( A ) Estimation of BID protein levels in TKO MEFs . DKO MEFs re - expressing human BAK , TKO MEFs , and two human leukaemia cell lines RS4 ; 11 and MV4 ; 11 , were lysed and analysed by Western blot for BID levels , using a rat monoclonal antibody raised against human BID ( anti - hBID clone 2D1 ) that also recognises mouse BID . Also present are samples of the human FL - BID protein added at the equivalent concentrations used in ( B ) and ( C ) . On the right gel , duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells ( from B and C ) showing that endogenous mouse BID is cytosolic in these cells . Data shown are representative of 2 independent experiments .

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(A) Estimation of BID protein levels in TKO MEFs. DKO MEFs re-expressing human BAK, TKO MEFs, and two human leukaemia cell lines RS4;11 and MV4;11, were lysed and analysed by Western blot for BID levels, using a rat monoclonal antibody raised against human BID (anti-hBID clone 2D1) that also recognises mouse BID. Also present are samples of the human FL-BID protein added at the equivalent concentrations used in (B) and (C). On the right gel, duplicate samples of DKO and TKO cells and permeabilised DKO and TKO cells (from B and C) showing that endogenous mouse BID is cytosolic in these cells. Data shown are representative of 2 independent experiments.


( B , C ) Partial cytochrome c release by FL - BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID . DKO MEFs re - expressing human BAK or TKO MEFs were permeabilised , and then the cells incubated with the indicated concentrations of Bim BH3 peptide , cBID or FL - BID , alone or in the indicated combinations , at 30oC for 30 min . Cells were then co - stained for cytochrome c and TOM20 and analysed by flow cytometry . Left hand panels show median fluorescence data from replicate experiments ( squares denote experiment 1 , circles denote experiment 2 ) . Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 ( treatments are as listed in the left - hand panels ) .

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(B, C) Partial cytochrome c release by FL-BID from mitochondria of TKO MEFs when combined with BIM BH3 peptide or cBID. DKO MEFs re-expressing human BAK or TKO MEFs were permeabilised, and then the cells incubated with the indicated concentrations of Bim BH3 peptide, cBID or FL-BID, alone or in the indicated combinations, at 30oC for 30 min. Cells were then co-stained for cytochrome c and TOM20 and analysed by flow cytometry. Left hand panels show median fluorescence data from replicate experiments (squares denote experiment 1, circles denote experiment 2). Right hand panels show histograms of cytochrome c staining and TOM20 staining from experiment 2 (treatments are as listed in the left-hand panels).


B RT - PCR analysis of RNA from mid - temporal autopsy brain sections obtained from NCI , MCI or AD subjects . Representative gel of results is shown . C Quantitation of RT - PCR products of all samples analyzed from post - mortem brain tissues ( mean s . e . m . ) . An omnibus significant effect was obtained using the one - way analysis of variance ( one - way ANOVA ) with Tukey post - test to determine the p - value . Error bars are s . e . m .

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B RT-PCR analysis of RNA from mid-temporal autopsy brain sections obtained from NCI, MCI or AD subjects. Representative gel of results is shown.C Quantitation of RT-PCR products of all samples analyzed from post-mortem brain tissues (mean s.e.m.). An omnibus significant effect was obtained using the one-way analysis of variance (one-way ANOVA) with Tukey post-test to determine the p-value. Error bars are s.e.m.


D RT - PCR analysis of RNA from the hippocampus of 1 - 2 and 3 - 7 month old non - transgenic ( WT ) or TgCRND8 ( AD ) mice . E Quantification of the relative ratio of exon 19 inclusion for TgCRND8 ( AD ) and WT mice ( mean s . e . m . ; one - way ANOVA with Tukey post - test ) . The number of samples analyzed ( n ) is indicated .

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D RT-PCR analysis of RNA from the hippocampus of 1-2 and 3-7 month old non-transgenic (WT) or TgCRND8 (AD) mice.E Quantification of the relative ratio of exon 19 inclusion for TgCRND8 (AD) and WT mice (mean s.e.m.; one-way ANOVA with Tukey post-test). The number of samples analyzed (n) is indicated.


B Top , diagram of ASOs tested below , mapped to their position of complementarity on ApoER2 . Bottom , RT - PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM . RNA spliced forms are labeled . Quantification of the percentage inclusion is shown below the image and calculated as : Inclusion ( % ) = [ exon 19 included / ( exon 19 included + exon 19 skipped ) ] x 100 .

