Photographic showing hair loss in mice at 14 days post - IR ( 15 Gy ) following treatment by daily topical application of acquosum ( carrier control ) or JAK inhibitors , ruxolitinib or tofacitinib . Chest neck hairs of mice in groups were clipped prior to irradiation to enhance drug absorption . of of ventral hair loss ( in pixels ) in mice in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , by two - Student ' s t ; two independent experiments .

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Photographic images showing hair loss in mice at 14 days post-IR (15 Gy) following treatment by daily topical application of acquosum (carrier control) or JAK inhibitors, ruxolitinib or tofacitinib. Chest and neck hairs of mice in all groups were clipped prior to irradiation to enhance drug absorption. Quantification of the area of ventral hair loss (in pixels) in mice in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, **P<0.01, by two-tailed Student's t test; two independent experiments.


UMAP plots depicting relative expression localization of Il1a Il1b mRNAs in irradiated naïve skin - derived clusters .

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UMAP plots depicting relative expression and localization of Il1a and Il1b mRNAs in irradiated and naïve skin-derived cell clusters.


Photographic showing hair loss 14 days post - IR ( 15 Gy ) in WT IL - 1R - / - mice . of ventral hair loss 14 days post - IR ( 15Gy ) in WT IL - 1R - / - mice , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by two - Mann - Whitney ( C ) five independent experiments .

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Photographic images showing hair loss 14 days post-IR (15 Gy) in WT and IL-1R-/- mice. Quantification of ventral hair loss area 14 days post-IR (15Gy) in WT and IL-1R-/- mice, (n = 4). Data information: Data represent mean ± SD. *P<0.05, by two-tailed Mann-Whitney test (C) five independent experiments.


Histochemical ( H & E ) immunostaining ( red ) showing neutrophil ( Ly6b ) infiltration in skin of mice in ( B ) . Scale bar , 50 μm . of epidermal thickness , neutrophils ( Ly6b ) T ( CD3 + ) in specimens in ( D ) , ( n = 4 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , by Student ' s t ( E ) ; five independent experiments .

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Histochemical (H&E) and immunostaining (red) showing morphology and neutrophil (Ly6b) infiltration in the skin of mice in (B). Scale bar, 50 μm. Quantification of epidermal thickness, neutrophils (Ly6b) and T cells (CD3+) in specimens in (D), (n = 4). Data information: Data represent mean ± SD. *P<0.05, by Student's t test (E); five independent experiments.


UMAP plots depicting relative expression localization of Ccr6 Ccl20 mRNAs in irradiated naïve skin - derived clusters . by real - time qPCR of Ccr6 Ccl20 mRNAs in whole skin of naïve irradiated ( 15Gy ) WT IL - 6 - / - mice at indicated times post - IR , ( n = 5 - 7 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney Two independent experiments .

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UMAP plots depicting relative expression and localization of Ccr6 and Ccl20 mRNAs in irradiated and naïve skin-derived cell clusters. Quantification by real-time qPCR analysis of Ccr6 and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) WT and IL-6-/- mice at indicated times post-IR, (n=5-7). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Photographic showing development of radiodermatitis alopecia in irradiated ( 15 Gy ) WT CCR6 - / - mice at indicated times post - IR . Escalating radiodermatitis , which manifests on day 7 - 10 as serous exudates on chin neck , is visible in both strains progresses in WT mice to alopecia by day 14 post - IR but resolves spontaneously in CCR6 - / - mice . ( lower right ) of of hair loss at 14 days post - IR , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney Two independent experiments .

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Photographic images showing the development of radiodermatitis and alopecia in irradiated (15 Gy) WT and CCR6-/- mice at indicated times post-IR. Escalating radiodermatitis, which manifests on day 7-10 as serous exudates on the chin and neck, is visible in both strains and progresses in WT mice to alopecia by day 14 post-IR but resolves spontaneously in the CCR6-/- mice. Quantification (lower right) of the area of hair loss at 14 days post-IR, (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Histochemical ( H & E ) immunostaining ( red ) show infiltration of neutrophils ( Ly6b + ) T ( CD3 + ) ( red arrows ) to skin to hair follicle ( HF ) ( dashed lines ) in WT or CCR6 - / - mice 14 days post - IR . Scale bars , 50 μm ( black ) 25 μm ( red ) . of epidermal thickness , neutrophils ( Ly6b + ) , total T ( CD3 + ) infiltration to skin , ( F ) CD3 + infiltration to hair follicle in ( D ) , ( n = 5 - 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - Student ' s t ( E - Ly6b , by Mann - Whitney Two independent experiments .

