C - D ) Mean interactions frequencies + / - 1Mb centered on all TAD boundaries for each dataset at 40kb resolution , as well as the log2 ratios of aXL ( left panel ) or bXL ( right panel ) samples . The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking .

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C-D) Mean interactions frequencies +/- 1Mb centered on all TAD boundaries for each dataset at 40kb resolution, as well as the log2 ratios of aXL (left panel) or bXL (right panel) samples. The TAD structures are not altered upon RNase A treatment in samples treated with RNase after crosslinking.


A ) Hi - C interaction heatmaps at 500kb ( left ) resolution showing all of chromosome 11 , and at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for control and 24hr ActD treated cells . The arrows point out to perturbed long - range interactions at shorter scales .

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A) Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for control and 24hr ActD treated cells. The arrows point out to perturbed long-range interactions at shorter scales.


B ) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4 , with the 1st principal component ( 1st PC ) below for control and 24hr ActD treated cells .

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B) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal component (1st PC) below for control and 24hr ActD treated cells.


C ) Violin plot showing the first eigenvalues ( calculated by the trans - chromosomal data ) in control and ActD treated cells . p - value : Wilcoxon rank - sum test . In the violin plots , the horizontal bands represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates .

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C) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and ActD treated cells. p-value: Wilcoxon rank-sum test. In the violin plots, the horizontal bands represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


D ) Saddle plots showing the compartmental interactions for both cis - and trans - contacts across the conditions .

D) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions.


E ) Scaling plot generated by 500kb resolution Hi - C data showing the interaction frequency as a function of genomic distance .

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E) Scaling plot generated by 500kb resolution Hi-C data showing the interaction frequency as a function of genomic distance.


A ) Interaction heatmaps at 40kb resolution zooming in chr9 : 104 - 114Mb and showing the TAD structures , as well as the log2 ratios , in control and transcriptionally inhibited datasets . Black arrow indicates the increase in inter - TAD interactions .

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A) Interaction heatmaps at 40kb resolution zooming in chr9:104-114Mb and showing the TAD structures, as well as the log2 ratios, in control and transcriptionally inhibited datasets. Black arrow indicates the increase in inter-TAD interactions.


B ) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells . p - value : Wilcoxon rank - sum test . The horizontal bands in the boxplots represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates .

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B) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


C ) Mean interaction frequencies centered on all TAD boundaries + / - 1Mb for each dataset at 40kb resolution , as well as the log2 ratios . The inter - TAD interactions are increased upon transcriptional inhibition .

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C) Mean interaction frequencies centered on all TAD boundaries +/- 1Mb for each dataset at 40kb resolution, as well as the log2 ratios. The inter-TAD interactions are increased upon transcriptional inhibition.


D ) Aggregate Peak Analysis based on the K562 loops coordinates The heatmaps are centered on the loop positions + / - 250kb using 25kb resolution Hi - C data . There is a ~ 50 % reduction of loop intensity as assessed by z - scores and log2 fold change .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Aggregate Peak Analysis based on the K562 loops coordinates The heatmaps are centered on the loop positions +/- 250kb using 25kb resolution Hi-C data. There is a ~50% reduction of loop intensity as assessed by z-scores and log2 fold change.


E ) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells for boundaries that are bound with differing numbers of CTCF binding sites . The reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding . p - value : Wilcoxon rank - sum test . The horizontal bands in the boxplots represent the median , the error bars represent + / - 1 . 5 x IQR values , and the outer shapes represent the density of the data points . The figures represent data generated from the pooled Hi - C datasets with 2 biological replicates .

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-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

E) Boxplot showing the TAD boundary scores in control and 24hr ActD treated cells for boundaries that are bound with differing numbers of CTCF binding sites. The reduction of TAD boundary scores upon ActD treatment is independent of CTCF binding. p-value: Wilcoxon rank-sum test. The horizontal bands in the boxplots represent the median, the error bars represent +/- 1.5 x IQR values, and the outer shapes represent the density of the data points. The figures represent data generated from the pooled Hi-C datasets with 2 biological replicates.


F ) Scaling plot generated by 40kb resolution Hi - C data showing the interaction frequency as a function of genomic distance , with upper distance limit of 3Mb . The 24hr ActD treated samples shows a slower rate of decay at shorter distances , correlating with the reduction of insulation scores .

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F) Scaling plot generated by 40kb resolution Hi-C data showing the interaction frequency as a function of genomic distance, with upper distance limit of 3Mb. The 24hr ActD treated samples shows a slower rate of decay at shorter distances, correlating with the reduction of insulation scores.


A . Exogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 . Data information : Data are representative of three biological replicates with similar results .

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A. Exogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3. Data information: Data are representative of three biological replicates with similar results.


B . Endogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 in mouse peritoneal macrophages . Data information : Data are representative of three biological replicates with similar results .

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B. Endogenous immunoprecipitation assay of the interaction between TRIM50 and NLRP3 in mouse peritoneal macrophages. Data information: Data are representative of three biological replicates with similar results.


C . TRIM50 and NLRP3 proteins were expressed and obtained from the in vitro translation system and their interaction were further analyzed . Data information : Data are representative of three biological replicates with similar results .

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C. TRIM50 and NLRP3 proteins were expressed and obtained from the in vitro translation system and their interaction were further analyzed. Data information: Data are representative of three biological replicates with similar results.


D . Co - IP assay of the interaction between NLRP3 and TRIM50 after stimulation with nigericin for 0 , 15 , 30 and 60 min . Data information : Data are representative of three biological replicates with similar results .

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D. Co-IP assay of the interaction between NLRP3 and TRIM50 after stimulation with nigericin for 0, 15, 30 and 60 min. Data information: Data are representative of three biological replicates with similar results.


E . Co - IP assay of the interaction between NLRP3 and TRIM50 in mouse peritoneal macrophages primed with LPS for 6h and treated with ATP for 30min . Data information : Data are representative of three biological replicates with similar results .

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E. Co-IP assay of the interaction between NLRP3 and TRIM50 in mouse peritoneal macrophages primed with LPS for 6h and treated with ATP for 30min. Data information: Data are representative of three biological replicates with similar results.


F . Confocal microscopy assay of the interaction between TRIM50 and NLRP3 in THP1 cells with stimulation by LPS and ATP . Scale bars : 10μm . Data information : Data are representative of three biological replicates with similar results .

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F. Confocal microscopy assay of the interaction between TRIM50 and NLRP3 in THP1 cells with stimulation by LPS and ATP. Scale bars: 10μm. Data information: Data are representative of three biological replicates with similar results.


G . Schematic diagram of human TRIM50 ( WT ) and its truncation mutants was presented ( top ) , and the interaction of TRIM50 ( TRIM50 truncation mutants ) and NLRP3 was detected by IP assay . Data information : Data are representative of three biological replicates with similar results .

G. Schematic diagram of human TRIM50 (WT) and its truncation mutants was presented(top), and the interaction of TRIM50 (TRIM50 truncation mutants) and NLRP3 was detected by IP assay. Data information: Data are representative of three biological replicates with similar results.


H , I . Schematic diagram of human NLRP3 ( WT ) and its truncation mutants ( top ) , and immunoprecipitation assay of the interaction of NLRP3 ( NLRP3 truncation mutants ) with TRIM50 in HEK293T cells . Data information : Data are representative of three biological replicates with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Schematic diagram of human NLRP3 (WT) and its truncation mutants (top), and immunoprecipitation assay of the interaction of NLRP3 (NLRP3 truncation mutants) with TRIM50 in HEK293T cells. Data information: Data are representative of three biological replicates with similar results.


