tagged WT ubiquitin (Ub) or mutants (K6-, K11-, K27-, K29-, K33-, K48- and K63-Only), together with empty vector or Myc-USP8, followed by MG132 (10 mM) treatment for 6 hrs.


( H ) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag - caspase - 1 , HA - K11 - Ub together with empty vector or Myc - NS1 , followed by two - step immunoprecipitation with anti - Flag beads and immunoblot analysis with anti - HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of extracts of WT or USP8 KO 293T cells transfected with Flag-caspase-1, HA-K11-Ub together with empty vector or Myc-NS1, followed by two-step immunoprecipitation with anti-Flag beads and immunoblot analysis with anti-HA.


( I ) Co - immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag - caspase - 1 and HA - K11 - Ub together with WT or inactive mutant C786A of USP8 .

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

(I) Co-immunoprecipitation and immunoblot analysis of extracts of USP8 KO 293T cells transfected with Flag-caspase-1 and HA-K11-Ub together with WT or inactive mutant C786A of USP8.


( A ) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA - NS1 together with Flag - caspae - 1 wild type ( WT ) and mutants ( K37R , K44R or K134R ) . Lower panel shows the intensity analysis of the bands from the three independent experiments .

(A) Immunoblot analysis of extracts of 293T cells transfected with empty vector or HA-NS1 together with Flag-caspae-1 wild type (WT) and mutants (K37R, K44R or K134R). Lower panel shows the intensity analysis of the bands from the three independent experiments.


( B ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA - K11 - ubiquitin ( Ub ) together with Flag - caspase - 1 ( WT , K37R , K44R , K134R ) .

(B) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with HA-K11-ubiquitin (Ub) together with Flag-caspase-1 (WT, K37R, K44R, K134R).


( C ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - NS1 . Lower panel shows the intensity analysis of the bands from the three independent experiments .

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

(C) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-NS1. Lower panel shows the intensity analysis of the bands from the three independent experiments.


( D ) Co - immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag - caspase - 1 ( WT or K134R ) and HA - K11 - Ub , together with empty vector or Myc - USP8 . Lower panel shows the intensity analysis of the bands from the three independent experiments .

(D) Co-immunoprecipitation and immunoblot analysis of extracts of 293T cells transfected with Flag-caspase-1 (WT or K134R) and HA-K11-Ub, together with empty vector or Myc-USP8. Lower panel shows the intensity analysis of the bands from the three independent experiments.


( A ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in wild type ( WT ) or caspase - 1 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(A) Relative qRT-PCR analysis of IFNB, IFIT2 and IFIT1 mRNA levels in wild type (WT) or caspase-1 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( B ) Relative qRT - PCR analysis of IFNB mRNA levels in THP - 1 cells left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) and ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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 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

(B) Relative qRT-PCR analysis of IFNB mRNA levels in THP-1 cells left untreated or treated with LPS (500 ng/ml, 3 hrs) and ATP (5 mM, 6 hrs), followed by ZIKV infection (MOI=1) for the indicated time points.


( C ) Relative qRT - PCR analysis of IFNB mRNA levels in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(C) Relative qRT-PCR analysis of IFNB mRNA levels in wild type (WT) or NLRP3 knockout (KO) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( D ) Relative qRT - PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag - NS1 - inducible THP - 1 cells treated with or without Dox then left untreated or treated with YVAD ( 20 μM ) for 3 hrs , followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(D) Relative qRT-PCR analysis of IFNB , IFIT2 and IFIT1 mRNA levels in Flag-NS1-inducible THP-1 cells treated with or without Dox then left untreated or treated with YVAD (20 μM) for 3 hrs, followed by ZIKV infection (MOI=1) for the indicated time points.


( E ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs .

(E) Relative qRT-PCR analysis of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV (MOI=1) for 36 hrs.


( F ) Relative qRT - PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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

(F) Relative qRT-PCR analysis of IFNB mRNA in PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


( G ) ELISA of IFN - β in the supernatants of PBMCs treated with DMSO or Ac - YVAD - cmk ( 20μM ) for 3hrs followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

(G) ELISA of IFN-β in the supernatants of PBMCs treated with DMSO or Ac-YVAD-cmk (20μM) for 3hrs followed by ZIKV infection (MOI=1) for 36 hrs.