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Histochemical staining (H&E) and immunostaining (red) show morphology and infiltration of neutrophils (Ly6b+) and T cells (CD3+) (red arrows) to the skin and to the hair follicle (HF) (dashed lines) in WT or CCR6-/- mice 14 days post-IR. Scale bars, 50 μm (black) and 25 μm (red). Quantification of epidermal thickness, neutrophils (Ly6b+), total T cell (CD3+) infiltration to the skin, and (F) CD3+ cells infiltration to the hair follicle in (D), (n = 5-6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t test (E-Ly6b, by Mann-Whitney test Two independent experiments.


MHC class I immunostaining ( red ) of skin thin sections prepared at 14 days post - IR from mice in ( C ) , ( right ) of MHC I positive hair follicles , ( n = 4 - 5 ) . Scale bar , 50 μm . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney Two independent experiments .

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MHC class I immunostaining (red) of skin thin sections prepared at 14 days post-IR from mice in (C), and quantification (right) of MHC I positive hair follicles, (n = 4-5). Scale bar, 50 μm. Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


by real - time qPCR of Il6 , Il17a , Ccl20 , Ccl3 mRNAs in skin of naïve WT irradiated ( 15Gy ) WT CCR6 - / - mice at 14 days post - IR , ( n = 3 - 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by Mann - Whitney Two independent experiments .

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Quantification by real-time qPCR analysis of Il6, Il17a, Ccl20, and Ccl3 mRNAs in the skin of naïve WT and irradiated (15Gy) WT and CCR6-/- mice at 14 days post-IR, (n = 3-5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by Mann-Whitney test Two independent experiments.


Photographic showing hair loss in control ( PBS ) cyclosporine ( CsA ) treated WT mice at 14 days post - IR ( 15 Gy ) . of of ventral hair loss in mice in ( A ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - Mann - Whitney ; two independent experiments .

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Photographic images showing hair loss in control (PBS) and cyclosporine (CsA) treated WT mice at 14 days post-IR (15 Gy). Quantification of the area of ventral hair loss in mice in (A), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


Histochemical ( H & E ) immunostaining ( red ) show T ( CD3 + ) neutrophil ( Ly6b + ) infiltration in skin of mice in ( A ) . Scale bar , 100 μm . ( of CD3 Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections . ) of epidermal thickness , T ( CD3 + ) neutrophil ( Ly6b + ) infiltration ( red ) in skin thin sections from ( C ) , ( n = 6 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - Mann - Whitney ; two independent experiments .

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Histochemical (H&E) and immunostaining (red) show T cell (CD3+) and neutrophil (Ly6b+) infiltration in the skin of mice in (A). Scale bar, 100 μm. (Images of CD3 and Ly6b immunostaining from CsA treated mice represent seperate analyses performed on serial thin sections.) Quantification of epidermal thickness, and T cell (CD3+) and neutrophil (Ly6b+) infiltration (red staining) in skin thin sections from (C), (n = 6). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


by real - time qPCR of Il6 , Il17a , II22 , Ccl3 , Ccr6 , Ccl20 mRNAs in whole skin of naïve irradiated ( 15Gy ) control ( PBS ) CSA treated WT mice at 14 days post - IR , ( n = 5 ) . Data information : Data represent mean ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - Mann - Whitney ; two independent experiments .

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Quantification by real-time qPCR analysis of Il6, Il17a, II22, Ccl3, Ccr6, and Ccl20 mRNAs in whole skin of naïve and irradiated (15Gy) control (PBS) and CSA treated WT mice at 14 days post-IR, (n = 5). Data information: Data represent mean ± SD. *P < 0.05, **P < 0.01 by two-tailed Mann-Whitney test; two independent experiments.


( A - B ) Immunofluorescence ( IF ) of IκBα in sections from murine small intestine of 2 month - old WT ( A ) Lgr5 - GFP reporter mice ( B ) . Scale bars , 50μm .

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(A-B) Immunofluorescence (IF) analysis of IκBα in sections from murine small intestine of 2 month-old WT (A) and Lgr5-GFP reporter mice (B). Scale bars, 50μm.


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

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

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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 according to their position in intestinal crypt from 200 crypts counted .

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


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

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(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 of murine small intestine sections with indicated antibodies . Scale bars , 50 μm .