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B Top, diagram of ASOs tested below, mapped to their position of complementarity on ApoER2. Bottom, RT-PCR of RNA isolated from HeLa cells transfected with the indicated ASO at a final concentration of 80 nM. RNA spliced forms are labeled. Quantification of the percentage inclusion is shown below the image and calculated as: Inclusion (%)=[exon 19 included/(exon 19 included+exon 19 skipped)] x 100.


D Top , RT - PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1 - specific siRNA . Control is a non - specific siRNA . Bottom , Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . E Quantification of exon 19 splicing in ( D ) . Error bars show s . e . m . P - value was determined using Student ' s two - tailed t - test .

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D Top, RT-PCR analysis of ApoER2 exon 19 splicing in HeLa cells depleted of SRSF1 using an SRSF1-specific siRNA. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.E Quantification of exon 19 splicing in (D). Error bars show s.e.m. P-value was determined using Student's two-tailed t-test.


F Top , RT - PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi . Control is a non - specific siRNA . Bottom , Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1 . β - actin is included as a control . G Quantification of exon 19 splicing in E . Error bars show s . e . m . P - value was calculated using Student ' s two - tailed t - test .

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F Top, RT-PCR analysis of exon 19 splicing in a primary mouse kidney cell line depleted of SRSF1 by RNAi. Control is a non-specific siRNA. Bottom, Immunoblot analysis of SRSF1 from HeLa lysates depleted of SRSF1. β-actin is included as a control.G Quantification of exon 19 splicing in E. Error bars show s.e.m. P-value was calculated using Student's two-tailed t-test.


A Top , ASOs mapped onto the mouse ApoER2 exon 19 gene region . Bottom , RT - PCR of RNA isolated from mouse cells transfected with different ASOs . Quantification of percent exon 19 inclusion is shown below the gel image .

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A Top, ASOs mapped onto the mouse ApoER2 exon 19 gene region. Bottom, RT-PCR of RNA isolated from mouse cells transfected with different ASOs. Quantification of percent exon 19 inclusion is shown below the gel image.


B ASO improves exon 19 inclusion in a dose - responsive manner . RT - PCR analysis of RNA from mouse cells transfected with increasing doses of ASO - 15 . The percent inclusion of exon 19 is shown below the gel image . The half - maximal effective concentration ( EC50 ) was calculated using GraphPad Prism version 6 . 0 ( GraphPad Software , San Diego , CA ) after fitting the data using nonlinear regression with normalized response and variable slope .

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B ASO improves exon 19 inclusion in a dose-responsive manner. RT-PCR analysis of RNA from mouse cells transfected with increasing doses of ASO-15. The percent inclusion of exon 19 is shown below the gel image. The half-maximal effective concentration (EC50) was calculated using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA) after fitting the data using nonlinear regression with normalized response and variable slope.


D RT - PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs . C refers to a non - specific control ASO . E Quantitation of ApoER2 exon 19 inclusion from D ( mean s . e . m . , one - way ANOVA with Bonferroni multiple comparison test .

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D RT-PCR analysis of RNA isolated from the hippocampus of mice treated as adults by ICV injection of indicated ASOs. C refers to a non-specific control ASO.E Quantitation of ApoER2 exon 19 inclusion from D (mean s.e.m., one-way ANOVA with Bonferroni multiple comparison test.


A RT - PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO - 21 or ASO - C at P1 - 2 . B Quantitation of exon 19 splicing . ( mean s . e . m . ; Kruskal - Wallis test with Dunn ' s multiple comparison test .

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A RT-PCR analysis of ApoER2 exon 19 splicing in RNA isolated from hippocampus of 4 month old AD and WT mice treated with ASO-21 or ASO-C at P1-2.B Quantitation of exon 19 splicing. (mean s.e.m.; Kruskal-Wallis test with Dunn's multiple comparison test.


C Immunoblot for exon 19 containing ApoER2 ( top ) and total ApoER2 ( bottom ) . β - actin was used as loading control . D Quantification of ApoER2 protein isoforms shown in C ( mean s . e . m . , student ' s t - test ) .