A . Immunoblot analysis of the NLRP3 protein level in the mouse peritoneal macrophages transfected with Si - Trim50 . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test ( A , Data are representative of three biological replicates with similar results . ( mean ±

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A. Immunoblot analysis of the NLRP3 protein level in the mouse peritoneal macrophages transfected with Si-Trim50. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test (A, Data are representative of three biological replicates with similar results. (mean ± SD


B . qRT - PCR analysis of Nlrp3 and IL - 1β mRNA levels in LPS - primed Si - Trim50 transfected mouse peritoneal macrophages . Data information : P - values are shown , two - way analysis of variance ( ANOVA ) ( B , Data are representative of three biological replicates with similar results . ( mean ±

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B. qRT-PCR analysis of Nlrp3 and IL-1β mRNA levels in LPS-primed Si-Trim50 transfected mouse peritoneal macrophages. Data information: P-values are shown, two-way analysis of variance (ANOVA) (B, Data are representative of three biological replicates with similar results. (mean ± SD


C , D . Immunoblot analysis of Caspase1 - P20 ( C ) and Gasdermin D ( D ) of WT or Trim50 - / - mouse peritoneal macrophages after LPS stimulation for 6h and ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

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C, D. Immunoblot analysis of Caspase1-P20 (C) and Gasdermin D (D) of WT or Trim50-/- mouse peritoneal macrophages after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


E , Immunoblot analysis of Caspase1 - P20 ( E ) of WT or Trim50 - / - BMDM cells after LPS stimulation for 6h and ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

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E, Immunoblot analysis of Caspase1-P20 (E) of WT or Trim50-/- BMDM cells after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


F . Immunoblot analysis of Gasdermin D ( F ) of WT or Trim50 - / - BMDM cells after LPS stimulation for 6h and ATP treatment for 30min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

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F. Immunoblot analysis of Gasdermin D (F) of WT or Trim50-/- BMDM cells after LPS stimulation for 6h and ATP treatment for 30min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


G , ELISA analysis of IL - 1β , TNF - α and IL - 6 in the supernatant of WT or Trim50 - / - peritoneal macrophages with the activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way analysis of variance ( ANOVA ) G , Data are representative of three biological replicates with similar results . ( mean ±

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G, ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatant of WT or Trim50-/- peritoneal macrophages with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) G, Data are representative of three biological replicates with similar results. (mean ± SD


H . ELISA analysis of IL - 1β , TNF - α and IL - 6 in the supernatant of WT or Trim50 - / - BMDM cells with the activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way analysis of variance ( ANOVA ) Data are representative of three biological replicates with similar results . ( mean ±

H. ELISA analysis of IL-1β, TNF-α and IL-6 in the supernatant of WT or Trim50-/- BMDM cells with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) Data are representative of three biological replicates with similar results. (mean ± SD


I . LDH analysis of WT or Trim50 - / - mouse peritoneal macrophages with the activation of NLRP3 inflammasome . n = 3 Data information : P - values are shown , two - way analysis of variance ( ANOVA ) I ) . Data are representative of three biological replicates with similar results . ( mean ±

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I. LDH analysis of WT or Trim50-/- mouse peritoneal macrophages with the activation of NLRP3 inflammasome. n=3 Data information: P-values are shown, two-way analysis of variance (ANOVA) I). Data are representative of three biological replicates with similar results. (mean ± SD


A . Immunoprecipitation analysis of the NLRP3 ubiquitination in HEK293T cells transfected with HA - ubiquitin , His - NLRP3 and Flag - TRIM50 plasmids . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

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A. Immunoprecipitation analysis of the NLRP3 ubiquitination in HEK293T cells transfected with HA-ubiquitin, His-NLRP3 and Flag-TRIM50 plasmids. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


B . Immunoprecipitation analysis of the endogenous NLRP3 ubiquitination in WT or Trim50 - / - BMDM cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

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B. Immunoprecipitation analysis of the endogenous NLRP3 ubiquitination in WT or Trim50-/- BMDM cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


C . Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3 , HA - Ubiquitin , TRIM50 or TRIM50 ( ΔRING ) mutant plasmids . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

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C. Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3, HA-Ubiquitin, TRIM50 or TRIM50 (ΔRING) mutant plasmids. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


D . Immunoblot analysis of the ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and the whole cell lysate ( soluble ) in HEK293T cells with nigericin treatment for 30 min . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

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D. Immunoblot analysis of the ASC oligomerization in cross-linked cytosolic pellets (insoluble) and the whole cell lysate (soluble) in HEK293T cells with nigericin treatment for 30 min. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


E . Immunoblot analysis of NLRP3 protein levels in cycloheximide ( CHX ) treated HEK293T cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

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E. Immunoblot analysis of NLRP3 protein levels in cycloheximide (CHX) treated HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


F . Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 or TRIM50 ( ΔRING ) mutant , followed with the further treatment with CHX for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

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F. Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 or TRIM50 (ΔRING) mutant, followed with the further treatment with CHX for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


G . Immunoblot analysis of NLRP3 in mouse peritoneal macrophages , followed with the further treatment with CHX for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA Data are representative of three biological replicates with similar results . ( mean ±

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G. Immunoblot analysis of NLRP3 in mouse peritoneal macrophages, followed with the further treatment with CHX for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA Data are representative of three biological replicates with similar results. (mean ± SD


A . SDD - AGE analysis of NLRP3 oligomerization in HEK293T cells transfected with NLRP3 and TRIM50 plasmids . Data information : Data are representative of three biological replicates with similar results .

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A. SDD-AGE analysis of NLRP3 oligomerization in HEK293T cells transfected with NLRP3 and TRIM50 plasmids. Data information: Data are representative of three biological replicates with similar results.


B . SDD - AGE analysis of NLRP3 oligomerization in NLRP3 and TRIM50 transfected HEK293T cells followed with nigericin treatment for 30min . Data information : Data are representative of three biological replicates with similar results .

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B. SDD-AGE analysis of NLRP3 oligomerization in NLRP3 and TRIM50 transfected HEK293T cells followed with nigericin treatment for 30min. Data information: Data are representative of three biological replicates with similar results.


C . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in HEK293T cells transfected with NLRP3 , ASC and TRIM50 plasmids followed with nigericin treatment for 30 min . Data information : Data are representative of three biological replicates with similar results .

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C. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in HEK293T cells transfected with NLRP3, ASC and TRIM50 plasmids followed with nigericin treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.


D . SDD - AGE analysis of NLRP3 oligomerization in WT or Trim50 - / - peritoneal macrophages with LPS treatment for 6h and ATP treatment for 30 min . Data information : Data are representative of three biological replicates with similar results .

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D. SDD-AGE analysis of NLRP3 oligomerization in WT or Trim50-/- peritoneal macrophages with LPS treatment for 6h and ATP treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.


E . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in WT or Trim50 - / - peritoneal macrophages by LPS stimulation for 6h and ATP treatment for 30min . Data information : Data are representative of three biological replicates with similar results .

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E. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in WT or Trim50-/- peritoneal macrophages by LPS stimulation for 6h and ATP treatment for 30min. Data information: Data are representative of three biological replicates with similar results.


F . SDD - AGE analysis of NLRP3 oligomerization in WT or Trim50 - / - BMDM cells with LPS treatment for 6h and ATP treatment for 30 min . Data information : Data are representative of three biological replicates with similar results .

F. SDD-AGE analysis of NLRP3 oligomerization in WT or Trim50-/- BMDM cells with LPS treatment for 6h and ATP treatment for 30 min. Data information: Data are representative of three biological replicates with similar results.


G . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in WT or Trim50 - / - BMDM cells by LPS stimulation for 6h and ATP treatment for 30min . Data information : Data are representative of three biological replicates with similar results .

G. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate (soluble) in WT or Trim50-/- BMDM cells by LPS stimulation for 6h and ATP treatment for 30min. Data information: Data are representative of three biological replicates with similar results.


H . SDD - AGE analysis of the oligomerization of NLRP3 in HEK293T cells transfected with His - NLRP3 and TRIM50 or TRIM50 ( ΔRING ) mutant . Data information : Data are representative of three biological replicates with similar results .

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H. SDD-AGE analysis of the oligomerization of NLRP3 in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50(ΔRING) mutant. Data information: Data are representative of three biological replicates with similar results.


I . In vitro NLRP3 oligomerization assay with in - vitro translated TRIM50 and NLRP3 proteins . Data information : Data are representative of three biological replicates with similar results .

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I. In vitro NLRP3 oligomerization assay with in-vitro translated TRIM50 and NLRP3 proteins. Data information: Data are representative of three biological replicates with similar results.


J . Immunoprecipitation analysis of NLRP3 ubiquitination in HA - ubiquitin , His - NLRP3 and Myc - TRIM50 transfected HEK293T cells with further treatment with tranilast or MCC950 for 24h . Data information : Data are representative of three biological replicates with similar results .

O O 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

J. Immunoprecipitation analysis of NLRP3 ubiquitination in HA-ubiquitin, His-NLRP3 and Myc-TRIM50 transfected HEK293T cells with further treatment with tranilast or MCC950 for 24h. Data information: Data are representative of three biological replicates with similar results.


A . Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3 , HA - Ubiquitin , TRIM50 or its truncation mutants . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoprecipitation analysis of NLRP3 ubiquitination in HEK293T cells transfected with NLRP3, HA-Ubiquitin, TRIM50 or its truncation mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


B . SDD - AGE analysis of NLRP3 oligomerization in NLRP3 together with TRIM50 or its coiled - coil domain - deleted mutants ( ΔC - C1 and ΔC - C2 ) transfected HEK293T cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SDD-AGE analysis of NLRP3 oligomerization in NLRP3 together with TRIM50 or its coiled-coil domain-deleted mutants (ΔC-C1 and ΔC-C2) transfected HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


C . SDD - AGE analysis of the oligomerization of TRIM50 in TRIM50 or its coiled - coil domain - deleted mutants ( ΔC - C1 and ΔC - C2 ) transfected HEK293T cells . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

C. SDD-AGE analysis of the oligomerization of TRIM50 in TRIM50 or its coiled-coil domain-deleted mutants (ΔC-C1 and ΔC-C2) transfected HEK293T cells. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


D . SDD - AGE analysis of the oligomerization of NLRP3 and TRIM50 in HEK293T cells overexpressing NLRP3 together with TRIM50 or its coiled - coil domain - deleted mutants . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SDD-AGE analysis of the oligomerization of NLRP3 and TRIM50 in HEK293T cells overexpressing NLRP3 together with TRIM50 or its coiled-coil domain-deleted mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


E . Immunoblot analysis of NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 coiled - coil domain - deleted mutants , followed with the cycloheximide ( CHX ) treatment for 0h , 4h or 8h . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . P - values are shown , two - way ANOVA ( E ) . Data are representative of three biological replicates with similar results . ( mean ± SD in E ) .

O O 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 O O O 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 NLRP3 in HEK293T cells transfected with NLRP3 plasmid and TRIM50 coiled-coil domain-deleted mutants, followed with the cycloheximide (CHX) treatment for 0h, 4h or 8h. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. P-values are shown, two-way ANOVA(E). Data are representative of three biological replicates with similar results. (mean ± SD in E).


F . Immunoblot analysis of ASC oligomerization in cross - linked cytosolic pellets ( insoluble ) and whole cell lysate ( soluble ) in HEK293T cells transfected with His - NLRP3 and TRIM50 or TRIM50 coiled - coil domain - deleted mutants . Data information : Band densities were quantitated by ' Image J ' software and normalized to GAPDH . Data are representative of three biological replicates with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoblot analysis of ASC oligomerization in cross-linked cytosolic pellets (insoluble) and whole cell lysate(soluble) in HEK293T cells transfected with His-NLRP3 and TRIM50 or TRIM50 coiled-coil domain-deleted mutants. Data information: Band densities were quantitated by 'Image J' software and normalized to GAPDH. Data are representative of three biological replicates with similar results.


A . Survival status of WT and Trim50 - / - mice after intraperitoneal injection with LPS ( n = 10 mice / group ) . Data information : P - values are shown , two - way ANOVA ( A ) Data are representative of three biological replicates with similar results . ( mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival status of WT and Trim50-/- mice after intraperitoneal injection with LPS (n=10 mice/group). Data information: P-values are shown, two-way ANOVA (A) Data are representative of three biological replicates with similar results. (mean ± SD


B . ELISA analysis of IL - 1β , TNF - α and IL - 6 in serum of WT or Trim50 - / - mice after i . p . injection with LPS for 6h ( n = 4 - 6 mice / group ) . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ELISA analysis of IL-1β, TNF-α and IL-6 in serum of WT or Trim50-/- mice after i.p. injection with LPS for 6h (n=4-6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


C . Histopathological analysis of the lung tissues by hematoxylin and eosin ( H & E ) staining . The quantified lung injury was depicted by defined clinical parameters in ALI score ( n = 6 mice / group ) . Scale bars : 50μm . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Histopathological analysis of the lung tissues by hematoxylin and eosin (H&E) staining. The quantified lung injury was depicted by defined clinical parameters in ALI score (n=6 mice/group). Scale bars: 50μm. Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


D . Flow cytometry analysis of the neutrophils ( left ) and Ly6C + monocytes ( right ) in peritoneum of WT or Trim50 - / - mice after i . p . injection with Alum . ( n = 6 mice / group ) . Data information : P - values are shown , two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

D. Flow cytometry analysis of the neutrophils (left) and Ly6C+ monocytes (right) in peritoneum of WT or Trim50-/- mice after i.p. injection with Alum. (n=6 mice/group). Data information: P-values are shown, two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


E . WT or Trim50 - / - mice were i . p . injected with FA for 36h , followed with immunoblot analysis of NLRP3 , pro - IL - 1β , Caspase1 p20 , IL - 1β in the kidney tissue samples . Data information : P - values are shown , two - way ANOVA ( A ) or two - tailed Student ' s t - test Data are representative of three biological replicates with similar results . ( mean ±

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

E. WT or Trim50-/- mice were i.p. injected with FA for 36h, followed with immunoblot analysis of NLRP3, pro-IL-1β, Caspase1 p20, IL-1β in the kidney tissue samples. Data information: P-values are shown, two-way ANOVA (A) or two-tailed Student's t-test Data are representative of three biological replicates with similar results. (mean ± SD


F . H & E staining of kidney tissue sections . Scale bars : 20μm .

F. H&E staining of kidney tissue sections. Scale bars: 20μm.


G . Periodic - acid - Schiff ( PAS ) staining of kidney tissue sections . Scale bars : 20μm .

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G. Periodic-acid-Schiff (PAS) staining of kidney tissue sections. Scale bars: 20μm.