( H ) ELISA of IL - 1β production in brain of WT or Nlrp3 - / - 1 - day - old neonatal mice ( n = 3 per group ) intracerebrally injected with 1 × 105 plaque - forming units ( PFU ) of ZIKV for 5 days .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ELISA of IL-1β production in brain of WT or Nlrp3-/- 1-day-old neonatal mice (n=3 per group) intracerebrally injected with 1×105 plaque-forming units (PFU) of ZIKV for 5 days.


( I ) Relative qRT - PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV

(I) Relative qRT-PCR analysis of IFNB mRNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection


( J ) ELISA of IFN - β production in brains of WT or Nlrp3 - / - neonatal

(J) ELISA of IFN-β production in brains of WT or Nlrp3-/- neonatal mice


( K ) Relative qRT - PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV

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

(K) Relative qRT-PCR analysis of ZIKV RNA levels in brain tissues of WT or Nlrp3-/- neonatal mice with or without ZIKV infection


( L ) Plaque titration of ZIKV in brain tissues of WT or Nlrp3 - / - neonatal

(L) Plaque titration of ZIKV in brain tissues of WT or Nlrp3-/- neonatal mice


( M ) Body weight gain of WT and Nlrp3 - / - neonatal mice ( n = 4 per group ) inoculated with ZIKV ( 1 × 105 PFUs ) .

(M) Body weight gain of WT and Nlrp3-/- neonatal mice (n=4 per group) inoculated with ZIKV (1×105 PFUs).


( A ) Relative qRT - PCR analysis of ZIKV RNA levels in wild type ( WT ) or cGAS knockout ( KO ) THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points .

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

(A) Relative qRT-PCR analysis of ZIKV RNA levels in wild type (WT) or cGAS knockout (KO) THP-1 cells after ZIKV infection (MOI=1) for indicated time points.


( B ) Relative qRT - PCR analysis of IFNΒ , IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP - 1 cells after ZIKV infection ( MOI = 1 ) for indicated time points .

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

(B) Relative qRT-PCR analysis of IFNΒ, IFIT2 and IFIT1 mRNA levels in WT or cGAS KO THP-1 cells after ZIKV infection (MOI=1) for indicated time points.


( C ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP , cGAS or NLRP3 and infected with ZIKV ( MOI = 1 ) for 36 hrs .

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

(C) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in PBMCs transduced with Cas9 and small guide RNA targeting GFP, cGAS or NLRP3 and infected with ZIKV (MOI=1) for 36 hrs.


( D ) Relative qRT - PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP - 1 cells treated with or without YVAD ( 20 μM ) , followed by ZIKV infection ( MOI = 1 ) for indicated times points .

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

(D) Relative qRT-PCR analysis of ZIKV RNA and IFNΒ mRNA levels in WT or cGAS KO THP-1 cells treated with or without YVAD (20 μM), followed by ZIKV infection (MOI=1) for indicated times points.


( E ) Immunoblot analysis of extracts of Flag - cGAS - overexpressed THP - 1 cells treated with or without YVAD ( 20 μM ) then infected with ZIKV ( MOI = 5 ) for 24 hrs or infected with HSV - 1 ( MOI = 1 ) for 24 hrs .

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

(E) Immunoblot analysis of extracts of Flag-cGAS-overexpressed THP-1 cells treated with or without YVAD (20 μM) then infected with ZIKV (MOI=5) for 24 hrs or infected with HSV-1 (MOI=1) for 24 hrs.


Immunoblot analysis of WT , NLRP3 KO ( F ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

Immunoblot analysis of WT, NLRP3 KO (F) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


Immunoblot analysis of WT , caspase - 1 KO ( G ) THP - 1 cells infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

Immunoblot analysis of WT, caspase-1 KO (G) THP-1 cells infected with ZIKV (MOI=1) for the indicated time points.


( H ) Immunoblot analysis of extracts of WT or Nlrp3 - / - BMDMs infected with ZIKV ( MOI = 1 ) for the indicated time points .

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

(H) Immunoblot analysis of extracts of WT or Nlrp3-/- BMDMs infected with ZIKV (MOI=1) for the indicated time points.


( I ) Immunoblot analysis of PBMCs from Donor # 1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for the indicated time points .

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

(I) Immunoblot analysis of PBMCs from Donor#1 transfected with control siRNA or NLRP3 siRNA followed by ZIKV infection (MOI=1) for the indicated time points.


( J ) Immunoblot analysis of extracts of 293T cells transfected with Myc - cGAS ( tagged on C - terminal ) , GFP - ASC , Flag - caspase - 1 , together with empty vector or HA - NS1 .