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C Immunoblot for exon 19 containing ApoER2 (top) and total ApoER2 (bottom). β-actin was used as loading control.D Quantification of ApoER2 protein isoforms shown in C (mean s.e.m., student's t-test).


A Input - output curves show hippocampal CA1 field excitatory postsynaptic potential ( fEPSP ) response amplitudes as a function of stimulus intensity delivered to Schaffer collateral afferent fibers . Two - way repeated - measures ANOVA revealed a significant effect of treatment ( F ( 2 , 119 ) = 31 . 62 , p < 0 . 0001 ) with the Bonferroni multiple comparisons test showing significant differences between ASO - C treated AD and age - matched ASO - C treated WT ( * p < 0 . 05 ) . Responses from ASO - 21 treated AD mice were not significantly different from ASO - C treated WT or AD mice .

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A Input-output curves show hippocampal CA1 field excitatory postsynaptic potential (fEPSP) response amplitudes as a function of stimulus intensity delivered to Schaffer collateral afferent fibers. Two-way repeated-measures ANOVA revealed a significant effect of treatment (F(2,119) = 31.62, p < 0.0001) with the Bonferroni multiple comparisons test showing significant differences between ASO-C treated AD and age-matched ASO-C treated WT (* p < 0.05). Responses from ASO-21 treated AD mice were not significantly different from ASO-C treated WT or AD mice.


B Representative paired - pulse facilitation ( PPF ) responses from ASO - C treated WT ( top ) , ASO - C treated AD ( middle ) and ASO - 21 treated AD ( bottom ) mice are shown to the left . Scale bars = 0 . 2 mV by 10 msec apply to all traces . Bar graph ( right ) summarizes the paired - pulse ratios . PPF was not significantly different in AD compared to WT samples , nor was it affected by ASO - 21 treatment .

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B Representative paired-pulse facilitation (PPF) responses from ASO-C treated WT (top), ASO-C treated AD (middle) and ASO-21 treated AD (bottom) mice are shown to the left. Scale bars = 0.2 mV by 10 msec apply to all traces. Bar graph (right) summarizes the paired-pulse ratios. PPF was not significantly different in AD compared to WT samples, nor was it affected by ASO-21 treatment.


C LTP was unaffected after ASO - 21 treatment and similar in AD compared to WT mice . To combine data from multiple slices , individually measured fEPSP rise slopes were normalized to the average baseline value taken 0 - 10 minutes before tetanic stimulation ( arrows ) . The normalized values are plotted as a function of time for ASO - C treated WT ( gray , left ) , ASO - C treated AD ( red , middle ) and ASO - 21 treated AD mice ( blue , right ) . Representative traces taken before and 50 - 60 minutes after tetanic stimulation are shown in the insets ( scale bars = 0 . 2 mV by 10 msec for all traces ) . D Graph summarizes LTP results as the ratio of mean fEPSP rise slope measured 50 - 60 minutes after tetanic stimulation divided by that taken 0 - 10 minutes before tetanic stimulation . There was no significant difference among groups . All data are shown as mean ± s . e . m and are from the same hippocampal brain slices ( n = 6 slices from 4 , 3 , and 4 WT / ASO - C , AD / ASO - C and AD / ASO - 21 , animals per group , respectively ) .

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C LTP was unaffected after ASO-21 treatment and similar in AD compared to WT mice. To combine data from multiple slices, individually measured fEPSP rise slopes were normalized to the average baseline value taken 0-10 minutes before tetanic stimulation (arrows). The normalized values are plotted as a function of time for ASO-C treated WT (gray, left), ASO-C treated AD (red, middle) and ASO-21 treated AD mice (blue, right). Representative traces taken before and 50-60 minutes after tetanic stimulation are shown in the insets (scale bars = 0.2 mV by 10 msec for all traces).D Graph summarizes LTP results as the ratio of mean fEPSP rise slope measured 50-60 minutes after tetanic stimulation divided by that taken 0-10 minutes before tetanic stimulation. There was no significant difference among groups. All data are shown as mean ± s.e.m and are from the same hippocampal brain slices (n=6 slices from 4, 3, and 4 WT/ASO-C, AD/ASO-C and AD/ASO-21, animals per group, respectively).