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


( H ) Western blot of ( CYT ) ( NUC ) extracts from isolated intestinal crypt .

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


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

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


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

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(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 indicated genomic regions obtained from ChIP - sequencing ( n = 4 ) .

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


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

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


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

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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.


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

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

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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 intestinal populations purified in sorting experiments .

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


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

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(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 . Data information : Scale bars , 25 μm .

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


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

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(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 indicated differentially expressed gene ( DEG ) sets . Values for DEG in adult ISC was obtained from Nusse et al , 2012 .

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(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 top 15 up - regulated genes in IκBα KO EphB2 - high signature including 11 genes in ISC signature ( in orange ) .

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(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 indicating enrichment of relevant ISC - related pathways .

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


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

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


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

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(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 of c - Rel RelA / p65 in P6 WT IκBα KO intestines . Scale bars in 25 μm .

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(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 associated with different genomic localizations was obtained merging two biological replicates per condition ( n = 2 P6 WT n = 2 P6 IκBα KO intestinal crypt ) . Data from experiments have been deposited at GSE131187 .

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

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


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

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


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

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(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 methylation - related genes ( PRC2 members KDM6 demethylase ) differentially express between IκBα WT KO EphB2 - high sorted ( - seq ) .

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(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 of IκBα IP from crypt extracts .

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


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

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(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 ) H3K27me3 ( n = 2 ) distribution ( from ChIP - sequencing ) expression ( from - sequencing ) in genomic region of Litaf .

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(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 IκBα KO - derived intestinal organoids . Scale bars in A , 100

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


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

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(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 of indicated ISC genes in WT 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 mean ( s . e . m ) . p values were derived from an unpaired t - , two - , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 .

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(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 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 adult organoids were obtained from Mustata et al , 2013 .

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(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 of indicated genes in WT 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 mean ( s . e . m ) . p values were derived from an unpaired t - , two - , * * * * p - value < 0 . 0001 , * * p - value < 0 . 005 , * p - value < 0 . 05 .

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(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 of control IκBα - deleted organoids using CRISPR - Cas9 technology .

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


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

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


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

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

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(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 of IκBα KO organoids untreated or treated with PRC2 inhibitor EPZ - 6438 . Data information Scale bars in 50 μm .

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(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 of indicated adult ISC genes ( D ) elements of Notch IFN pathway ( E ) ( differentially expressed in IκBα KO organoids ) in IκBα KO organoids untreated or treated with 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 mean ( s . e . m ) ; p values were derived from an unpaired t - , two - , * * * * p - value < 0 . 0001 , * * * p - value < 0 . 0005 , * * p - value < 0 . 005 , * p - value < 0 . 05 , n . s . no significant .

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

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

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

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

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

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

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

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

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

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

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

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

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(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 in whole lysates of HBV - expressing 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 for β - actin indicates equivalent amount of lysates for IP ( C D ) .

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(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 in whole lysates extracted from HepAD38 transfected with non - targeting ( NT ) 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 ) . expression was analyzed by Western blotting with antibodies specific to Mfn2 , Parkin , β - actin . (

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

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

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(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 lysates extracted from Huh7 with empty vector ( Mock ) those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to indicated .

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

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(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 C ) of number of total GFP - LC3 ( B ) GFP - LC3 colocalized with TOM20 ( C ) in panel ( A ) . ( D ) Quantitative of of GFP - LC3 ( ) representing merge of GFP - LC3 , TOM20 , Parkin in panel ( A ) .

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

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(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 of signal targeted to mitochondria in panel ( B ) .

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


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

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(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 HepAD38 grown in absence or presence of tetracycline were transfected with non - targeting ( NT ) Parkin siRNA , respectively , for 72 h . were used for Western blot using antibodies specific to indicated ( A ) , caspase - 3 / 7 activity ( B ) , TUNEL ( C )

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

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(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 panels , values are means ± s . e . m . of three independent experiments carried out in triplicate . * P0 . 05 , * * P0 . 01 , * * * P0 . 005 Student ' s t - ; other comparisons are not significant ( n . s . ) . Representative maximum - projections of serial confocal optical sections are shown . Uncropped of blots are shown in Supplementary Fig . S7 .

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

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(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 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 . comparisons are with control within each treatment condition , * P0 . 05 , * * * P0 . 005 Student ' s t - ; n . s . not significant . Uncropped of blots are shown in Supplementary Fig . S7 .