Representative time - lapse image series of exponentially growing M . smegmatis P2P1recA - GFPwt ( C ) and P2P1recA - GFPdes ( D ) reporters . Phase - contrast and green fluorescence are merged . Numbers represent hours . Scale bar , 10 µm . Histograms showing the distributions of single - cell fluorescence ( n = 204 ) averaged over the lifetime of the cell ( right panels ) . Black line indicates fitting of the data with a Lognormal function ( C ) or with the sum of three Gaussians ( D ) . Insets : P2P1recA - GFPwt population ' s geometric mean ( Gµ ) calculated by computing the logarithm of the single - cell fluorescence values , the mean of the logarithms and its antilog , P2P1recA - GFPdes subpopulations ' means ( µ ) , and R2 values . The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations , which are masked by using the stable GFPwt variant . E Spearman ' s correlation between single - cell P2P1recA - GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell ( n = 204 ) . The data shown are from 3 independent experiments . Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single - cells . Colors denote cells belonging to underdispersed ( gray ) , dispersed ( dark green ) , and highly dispersed ( light green ) subpopulations . F P2P1recA - GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR ( 40 < n < 112 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis and Dunn ' s multiple comparison test : * * * * p < 0 . 0001 . G Single - cell time traces of P2P1recA - GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR . The average fluorescence ( solid black lines ) and half of the generation time ( dashed lines ) are indicated . Population statistics ( n = 204 ) : 38 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 18 % ( VMR > 10 ) . The data shown are from 3 independent experiments . H Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time . Dark green bars indicate moderately pulsing cells ( n = 165 ) . Light green bars indicate highly pulsing cells ( n = 66 ) . Significance by Mann - Whitney test . The data shown are from 3 independent experiments . Peaks mainly occur between 40 % and 70 % of the generation time .

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

Representative time-lapse image series of exponentially growing M. smegmatis P2P1recA-GFPwt (C) and P2P1recA-GFPdes (D) reporters. Phase-contrast and green fluorescence are merged. Numbers represent hours. Scale bar, 10 µm. Histograms showing the distributions of single-cell fluorescence (n = 204) averaged over the lifetime of the cell (right panels). Black line indicates fitting of the data with a Lognormal function (C) or with the sum of three Gaussians (D). Insets: P2P1recA-GFPwt population's geometric mean (Gµ) calculated by computing the logarithm of the single-cell fluorescence values, the mean of the logarithms and its antilog, P2P1recA-GFPdes subpopulations' means (µ), and R2 values. The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations, which are masked by using the stable GFPwt variant. E Spearman's correlation between single-cell P2P1recA-GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell (n = 204). The data shown are from 3 independent experiments. Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single-cells. Colors denote cells belonging to underdispersed (gray), dispersed (dark green), and highly dispersed (light green) subpopulations. F P2P1recA-GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR (40 < n < 112). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001. G Single-cell time traces of P2P1recA-GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 204): 38% (0<VMR≤1); 44% (1<VMR≤10); 18% (VMR>10). The data shown are from 3 independent experiments. H Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Dark green bars indicate moderately pulsing cells (n = 165). Light green bars indicate highly pulsing cells (n = 66). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 40% and 70% of the generation time.


Color - coded single - cell time traces of the four reporters expressed as percent of the generation time ( B ) . Population coefficients of variation of fluorescence ( % ) are in color - coded boxes . Single - cell VMR of fluorescence ( C ) and fluorescence averaged over the lifetime of the cell ( D ) in exponential phase . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments ( n = 105 cells per strain ) . Single - cell fluorescence decreases but variation does not change in strains devoid of positive regulation . Single - cell fluorescence increases and variation decreases in the strain devoid of negative regulation .

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

Color-coded single-cell time traces of the four reporters expressed as percent of the generation time (B). Population coefficients of variation of fluorescence (%) are in color-coded boxes. Single-cell VMR of fluorescence (C) and fluorescence averaged over the lifetime of the cell (D) in exponential phase. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ***p = 0.0002; ****p < 0.0001. The data shown are from 2 independent experiments (n = 105 cells per strain). Single-cell fluorescence decreases but variation does not change in strains devoid of positive regulation. Single-cell fluorescence increases and variation decreases in the strain devoid of negative regulation.


Color - coded time - traces of fluorescence ( left panel ) averaged for 50 individual cells per strain ( mean ± SD ) treated with MMC ( 0 . 5 μg / ml ) for 2 hours ( gray shading ) . Fluorescence induction during 6 hours from MMC exposure ( right panel ) . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * * * p < 0 . 0001 . The data shown are from 2 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Color-coded time-traces of fluorescence (left panel) averaged for 50 individual cells per strain (mean ± SD) treated with MMC (0.5 μg/ml) for 2 hours (gray shading). Fluorescence induction during 6 hours from MMC exposure (right panel). Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.


Single - cell analysis of independent lineages separated by black vertical lines . Color - coded traces represent single - cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours ( n = 20 ) . White and gray vertical bands represents cell divisions . The data shown are from 2 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

Single-cell analysis of independent lineages separated by black vertical lines. Color-coded traces represent single-cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours (n = 20). White and gray vertical bands represents cell divisions. The data shown are from 2 independent experiments.


Aligned pulse profiles averaged for each reporter strain ( color - coded as in A ) . Error bars are SD ( 4 < n < 37 ) . The data shown are from 2 independent experiments . Pulses are abrogated in the strain devoid of negative regulation .

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

Aligned pulse profiles averaged for each reporter strain (color-coded as in A). Error bars are SD (4 < n < 37). The data shown are from 2 independent experiments. Pulses are abrogated in the strain devoid of negative regulation.


Analysis of pulse characteristics . Black lines indicate means and SD ( 52 < n < 88 ) . Significance by Kruskal - Wallis nd Dunn ' s multiple comparison test : ns , not significant ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments .

Analysis of pulse characteristics. Black lines indicate means and SD (52 < n < 88). Significance by Kruskal-Wallis nd Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.


Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time ( 52 < n < 88 ) . Different strains are color - coded . Statistical analysis by one - way ANOVA and Holm - Sidak ' s multiple comparisons test . Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time .

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

Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time (52 < n < 88). Different strains are color-coded. Statistical analysis by one-way ANOVA and Holm-Sidak's multiple comparisons test. Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time.


Spearman ' s correlation between green ( A - D ) or yellow ( E ) fluorescence range and red fluorescence range over the lifetime of the cell ( 105 < n < 153 ) . The data shown are from 2 independent experiments . On the x - axes P2P1recA - GFPdes ( A - D ) or a C - term translational reporter of SSBa fused to mCitrinewt ( E ) . On the y - axes , a C - term translational reporter of RecA fused to mCherrywt ( A and E ) , transcriptional reporters of ruvC ( B ) and radA ( C ) , and mCherrydes expressed from a constitutive control promoter ( D ) . Colors ( A - D ) indicate subpopulations segregated by VMR of P2P1recA - GFPdes : gray ( 0 < VMR ≤ 1 ) , dark green ( 1 < VMR ≤ 10 ) , and light green ( VMR > 10 ) . Colors ( E ) indicate subpopulations segregated by VMR of RecA - mCherrywt : gray ( 0 < VMR ≤ 1 ) , plum ( 1 < VMR ≤ 10 ) , and magenta ( VMR > 10 ) .

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Spearman's correlation between green (A-D) or yellow (E) fluorescence range and red fluorescence range over the lifetime of the cell (105 < n < 153). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (A-D) or a C-term translational reporter of SSBa fused to mCitrinewt (E). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (A and E), transcriptional reporters of ruvC (B) and radA (C), and mCherrydes expressed from a constitutive control promoter (D). Colors (A-D) indicate subpopulations segregated by VMR of P2P1recA-GFPdes: gray (0<VMR≤1), dark green (1<VMR≤10), and light green (VMR>10). Colors (E) indicate subpopulations segregated by VMR of RecA-mCherrywt: gray (0<VMR≤1), plum (1<VMR≤10), and magenta (VMR>10).