A Representative water - maze pathway traces of the last four trials of day 3 . Trial 1 is shown in red , trial 2 in green , trial 3 in blue and trial 4 in black . B Morris water maze ( MWM ) analysis of acquisition performance of male ( left ) and female ( right ) mice represented as the mean distance ( cm ) that mice traveled to find the submerged platform plotted per day ( mean s . e . m . ) Symbols represent statistically significant difference within the same genotype ( * AD ASO - C vs AD ASO - 21 and # WT ASO - C vs WT ASO - 21 ) or between groups with same treatment ( ΔAD ASO - C vs WT ASO - C and ^ AD ASO - 21 vs WT ASO - 21 ) on the corresponding day ( two - way repeated - measures ANOVA with Tukey multiple comparisons test ; * * p < 0 . 01 , ^ p < 0 . 05 , ΔΔΔΔp < 0 . 0001 ) . See source data for statistical results . C Morris water maze analysis integrated distance ( Zhang et al , 2014 ) ( area under the curve , AUC ) for male ( left ) and female ( right ) WT and AD mice treated with control ASO ( ASO - C ) or ASO - 21 . ( mean s . e . m . ; one - way ANOVA , Tukey post - test . D , E Correlation analysis of performance on day 3 of the MWM with exon 19 skipping among all groups of mice ( D ) or between AD mice treated with ASO - C or ASO - 21 ( E ) . Male and female mice are indicated . Pearson correlation coefficient ( r ) , covariance ( σ ) and two - tailed p - value are shown .

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A Representative water-maze pathway traces of the last four trials of day 3. Trial 1 is shown in red, trial 2 in green, trial 3 in blue and trial 4 in black.B Morris water maze (MWM) analysis of acquisition performance of male (left) and female (right) mice represented as the mean distance (cm) that mice traveled to find the submerged platform plotted per day (mean s.e.m.) Symbols represent statistically significant difference within the same genotype (*AD ASO-C vs AD ASO-21 and #WT ASO-C vs WT ASO-21) or between groups with same treatment (ΔAD ASO-C vs WT ASO-C and ^AD ASO-21 vs WT ASO-21) on the corresponding day (two-way repeated-measures ANOVA with Tukey multiple comparisons test; **p<0.01, ^p<0.05, ΔΔΔΔp<0.0001). See source data for statistical results.C Morris water maze analysis integrated distance (Zhang et al, 2014) (area under the curve, AUC) for male (left) and female (right) WT and AD mice treated with control ASO (ASO-C) or ASO-21. (mean s.e.m.; one-way ANOVA, Tukey post-test.D,E Correlation analysis of performance on day 3 of the MWM with exon 19 skipping among all groups of mice (D) or between AD mice treated with ASO-C or ASO-21 (E). Male and female mice are indicated. Pearson correlation coefficient (r), covariance (σ) and two-tailed p-value are shown.


B ) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells .

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B) Agarose gel electrophoresis of RNA extracted from RNase treated or control K562 cells.


D ) Agarose gel electrophoresis of RNA extracted from control , 4hr and 24hr ActD treated K562 cells .

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D) Agarose gel electrophoresis of RNA extracted from control, 4hr and 24hr ActD treated K562 cells.


E ) Barplots showing qRT - PCR gene expression levels ( mean + / - S . D . ) of PTEN , FNDC3B , and STAM genes in control , 4hr and 24hr actinomycin D treated K562 cells . p - value : two - tailed t - test , ( * * * p < 0 . 05 ) . The results represent data from 3 technical replicates from 3 independent biological preparations .

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E) Barplots showing qRT-PCR gene expression levels (mean +/- S.D.) of PTEN, FNDC3B, and STAM genes in control, 4hr and 24hr actinomycin D treated K562 cells. p-value: two-tailed t-test, (*** p < 0.05). The results represent data from 3 technical replicates from 3 independent biological preparations.


Hi - C interaction heatmaps at 500kb ( left ) resolution showing all of chromosome 11 , and at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for aXL control and RNase A treated cells . The arrows represent similar patterning of long - range interactions in samples treated with RNase after crosslinking . Hi - C interaction heatmaps at 500kb ( left ) resolution showing all of chromosome 11 , and at 100kb resolution ( right ) showing chr11 : 79 - 115Mb for bXL control and RNase A treated cells . Black arrow indicates the reduction in long - range interactions in the bXL RNase A dataset . The arrows represent perturbed patterning of long - range interactions in samples treated with RNase before crosslinking .