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

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(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 allowed to starve using three different protocols ( see Methods ) with or without subsequent recovery .

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(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 mTORC1 activity . pHi was titrated in full tissue - culture medium containing nigericin , which enables changes in pHi to be forced by altering pH in medium , followed by immunostaining ( b , c ) or western blotting ( d ) using antibodies as shown .

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(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 KIF2 were overexpressed or knocked down in HeLa followed by pHi measurement .

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(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 pHi affect of ARL8 KIF2 in lysosomal fractions . their in total lysates or in isolated lysosomal fractions from HeLa 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 .

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

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

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(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 were incubated for 48 h , then left untreated or incubated with bafilomycin A1 . LC3 - II versus actin were quantified ( bottom graphs ) . Asterisk : nonspecific band .

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(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 degradation . HeLa overexpressing ARL8B or empty vector were analysed as in ( c ) .

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


( e ) HeLa were co - transfected with either ARL8B overexpression construct or siRNA ( non - targeting siRNA empty peGFP vector were transfection controls ) together with mCherry - LC3 for 48 h . After fixation , were stained for endogenous LAMP1 ( DAPI ) . Representative maximum - projections of serial confocal optical sections are shown . Co - localization panels show overlapping mCherry - LC3 LAMP1 signals . ( f - h ) of autophagosome - lysosome fusion in HeLa . Percentages of autolysosomes ( positive for both mCherry - LC3 LAMP1 ) to autophagosomes ( positive for mCherry - LC3 negative for LAMP1 ) were quantified . In ( h ) , we analysed treated for 2 h with nocodazole before fixation , which dispersed perinuclear lysosomal cluster ( see Fig . 2a ) . ( i ) Autophagosomal autolysosomal numbers in tfLC3 - expressing after ARL8B overexpression knockdown ( Supplementary Fig . S5o shows representative . ) . In ( f - i ) we analysed 20 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 - ; other comparisons not significant ( n . s . ) . Uncropped of blots are shown in Supplementary Fig . S7 .

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(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 ( UT ) or LPS - primed ( 500 ng / ml , 3 hrs ) THP - 1 then infected with ZIKV ( MOI = 1 ) for 36 hrs . Uninfected serves as mock control .

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

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(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 supernatants of wild type ( WT ) , NLRP3 knockout ( KO ) # 1 or AIM2 KO # 1 THP - 1 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 ) .

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(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 supernatants of two clones of NLRP3 KO THP - 1 with or without ZIKV infection ( MOI = 1 ) at indicated time points .

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(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 supernatants of PBMCs from two donors transfected with control ( ctrl ) siRNA or NLRP3 siRNA followed by ZIKV infection ( MOI = 1 ) for 36 hrs .

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(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 supernatant of WT , NLRP3 KO or AIM2 KO THP - 1 with ZIKV ( MR766 or GZ01 ) ( MOI = 1 ) or DENV - 2 ( MOI = 1 ) infection for 36 hrs .

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

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

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(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 of supernatants extracts of 293T transfected with plasmids expressing NLRP3 , ASC , pro - caspase - 1 pro - IL - 1β together with NS1 .

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

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(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 supernatants of Flag - NS1 - inducible THP - 1 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 .

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(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 of supernatants ( Sup ) extracts ( Lys ) of Flag - NS1 - inducible THP - 1 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O 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 of ZIKV in THP - 1 left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) ATP ( 5 mM , 6 hrs ) , followed by 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 B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O

(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 of ZIKV in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 infected with ZIKV ( MOI = 1 ) for indicated time points .

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) 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 of extracts of WT or Nlrp3 - / - BMDMs by indicated antibodies .

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

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


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

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

(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 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.


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

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

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


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

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

(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 of ZIKV in NS - 1 - inducible THP - 1 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 indicated time points .

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 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

(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 of NLRP3 knockdown efficiency in PBMCs from two donors .

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

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


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

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

(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 of ZIKV 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 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 immunoblot of extracts of 293T transfected with HA - NS1 together with Flag - NLRP3 , Flag - ASC , Flag - caspase - 1 Flag - IL - 1β . WCL , whole lysates .

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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 immunoblot of extracts of NS1 - inducible THP - 1 , which expressed Flag - tagged NS1 , treated with or without doxycycline ( Dox ) ( 100 ng / ml ) .

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-MEASURED_VAR 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 of extracts of THP - 1 treated with LPS ( 500 ng / ml , 3 hrs ) 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

(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 of extracts of 293T 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 .