Pearson ' s correlation between green ( F - I ) or yellow ( J ) fluorescence and red fluorescence , measured 3 hours after MMC exposure ( 203 < n < 314 ) . The data shown are from 2 independent experiments . On the x - axes P2P1recA - GFPdes ( F - I ) or a C - term translational reporter of SSBa fused to mCitrinewt ( J ) . On the y - axes , a C - term translational reporter of RecA fused to mCherrywt ( F and J ) , transcriptional reporters of ruvC ( G ) and radA ( H ) , and mCherrydes expressed from a constitutive control promoter ( I ) . Positive correlations between recA and other DNA - damage markers both under normal growth conditions , where spontaneous DNA - damage events occur at the subpopulation level , and in the presence of an alkylating agent that induces double - strand breaks in the entire population , validate recA as a reliable proxy for DNA damage in single 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Pearson's correlation between green (F-I) or yellow (J) fluorescence and red fluorescence, measured 3 hours after MMC exposure (203 < n < 314). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (F-I) or a C-term translational reporter of SSBa fused to mCitrinewt (J). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (F and J), transcriptional reporters of ruvC (G) and radA (H), and mCherrydes expressed from a constitutive control promoter (I). Positive correlations between recA and other DNA-damage markers both under normal growth conditions, where spontaneous DNA-damage events occur at the subpopulation level, and in the presence of an alkylating agent that induces double-strand breaks in the entire population, validate recA as a reliable proxy for DNA damage in single cells.


Representative time - lapse image series of the merodiploid recA reporter strain ( P2P1recA - GFPdes _ RecA - mCherrywt ) cultured in fresh ( A ) or spent ( B ) flow medium ( see Materials and Methods ) . Fresh 7H9 medium is unlabeled ; spent medium ( S ) , ciprofloxacin 1 µg / ml in fresh ( CIP ) or spent ( S - CIP ) medium , and SYTOX 0 . 5 µM ( SX ) are labeled . Phase - contrast ( gray ) , GFPdes ( green ) , and mCherrywt ( magenta ) fluorescence are merged . Fluorescence images of each channel are scaled to the brightest frame . Light - green background in the first 4 frames ( B ) is due to the autofluorescence of spent medium . Cells were imaged at 30 - min intervals and numbers represent hours . Scale bar , 10 µm .

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

Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm.


Representative time traces of individual lineages of cells fed with fresh medium ( C ) or spent medium ( D ) . Different styles are used for lineages with different fates : death and lysis ( L ) ; SYTOX - positive ( SX + ) ; and survival ( S ) . Line breaks represent division events in the same lineage , where a single random daughter cell is tracked for clarity . Vertical bars represent fresh ( white ) or spent ( dark shading ) 7H9 medium , CIP ( yellow ) , and SX ( 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

Representative time traces of individual lineages of cells fed with fresh medium (C) or spent medium (D). Different styles are used for lineages with different fates: death and lysis (L); SYTOX-positive (SX+); and survival (S). Line breaks represent division events in the same lineage, where a single random daughter cell is tracked for clarity. Vertical bars represent fresh (white) or spent (dark shading) 7H9 medium, CIP (yellow), and SX (blue).


Comparison of single - cell growth rates prior to CIP exposure for " D " cells that die and " S " cells that survive and regrow after CIP washout ( E ) , and lag time of regrowth ( F ) . Cells were cultured in fresh ( EXP ) or spent ( STAT ) flow medium . Gray and black lines indicate mean ± SD . Significance by Kruskal - Wallis and Dunn ' s multiple comparison test ( E ) and unpaired Mann - Whitney test ( F ) : ns , not significant ; * * * * p < 0 . 0001 ; 6 < n < 84 . The data shown are from 2 independent experiments . Single - cell fate is independent of the growth rate before exposure to the drug . However , cells cultured in spent medium ( STAT ) grow at significantly slower rates , survive at higher frequencies , and recover faster after CIP washout compared to cells cultured in fresh medium ( EXP ) .

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

Comparison of single-cell growth rates prior to CIP exposure for "D" cells that die and "S" cells that survive and regrow after CIP washout (E), and lag time of regrowth (F). Cells were cultured in fresh (EXP) or spent (STAT) flow medium. Gray and black lines indicate mean ± SD. Significance by Kruskal-Wallis and Dunn's multiple comparison test (E) and unpaired Mann-Whitney test (F): ns, not significant; ****p < 0.0001; 6 < n < 84. The data shown are from 2 independent experiments. Single-cell fate is independent of the growth rate before exposure to the drug. However, cells cultured in spent medium (STAT) grow at significantly slower rates, survive at higher frequencies, and recover faster after CIP washout compared to cells cultured in fresh medium (EXP).


Representative GFPdes ( G ) and mCherrywt ( H ) fluorescence time - traces for single - cell lineages of cells cultured in fresh medium ( EXP ) and cells cultured in spent medium ( STAT ) , and having different fates . Vertical bars are color - coded as in C and D .

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Representative GFPdes (G) and mCherrywt (H) fluorescence time-traces for single-cell lineages of cells cultured in fresh medium (EXP) and cells cultured in spent medium (STAT), and having different fates. Vertical bars are color-coded as in C and D.


Single - cell fate as a function of VMR of P2P1recA - GFPdes ( I ) and RecA - mCherrywt ( J ) fluorescence prior to CIP exposure . Significance by Chi - square test of independence for cells that die ( n = 84 ) and cells that survive ( n = 54 ) . The data shown are from 2 independent experiments . Cells that do not experience DNA damage prior to drug exposure ( RecA - non - pulsing cells ) survive more than cells that do experience DNA damage ( RecA - pulsing cells ) .

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Single-cell fate as a function of VMR of P2P1recA-GFPdes (I) and RecA-mCherrywt (J) fluorescence prior to CIP exposure. Significance by Chi-square test of independence for cells that die (n = 84) and cells that survive (n = 54). The data shown are from 2 independent experiments. Cells that do not experience DNA damage prior to drug exposure (RecA-non-pulsing cells) survive more than cells that do experience DNA damage (RecA-pulsing cells).


Spearman ' s correlation between single - cell RecA - mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell ( n = 360 ) . The data shown are from 3 independent experiments . Dashed lines separate different levels of dispersion , and colors denote subpopulations that are underdispersed ( gray ) , dispersed ( plum ) , or highly dispersed ( magenta ) . RecA - mCherrywt fluorescence of subpopulations segregated by fluorescence VMR ( 58 < n < 159 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis and Dunn ' s multiple comparison test : * * * * p < 0 . 0001 .

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Spearman's correlation between single-cell RecA-mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell (n = 360). The data shown are from 3 independent experiments. Dashed lines separate different levels of dispersion, and colors denote subpopulations that are underdispersed (gray), dispersed (plum), or highly dispersed (magenta). RecA-mCherrywt fluorescence of subpopulations segregated by fluorescence VMR (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001.


Single - cell time traces of RecA - mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR . The average fluorescence ( solid black lines ) and half of the generation time ( dashed lines ) are indicated . Population statistics ( n = 360 ) : 40 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 16 % ( VMR > 10 ) . The data shown are from 3 independent experiments .

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Single-cell time traces of RecA-mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 360): 40% (0<VMR≤1); 44% (1<VMR≤10); 16% (VMR>10). The data shown are from 3 independent experiments.


Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time . Bars shaded in plum indicate moderately pulsing cells ( n = 185 ) and magenta bars indicate highly pulsing cells ( n = 57 ) . Significance by Mann - Whitney test . The data shown are from 3 independent experiments . Peaks mainly occur between 80 % and 100 % of the generation time .

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Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Bars shaded in plum indicate moderately pulsing cells (n = 185) and magenta bars indicate highly pulsing cells (n = 57). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 80% and 100% of the generation time.


Single - cell size ( E ) and growth rate ( F ) averaged over the lifetime of the cell ( 58 < n < 159 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * p = 0 . 002 ; * * * * p < 0 . 0001 . Cells experiencing more DNA damage ( VMR > 10 ) are slightly larger and grow at slower rates .

Single-cell size (E) and growth rate (F) averaged over the lifetime of the cell (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; **p = 0.002; ****p < 0.0001. Cells experiencing more DNA damage (VMR>10) are slightly larger and grow at slower rates.


Representative time - lapse image series of the dual reporter of RecA and rRNA expression in exponential phase . Pre - CIP growing ( PG - S ) and arrested ( PA - S ) survivors and resistant ( R ) cells are indicated by arrows . CIP exposure ( 1 µg / ml , 4X - MIC ) and SX ( 0 . 5 µM ) death assay ( blue line ) are indicated . Phase - contrast ( gray ) , Prrn - GFPdes ( green ) , and RecA - mCherrywt ( magenta ) fluorescence are merged . Fluorescence images of each channel are scaled to the brightest frame . Cells were imaged at 3 - hour intervals and numbers indicate days . Scale bar , 5 µm .

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Representative time-lapse image series of the dual reporter of RecA and rRNA expression in exponential phase. Pre-CIP growing (PG-S) and arrested (PA-S) survivors and resistant (R) cells are indicated by arrows. CIP exposure (1 µg/ml, 4X-MIC) and SX (0.5 µM) death assay (blue line) are indicated. Phase-contrast (gray), Prrn-GFPdes (green), and RecA-mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Cells were imaged at 3-hour intervals and numbers indicate days. Scale bar, 5 µm.


Representative time traces of cell lineages with different fates . Vertical bars represent fresh 7H9 medium ( white ) , CIP ( yellow ) , and SX ( blue ) .

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Representative time traces of cell lineages with different fates. Vertical bars represent fresh 7H9 medium (white), CIP (yellow), and SX (blue).


Comparison of growth rate ( C ) , cell size averaged over the lifetime of the cell ( D ) , microcolony size ( E ) before CIP exposure Gray and black lines indicate mean ± SD . The data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney test : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA - mCherrywt both before and after drug exposure .

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

Comparison of growth rate (C), cell size averaged over the lifetime of the cell (D), microcolony size (E) before CIP exposure Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.


fluorescence range over the lifetime of the cell before CIP exposure ( F ) , and mean fluorescence during CIP exposure ( G ) of cells that died ( n = 174 ) and cells that survived ( n = 164 ) . Gray and black lines indicate mean ± SD . The data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney test : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA - mCherrywt both before and after drug exposure .

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fluorescence range over the lifetime of the cell before CIP exposure (F), and mean fluorescence during CIP exposure (G) of cells that died (n = 174) and cells that survived (n = 164). Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.


Single - cell fate as a function of VMR of RecA - mCherrywt fluorescence prior to CIP exposure . Significance by Chi - square test of independence : cells that died ( n = 174 ) , cells that survived ( n = 155 ) , and resistant cells ( n = 9 ) . The data shown are from 2 independent experiments . More survivors arise from the subpopulation that does not experience DNA damage before drug exposure .

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Single-cell fate as a function of VMR of RecA-mCherrywt fluorescence prior to CIP exposure. Significance by Chi-square test of independence: cells that died (n = 174), cells that survived (n = 155), and resistant cells (n = 9). The data shown are from 2 independent experiments. More survivors arise from the subpopulation that does not experience DNA damage before drug exposure.


B Measured free PF - 670 exposure in the brain tissue of mice and NHPs . Drug exposure in NHPs after administration of 10 mpk PF - 670 ( AUC = 3 . 6μMh ) is ~ 7 - fold higher than that in mice given 32 mpk PF - 670 ( AUC = 0 . 5 μMh ) ( mean ± SEM ) .

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B Measured free PF-670 exposure in the brain tissue of mice and NHPs. Drug exposure in NHPs after administration of 10 mpk PF-670 (AUC=3.6μMh) is ~7-fold higher than that in mice given 32 mpk PF-670 (AUC=0.5 μMh) (mean±SEM).


C , D Double - plotted actograms of NHPs ' activity for 16 days . NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at the same solar time of day : 4 pm for the DD experiment ( C ) and 4 : 30 pm ( ZT11 ) for the LD experiment ( D ) , respectively , which are highlighted as red lines . White and black rectangles indicate the times of light going on and off ( LD 12 : 12 ) .

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C, D Double-plotted actograms of NHPs' activity for 16 days. NHPs were treated with 10 mpk PF-670 for 3 consecutive days at the same solar time of day: 4 pm for the DD experiment (C) and 4:30 pm (ZT11) for the LD experiment (D), respectively, which are highlighted as red lines. White and black rectangles indicate the times of light going on and off (LD 12:12).


E , F Phase delay induced by the 3 - day dosing under DD ( E ) or LD ( F ) . The phase of activity onset in NHPs ( 10 mpk ) is more delayed than that in mice ( 32 mpk ) under DD ( E ) , but not under LD ( F ) ( P = 0 . 03 , one - way analysis of variance ( ANOVA ) ; n . s . , no significant difference ) . Veh denotes vehicle . The error bars represent mean ± SEM . The mouse data are adopted from ( Kim et al , 2013 ) . G The quantification of ( E ) and ( F ) indicates that light has a stronger attenuating effect on the PF - 670 - induced phase delay in NHPs than in mice ( P = 0 . 036 , two - way ANOVA ) .

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E, F Phase delay induced by the 3-day dosing under DD (E) or LD (F). The phase of activity onset in NHPs (10 mpk) is more delayed than that in mice (32 mpk) under DD (E), but not under LD (F) (P=0.03, one-way analysis of variance (ANOVA); n.s., no significant difference). Veh denotes vehicle. The error bars represent mean±SEM. The mouse data are adopted from (Kim et al, 2013). G The quantification of (E) and (F) indicates that light has a stronger attenuating effect on the PF-670-induced phase delay in NHPs than in mice (P=0.036, two-way ANOVA).


C Double - plotted actogram of NHPs ' activity for 16 days . The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm ( i . e . LD is delayed by 2 h ) .

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C Double-plotted actogram of NHPs' activity for 16 days. The light is on at 6 am and off at 6 pm except for Days 8 to 10 when the light is on at 8 am and off at 8 pm (i.e. LD is delayed by 2 h).


D The model successfully predicts the phase shift induced by 2 h jet lag ( Days 8 - 10 ) and its recovery after jet lag ( Days 11 - 13 ) ( n = 7 ; mean ±

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D The model successfully predicts the phase shift induced by 2 h jet lag (Days 8-10) and its recovery after jet lag (Days 11-13) (n=7; mean±SEM


E NHPs were treated with 10 mpk PF - 670 for 3 consecutive days at ZT4 in LD 12 : 12 , which are highlighted as the red line .

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E NHPs were treated with 10 mpk PF-670 for 3 consecutive days at ZT4 in LD 12:12, which are highlighted as the red line.