(J) Immunoblot analysis of extracts of 293T cells transfected with Myc-cGAS (tagged on C-terminal), GFP-ASC, Flag-caspase-1, together with empty vector or HA-NS1.


( K ) Relative qRT - PCR analysis of ZIKV RNA and IFNB mRNA levels in WT , cGAS KO THP - 1 cells as well as cGAS KO THP - 1 cells reconstituted with WT or D140A / D157A mutant of cGAS , followed by ZIKV infection ( MOI = 1 ) for 24 hrs .

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

(K) Relative qRT-PCR analysis of ZIKV RNA and IFNB mRNA levels in WT, cGAS KO THP-1 cells as well as cGAS KO THP-1 cells reconstituted with WT or D140A/D157A mutant of cGAS, followed by ZIKV infection (MOI=1) for 24 hrs.


Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA - mediated p97 depletion under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( A ) ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA-mediated p97 depletion under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (A) (***P<0.001; unpaired t-test, n=3, mean +SEM).


Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition ( CB5083 , 10µM for 6h ) under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( C ) ( * P < 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10µM for 6h) under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (C) (*P<0.05, ****P<0.0001; unpaired t-test, n=3, mean +SEM).


Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline - induced mild expression of the p97EQ variant under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( E ) ( * * P < 0 . 01 , * * * * P < 0 . 0001 ; unpaired t - test , n = 4 , mean + SEM ) .

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

Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-induced mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (E) (**P<0.01, ****P<0.0001; unpaired t-test, n=4, mean +SEM).


Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA - mediated p97 depletion . Graph represents the quantifications of ( H ) ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) and Western blot for efficacy of siRNA depletion of p97 ( right ) .

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

Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion.   Graph represents the quantifications of (H) (***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM) and Western blot for efficacy of siRNA depletion of p97 (right).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing K48 - linked hyper - ubiquitination of RNF8 after siRNA - mediated p97 depletion .

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing K48-linked hyper-ubiquitination of RNF8 after siRNA-mediated p97 depletion.


Western blot analysis of Strep - p97 Co - IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( K ) ( * P < 0 . 05 ; unpaired t - test , n = 2 , mean + SEM ) .

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

Western blot analysis of Strep-p97 Co-IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (K) (*P<0.05; unpaired t-test, n=2, mean +SEM).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing the ubiquitination pattern of RNF8 - WT and RNF8 - RING * variant , under siRNA - mediated luciferase ( siLuc ) or p97 - depleted conditions ( sip97 ) .

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

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97).


Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition ( MG132 , 10µM ) . Graph represents the quantifications of ( B ) ( ns ; not significant , P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) .

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

Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).


Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing hyper - ubiquitination of RNF8 after proteasome inhibition ( MG132 , 10µM for 6h ) .

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

Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h).


Western blot analysis of CHX chase kinetics in U2OS cells , comparing the degradation rate of Flag - RNF8 - WT and Flag - RNF8 - RING * . Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8 . Graph represents the quantifications of ( E ) ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) .

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

Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.   Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).


Western blot analysis of Strep - p97 Co - IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR ( 10 Gy ) .

Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy).


Western blot analysis of Flag - ATX3 Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and PSα6 under physiological conditions and after IR ( 10 Gy ) .

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

Western blot analysis of Flag-ATX3 Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and PSα6 under physiological conditions and after IR (10 Gy).


Western blot analysis of Flag - ATX3 WT or Flag - ATX3 VBM * Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and p97 core co - factors Npl4 - Ufd1 . ( * represents unspecific bands ) .

Western blot analysis of Flag-ATX3 WT or Flag-ATX3 VBM* Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and p97 core co-factors Npl4-Ufd1. (* represents unspecific bands).


Western blot analysis of Strep - p97EQ Co - IP in HEK293 cells , in presence or absence ( CRISPR knockout ; ∆ ATX3 ) of ATX3 , showing increased interaction of RNF8 with p97 in absence of ATX3 , under physiological conditions and after IR ( 10 Gy ) . Graph represents quantifications of ( F ) ( * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) .

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

Western blot analysis of Strep-p97EQ Co-IP in HEK293 cells, in presence or absence (CRISPR knockout; ∆ATX3) of ATX3, showing increased interaction of RNF8 with p97 in absence of ATX3, under physiological conditions and after IR (10 Gy).   Graph represents quantifications of (F) (*P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).