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Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for aXL control and RNase A treated cells. The arrows represent similar patterning of long-range interactions in samples treated with RNase after crosslinking.   Hi-C interaction heatmaps at 500kb (left) resolution showing all of chromosome 11, and at 100kb resolution (right) showing chr11: 79-115Mb for bXL control and RNase A treated cells. Black arrow indicates the reduction in long-range interactions in the bXL RNase A dataset. The arrows represent perturbed patterning of long-range interactions in samples treated with RNase before crosslinking.


C - D ) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4 , with the 1st principal components ( 1st PC ) below for aXL ( C ) and bXL ( D ) samples . The bXL RNase A sample shows reduced Pearson correlations across the genome .

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C-D) Pearson correlation heatmaps showing the genomic interactions at 500kb resolution for chromosome 4, with the 1st principal components (1st PC) below for aXL (C) and bXL (D) samples. The bXL RNase A sample shows reduced Pearson correlations across the genome.


E ) Violin plot showing the first eigenvalues ( calculated by the trans - chromosomal data ) in control and RNase treated cells before and after crosslinking . The bXL RNase A sample show reduction of the negative eigenvalues , indicative of perturbation of B - type compartments . 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 .

O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Violin plot showing the first eigenvalues (calculated by the trans-chromosomal data) in control and RNase treated cells before and after crosslinking. The bXL RNase A sample show reduction of the negative eigenvalues, indicative of perturbation of B-type compartments. 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.


F ) Saddle plots showing the compartmental interactions for both cis - and trans - contacts across the conditions . The bXL RNase A samples display reduced B - B type interactions .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O

F) Saddle plots showing the compartmental interactions for both cis- and trans-contacts across the conditions. The bXL RNase A samples display reduced B-B type interactions.


Interaction heatmaps at 40kb resolution zooming in chr21 : 19 . 5 - 33Mb and showing the TAD structures in aXL and bXL datasets . The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps .

B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

Interaction heatmaps at 40kb resolution zooming in chr21:19.5-33Mb and showing the TAD structures in aXL and bXL datasets. The aXL and bXL RNase A / control log2 ratios are shown below the heatmaps.


Boxplot showing the TAD boundary scores for the aXL and bXL datasets . p - value : Wilcoxon rank - sum test . ( * * * p < 0 . 05 ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Boxplot showing the TAD boundary scores for the aXL and bXL datasets. p-value: Wilcoxon rank-sum test. (*** p < 0.05). 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 - 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 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O

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 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O

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 .

O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

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 .

O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O

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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

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 .

O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O O O O O O 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-EXP_ASSAY O

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 .

O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O

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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O 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-EXP_ASSAY O O O O O O

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 B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY 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-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 .

O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

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 .

O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

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 .

O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O

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 .

O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE 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. 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

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 .

O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

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 B-ORGANISM B-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-CELL_LINE 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 ±

O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Immunoblot analysis of 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 ±

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ±

O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O 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, 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 ±

O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O 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 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 ±

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O 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 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 ±

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G, 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 ±

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. ELISA 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 ±

O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I. 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 .

O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

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 ±

O O B-EXP_ASSAY O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O 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 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 ±

O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O 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 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 ±

O O B-EXP_ASSAY O O B-GENEPROD O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

G. 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE 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 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 .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O

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 .

O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD 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 .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O 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-GENEPROD 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 B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O 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-GENEPROD O O O O O O O O O O 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 B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD 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-GENEPROD 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 B-EXP_ASSAY O O O O B-GENEPROD O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O 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 B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O O O O O O O O O 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 B-EXP_ASSAY O O O B-TISSUE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-DISEASE I-DISEASE O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O 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 ±

O O B-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE O O O O B-GENEPROD O B-CELL_TYPE O O O O B-TISSUE O O O B-GENEPROD O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O 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. 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 B-GENEPROD O O O B-ORGANISM O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O 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 .

O O O O O B-EXP_ASSAY O B-TISSUE O O O O O O O O

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


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

O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-EXP_ASSAY O B-TISSUE O O O O O O O O

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 B-EXP_ASSAY O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O B-EXP_ASSAY O O O 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-EXP_ASSAY O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O O O O O O O O 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 .

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

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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O 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-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O 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-EXP_ASSAY O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE 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 .

O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O

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).