B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones ( A / D = 1 . 25 ) than that of the experimentally measured nocturnal mouse PRC ( A / D = 0 . 41 Here , the red line and red range represent the mean ± SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly , the light PRC of diurnal humans has a higher A / D ratio than that of nocturnal

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B The simulated PRCs of diurnal NHPs have a higher ratio of maximal magnitudes between advance and delay zones (A/D=1.25) than that of the experimentally measured nocturnal mouse PRC (A/D=0.41 Here, the red line and red range represent the mean±SD of the simulated 12 h light PRCs of the NHP model with different gating and adaptation Similarly, the light PRC of diurnal humans has a higher A/D ratio than that of nocturnal mice


E Consistently , higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF - 670 - induced phase delay which was also captured by the NHP model when 20 mpk dosing was used .

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E Consistently, higher experimentally measured PER2 levels in the SCN at the dosing time leads to a larger PF-670-induced phase delay which was also captured by the NHP model when 20 mpk dosing was used.


( b ) MAPPIT screen identified RAL - interacting proteins . A MAPPIT array covering close to 8 , 500 preys was screened with RALA - G23V as bait . Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1 .

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(b) MAPPIT screen identified RAL-interacting proteins. A MAPPIT array covering close to 8,500 preys was screened with RALA-G23V as bait. Data are shown in a volcano plot of the log ratio of normalized MAPPIT luciferase activity versus P value Source data can be found in Supplementary Table S1.


( c ) MAPPIT assay confirmed interaction between the RAL proteins and USP33 . RALA - G23V or RALB - G23V baits were introduced into HEK293T cells together with the indicated preys . The results are expressed as a mean of normalized luciferase activity ± s . d ( leptin - treated cells versus leptin - untreated cells ) for three independent experiments . Statistics source data can be found in Supplementary Table S1 .

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(c) MAPPIT assay confirmed interaction between the RAL proteins and USP33. RALA-G23V or RALB-G23V baits were introduced into HEK293T cells together with the indicated preys. The results are expressed as a mean of normalized luciferase activity ± s.d (leptin-treated cells versus leptin-untreated cells) for three independent experiments. Statistics source data can be found in Supplementary Table S1.


( d , e ) USP33 interacts with both RALA and RALB . At 48 h post - transfection with tagged USP33 and RAL expression constructs , the indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) or anti - HA - agarose , followed by immunoblotting using anti - Flag or anti - HA antibodies . WCL , whole - cell lysate ; IP , immunoprecipitates .

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(d,e) USP33 interacts with both RALA and RALB. At 48 h post-transfection with tagged USP33 and RAL expression constructs, the indicated proteins were then immunoprecipitated with anti-Flag (M2) or anti-HA-agarose, followed by immunoblotting using anti-Flag or anti-HA antibodies. WCL, whole-cell lysate; IP, immunoprecipitates.


( f ) Endogenous USP33 interacts with RALB . Flag - RALB was immunoprecipitated using anti - Flag ( M2 ) agarose and analysed for co - precipitation with endogenous USP33 using anti - USP33 antibody . Cells expressing USP33 shRNA were used as a negative control .

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(f) Endogenous USP33 interacts with RALB. Flag-RALB was immunoprecipitated using anti-Flag (M2) agarose and analysed for co-precipitation with endogenous USP33 using anti-USP33 antibody. Cells expressing USP33 shRNA were used as a negative control.


( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The GST - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The GST-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.


( h ) USP33 affects ubiquitylation of RALB , but not RALA . 6xHis - tagged ubiquitin and Flag - RAL - G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA . Ubiquitylated RAL proteins were purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALA or RALB . Uncropped images of blots are shown in Supplementary Fig . S7 .

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(h) USP33 affects ubiquitylation of RALB, but not RALA. 6xHis-tagged ubiquitin and Flag-RAL-G23V mutants were introduced into HEK293T cells stably expressing GFP shRNA or USP33 shRNA. Ubiquitylated RAL proteins were purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALA or RALB. Uncropped images of blots are shown in Supplementary Fig. S7.


( g ) GTP binding is not required for interaction between USP33 and the RAL proteins . The USP33 - tagged RALA and RALB were isolated with glutathione - agarose and mixed with lysates derived from HEK293T cells expressing Flag - USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag - specific antibody .

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(g) GTP binding is not required for interaction between USP33 and the RAL proteins. The USP33-tagged RALA and RALB were isolated with glutathione-agarose and mixed with lysates derived from HEK293T cells expressing Flag-USP33 in the presence of GTPγS or excess of GDP followed by immunoblotting with Flag-specific antibody.


( a ) Tandem affinity purification of ubiquitylated RALB . 6xHis - ubiquitin and Flag - RALB - G23V were co - transfected into HEK293T cells , and ubiquitylated RALB was purified using anti - Flag resin followed by Co2 + metal affinity chromatography . Isolated RALB was visualized by immunoblotting using anti - RALB antibody .

(a) Tandem affinity purification of ubiquitylated RALB. 6xHis-ubiquitin and Flag-RALB-G23V were co-transfected into HEK293T cells, and ubiquitylated RALB was purified using anti-Flag resin followed by Co2+ metal affinity chromatography. Isolated RALB was visualized by immunoblotting using anti-RALB antibody.


( b ) RALB undergoes ubiquitylation on multiple sites . Schematic diagram of the RALB domains ( upper panel ) . RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63 % of the RALB amino acid sequence ( Supplementary Fig . S4a ) . The major identified ubiquitylation sites , Lys 47 and Lys 160 , are located in the effector - interacting domain ( green ) and the G5 box ( red ) , respectively .

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(b) RALB undergoes ubiquitylation on multiple sites. Schematic diagram of the RALB domains (upper panel). RALB ubiquitylation sites were identified in two independent tandem mass spectrometry analyses with a total coverage of 63% of the RALB amino acid sequence (Supplementary Fig. S4a). The major identified ubiquitylation sites, Lys 47 and Lys 160, are located in the effector-interacting domain (green) and the G5 box (red), respectively.


( c ) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB . (

(c) Substitution of arginine for lysine at position 47 significantly inhibits ubiquitylation of RALB. (


( d ) USP33 regulates RALB ubiquitylation at Lys 47 . ( e ) The GTP - GDP status of RALB affects RALB ubiquitylation at Lys 47 . For c - e , 6xHis - tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA . Ubiquitylated RALB was purified by Co2 + metal affinity chromatography and detected by antibodies specific to RALB . WT , wild type .

(d) USP33 regulates RALB ubiquitylation at Lys 47. (e) The GTP-GDP status of RALB affects RALB ubiquitylation at Lys 47. For c-e, 6xHis-tagged ubiquitin and the indicated RALB mutants were introduced into HEK293T cells expressing GFP shRNA or USP33 shRNA. Ubiquitylated RALB was purified by Co2+ metal affinity chromatography and detected by antibodies specific to RALB. WT, wild type.


( f ) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5 . The indicated Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - SEC5 antibody .

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(f) Lack of ubiquitylation at Lys 47 inhibits RALB binding to SEC5. The indicated Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-SEC5 antibody.


g ) Ubiquitylation at Lys 47 impairs RALB binding to EXO84 . Flag - tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - EXO84 antibody .

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g) Ubiquitylation at Lys 47 impairs RALB binding to EXO84. Flag-tagged RALB mutants were overexpressed in HEK293T cells and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-EXO84 antibody.


( h ) Ubiquitylation at Lys 47 does not affect RALB activity . The activity of RALB mutants was determined by a RALBP1 - RBD binding assay .

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(h) Ubiquitylation at Lys 47 does not affect RALB activity. The activity of RALB mutants was determined by a RALBP1-RBD binding assay.