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

(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 of extracts of 293T 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 B-CONTROLLED_VAR 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 of extracts of 293T transfected with Flag - caspase - 1 together with empty vector or HA - NS1 followed by cycloheximide ( CHX ) treatment ( 100 μg / ml ) 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 of extracts of 293T 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 B-CONTROLLED_VAR 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 immunoblot of extracts of Flag - NS1 - inducible THP - 1 treated with or without Dox ( 100 ng / ml ) .

(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 immunoblot of extracts of 293T transfected with plasmids expressing Flag - caspase - 1 indicated HA - tagged WT ubiquitin or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - 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 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 B-CONTROLLED_VAR 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 of extracts of wild type ( WT ) USP8 knockout ( KO ) 293T transfected with Flag - caspase - 1 together with empty vector or Myc - NS1 .

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 B-CONTROLLED_VAR 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 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 B-CONTROLLED_VAR 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 immunoblot of extracts of Flag - NS1 - inducible THP - 1 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 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 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 immunoblot of extracts of 293T transfected with Flag - caspase - 1 Myc - USP8 together with HA - NS1 followed by MG132 treatment ( 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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

(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 immunoblot of extracts of THP - 1 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

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


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

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

(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 immunoblot of extracts of 293T transfected with plasmids expressing Flag - caspase - 1 indicated HA - tagged WT ubiquitin ( Ub ) or mutants ( K6 - , K11 - , K27 - , K29 - , K33 - , K48 - 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 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 B-CONTROLLED_VAR 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 of extracts of WT or USP8 KO 293T transfected with Flag - caspase - 1 , HA - K11 - Ub together with empty vector or Myc - NS1 , followed by two - step immunoprecipitation with anti - Flag beads immunoblot with anti - HA .

O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 immunoblot of extracts of USP8 KO 293T transfected with Flag - caspase - 1 HA - K11 - Ub together with WT or inactive mutant C786A of USP8 .

O O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 of extracts of 293T transfected with empty vector or HA - NS1 together with Flag - caspae - 1 wild type ( WT ) mutants ( K37R , K44R or K134R ) . Lower panel shows of bands from three independent experiments .

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

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

O 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

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

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 B-CONTROLLED_VAR 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 immunoblot of extracts of 293T transfected with Flag - caspase - 1 ( WT or K134R ) HA - K11 - Ub , together with empty vector or Myc - USP8 . Lower panel shows of bands from 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 of IFNB , IFIT2 IFIT1 mRNA in wild type ( WT ) or caspase - 1 knockout ( KO ) THP - 1 infected with ZIKV ( MOI = 1 ) for indicated time points .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O 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 of IFNB mRNA in THP - 1 left untreated or treated with LPS ( 500 ng / ml , 3 hrs ) ATP ( 5 mM , 6 hrs ) , followed by ZIKV infection ( MOI = 1 ) for indicated time points .

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 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) 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 of IFNB mRNA in wild type ( WT ) or NLRP3 knockout ( KO ) THP - 1 infected with ZIKV ( MOI = 1 ) for indicated time points .

O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O 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 of IFNB , IFIT2 IFIT1 mRNA in Flag - NS1 - inducible THP - 1 treated with or without Dox then left untreated or treated with YVAD ( 20 μM ) for 3 hrs , followed by ZIKV infection ( MOI = 1 ) for indicated time points .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR 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 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 of IFNB mRNA in PBMCs transfected with control siRNA or NLRP3 siRNA then infected with ZIKV ( MOI = 1 ) for 36 hrs .

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

(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 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 B-MEASURED_VAR 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 supernatants of 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

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

(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 of IFNB mRNA in brain tissues of WT or Nlrp3 - / - neonatal mice with or without ZIKV

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

(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

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

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


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

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

(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

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

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


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

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

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


( A ) Relative qRT - PCR of ZIKV in wild type ( WT ) or cGAS knockout ( KO ) THP - 1 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 B-CONTROLLED_VAR 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 of IFNΒ , IFIT2 IFIT1 mRNA in WT or cGAS KO THP - 1 after ZIKV infection ( MOI = 1 ) for indicated time points .

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

(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 of ZIKV IFNB mRNA in PBMCs transduced with Cas9 small guide targeting GFP , cGAS or NLRP3 infected with ZIKV ( MOI = 1 ) for 36 hrs .

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

(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.