( i ) The Rosetta v3 protocol described in ref . was used for ubiquitin - docking simulations . The plot represents the energies of the produced decoys distributed by the root - mean - square deviation ( RMSD ) of ubiquitin position relative to RALA from an arbitrarily chosen decoy .

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(i) The Rosetta v3 protocol described in ref. was used for ubiquitin-docking simulations. The plot represents the energies of the produced decoys distributed by the root-mean-square deviation (RMSD) of ubiquitin position relative to RALA from an arbitrarily chosen decoy.


( j ) Rosetta modelling of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA - EXO84 - RBD complexes . Superposition of the top ten refined solutions of ubiquitylated - RALA - SEC5 - RBD complex , selected by combined scores of two independent docking simulations of ubiquitylated - RALA - SEC5 - RBD and ubiquitylated - RALA ( left ) . Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA - EXO84 complex ( right ) . Uncropped images of blots are shown in Supplementary Fig . S7 .

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(j) Rosetta modelling of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA-EXO84-RBD complexes. Superposition of the top ten refined solutions of ubiquitylated-RALA-SEC5-RBD complex, selected by combined scores of two independent docking simulations of ubiquitylated-RALA-SEC5-RBD and ubiquitylated-RALA (left). Simulated ubiquitin positions linked to Lys 47 of RALA were combined with the RALA-EXO84 complex (right). Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) USP33 does not affect RALB activity . The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector ( V ) or HA - tagged USP33 by RALBP1 - RBD binding assay .

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(a) USP33 does not affect RALB activity. The activity of RALB was determined in HEK293T cells expressing GFP shRNA or USP33 shRNA and empty vector (V) or HA-tagged USP33 by RALBP1-RBD binding assay.


( b ) USP33 modulates interaction between RALB and SEC5 . Flag - tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA , USP33 shRNA , empty vector ( V ) or HA - tagged USP33 . The indicated proteins were then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using anti - Flag or anti - SEC5 antibodies .

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(b) USP33 modulates interaction between RALB and SEC5. Flag-tagged RALB was overexpressed in HEK293T cells expressing GFP shRNA, USP33 shRNA, empty vector (V) or HA-tagged USP33. The indicated proteins were then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using anti-Flag or anti-SEC5 antibodies.


( c ) USP33 regulates RALB binding to EXO84 . Flag - tagged RALB was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - HA or anti - EXO84 antibody .

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(c) USP33 regulates RALB binding to EXO84. Flag-tagged RALB was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-HA or anti-EXO84 antibody.


( d ) Activated RALB co - localizes with both EXO84 and SEC5 . At 24 h after co - transfection with RALB - G23V - EGFP and HA - EXO84 , HeLa cells were immunostained with anti - HA and anti - SEC5 antibodies . Scale bars , 10 μm . The outlined areas are shown at higher magnification in the bottom panels .

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(d) Activated RALB co-localizes with both EXO84 and SEC5. At 24 h after co-transfection with RALB-G23V-EGFP and HA-EXO84, HeLa cells were immunostained with anti-HA and anti-SEC5 antibodies. Scale bars, 10 μm. The outlined areas are shown at higher magnification in the bottom panels.


( e ) RALB ubiquitylation controls interaction between EXO84 and SEC5 . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells overexpressing RALB - G23V or RALB - G23V - K47R . The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti - EXO84 antibody .

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(e) RALB ubiquitylation controls interaction between EXO84 and SEC5. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells overexpressing RALB-G23V or RALB-G23V-K47R. The presence of EXO84 in SEC5 complexes was analysed by immunoblotting using anti-EXO84 antibody.


( f ) USP33 regulates EXO84 - SEC5 complex formation . Flag - tagged SEC5 was immunoprecipitated using anti - Flag ( M2 ) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti - EXO84 antibody .

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(f) USP33 regulates EXO84-SEC5 complex formation. Flag-tagged SEC5 was immunoprecipitated using anti-Flag (M2) agarose from HEK293T cells expressing the indicated constructs followed by immunoblotting using anti-EXO84 antibody.


( g ) Suppression of SEC5 affects RALB binding to EXO84 . HA - tagged RALB - G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs . The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti - EXO84 antibody . For a - c , f , g immunoblotting using antibodies specific for USP33 , anti - Flag ( M2 ) or anti - HA determined the levels of USP33 suppression or overexpression . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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(g) Suppression of SEC5 affects RALB binding to EXO84. HA-tagged RALB-G23V was immunoprecipitated from HEK293T cells expressing the indicated constructs. The presence of EXO84 in RALB complexes was analysed by immunoblotting using anti-EXO84 antibody. For a-c,f,g immunoblotting using antibodies specific for USP33, anti-Flag (M2) or anti-HA determined the levels of USP33 suppression or overexpression. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


( a ) Suppression of USP33 facilitates TBK1 - SEC5 complex formation . Flag - tagged SEC5 was introduced into HEK293T cells expressing empty vector ( V ) or HA - tagged RALB - G23V together with GFP shRNA or USP33 shRNA as indicated , and then immunoprecipitated with anti - Flag ( M2 ) agarose followed by immunoblotting using antibody specific for TBK1 .

(a) Suppression of USP33 facilitates TBK1-SEC5 complex formation. Flag-tagged SEC5 was introduced into HEK293T cells expressing empty vector (V) or HA-tagged RALB-G23V together with GFP shRNA or USP33 shRNA as indicated, and then immunoprecipitated with anti-Flag (M2) agarose followed by immunoblotting using antibody specific for TBK1.


( c ) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB - SEC5 complex in response to poly ( I : C ) treatment . Flag - tagged SEC5 and the indicated HA - tagged RALB mutants were introduced into 293 - hTLR3 cells . At 48 h after transfection cells were treated 100 μg ml − 1 of poly ( I : C ) for 6 h . Flag - tagged SEC5 was immunoprecipitated with anti - Flag ( M2 ) , and analysed for co - precipitation by immunoblotting using anti - HA antibody .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RALB ubiquitylation at Lys 47 is crucial for formation of the RALB-SEC5 complex in response to poly(I:C) treatment. Flag-tagged SEC5 and the indicated HA-tagged RALB mutants were introduced into 293-hTLR3 cells. At 48 h after transfection cells were treated 100 μg ml−1 of poly(I:C) for 6 h. Flag-tagged SEC5 was immunoprecipitated with anti-Flag (M2), and analysed for co-precipitation by immunoblotting using anti-HA antibody.


( d ) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3 . At 48 h after transfection with empty vector ( V ) , Flag - RALB - G23V and Flag - RALB - G23V - K47R , 293 - hTLR3 cells were treated with 10 μg ml − 1 of poly ( I : C ) for 6 h . Immunoblot analysis was performed using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S7 .

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(d) RALB ubiquitylation at Lys 47 affects activation of TBK1 and IRF3. At 48 h after transfection with empty vector (V), Flag-RALB-G23V and Flag-RALB-G23V-K47R, 293-hTLR3 cells were treated with 10 μg ml−1 of poly(I:C) for 6 h. Immunoblot analysis was performed using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S7.


( a - d ) USP33 inhibits assembly of SEC5 - beclin - 1 complexes . The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti - Flag ( M2 ) , anti - HA or anti - SEC5 antibody and analysed for co - precipitation by immunoblotting using specific antibodies as indicated .

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(a-d) USP33 inhibits assembly of SEC5-beclin-1 complexes. The indicated proteins were immunoprecipitated from cells expressing GFP shRNA or USP33 shRNA with anti-Flag (M2), anti-HA or anti-SEC5 antibody and analysed for co-precipitation by immunoblotting using specific antibodies as indicated.