G . FM1 - 43 uptake by Syt11 KD ( RFP - positive ) and control ( RFP - negative ) hippocampal neurons stimulated by 100 mM K + for 2 min . Quantitative data are shown in the lower - right panel . Scale bars , 20 μm .

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G. FM1-43 uptake by Syt11 KD (RFP-positive) and control (RFP-negative) hippocampal neurons stimulated by 100 mM K+ for 2 min. Quantitative data are shown in the lower-right panel. Scale bars, 20 μm.


H . Representative images showing the preloaded FM4 - 64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz . Scale bars , 5 μm .

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H. Representative images showing the preloaded FM4-64 fluorescence in presynaptic boutons of hippocampal neurons before and after 800 stimuli at 40 Hz. Scale bars, 5 μm.


I . Time - course of FM4 - 64 unloading from control , Syt11 KD , and rescued nerve terminals within the same field of view ( 6 coverslips from at least 3 biological repeats each ) .

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I. Time-course of FM4-64 unloading from control, Syt11 KD, and rescued nerve terminals within the same field of view (6 coverslips from at least 3 biological repeats each).


A . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynasore . Endocytic inhibition by dynasore ( Dyna ) was estimated by comparison of Cm traces recorded during 1 - 3 min ( KD ) and 5 - 10 min ( KD + Dyna and Ctrl + Dyna ) after whole - cell dialysis .

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A. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynasore. Endocytic inhibition by dynasore (Dyna) was estimated by comparison of Cm traces recorded during 1-3 min (KD) and 5-10 min (KD + Dyna and Ctrl + Dyna) after whole-cell dialysis.


B , C . Endo - 5s and Cm jumps recorded as in ( A ) .

B, C. Endo-5s and Cm jumps recorded as in (A).


D . Averaged ΔCm traces induced by 200 - ms depolarization in the presence and absence of dynole - 34 - 2TM ( Dynole ) . Cm traces and endocytic rate were measured as in ( A ) except that 10 μM dynole - 34 - 2TM with 0 . 1 % DMSO was dialyzed into patched cells .

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D. Averaged ΔCm traces induced by 200-ms depolarization in the presence and absence of dynole-34-2TM (Dynole). Cm traces and endocytic rate were measured as in (A) except that 10 μM dynole-34-2TM with 0.1% DMSO was dialyzed into patched cells.


E , F . Endo - 5s and Cm jumps as in ( D ) .

E, F. Endo-5s and Cm jumps as in (D).


G , H . Averaged ∆ Cm traces and Endo - 5s recorded from KD neurons under control conditions ( 0 . 1 % DMSO ) .

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G, H. Averaged ∆Cm traces and Endo-5s recorded from KD neurons under control conditions (0.1 % DMSO).


I . KCl - evoked FM 1 - 43 uptake in DRG neurons after 10 - min pre - incubation with 0 . 1 % DMSO ( control ) or with 100 μM dynasore .

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I. KCl-evoked FM 1-43 uptake in DRG neurons after 10-min pre-incubation with 0.1% DMSO (control) or with 100 μM dynasore.


A . DRG neurons expressing Myc - Syt11 were immunostained for Myc - Syt11 , dynamin ( Dyn ) , AP - 2 , clathrin ( CL ) , TGN46 , synaptobrevin 2 ( Syb2 ) , and LAMP1 . For dynamin , the box along the plasma membrane was straightened , enlarged , and is shown in the upper right panels with arrows indicating co - localized puncta . An enlarged inset of the inside of the cell ( box ) is shown in the lower panel . For localization of the transferrin receptor , EGFP - transferrin receptor ( TfR ) was expressed in DRG neurons . Scale bars , 10 μm .

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A. DRG neurons expressing Myc-Syt11 were immunostained for Myc-Syt11, dynamin (Dyn), AP-2, clathrin (CL), TGN46, synaptobrevin 2 (Syb2), and LAMP1. For dynamin, the box along the plasma membrane was straightened, enlarged, and is shown in the upper right panels with arrows indicating co-localized puncta. An enlarged inset of the inside of the cell (box) is shown in the lower panel. For localization of the transferrin receptor, EGFP-transferrin receptor (TfR) was expressed in DRG neurons. Scale bars, 10 μm.


B . Analysis of co - localization of Myc - Syt11 with dynamin , AP - 2 , clathrin , TGN46 , synaptobrevin 2 , transferrin receptor , and LAMP1 . N = 3 independent experiments .

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B. Analysis of co-localization of Myc-Syt11 with dynamin, AP-2, clathrin, TGN46, synaptobrevin 2, transferrin receptor, and LAMP1. N = 3 independent experiments.


A . Transferrin ( Tf , red ) uptake in Syt11 KD ( GFP - positive ) and control neurons . Quantitative data are on the right . Scale bars , 20 μm .

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A. Transferrin (Tf, red) uptake in Syt11 KD (GFP-positive) and control neurons. Quantitative data are on the right. Scale bars, 20 μm.


B . Tf uptake in Syt11 over - expressing and control neurons . Quantitative data are on the right . Scale bars , 20 μm .

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B. Tf uptake in Syt11 over-expressing and control neurons. Quantitative data are on the right. Scale bars, 20 μm.


C . Normalized ∆ Cm induced by 200 - ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double - exponential decay function ( solid black and red , fitted curves ) .

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C. Normalized ∆Cm induced by 200-ms depolarization of a Syt11 KD neuron in the presence of 100 μM MDC was fitted to a double-exponential decay function (solid black and red, fitted curves).


D . Fast and slow time - constants of endocytosis in Syt11 KD neurons recorded as in ( C ) .

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D. Fast and slow time-constants of endocytosis in Syt11 KD neurons recorded as in (C).


E . KCl - evoked FM 1 - 43 uptake in DRG neurons after 1 - h pre - incubation with 0 . 1 % DMSO or 100 μM MDC in the bath solution .

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E. KCl-evoked FM 1-43 uptake in DRG neurons after 1-h pre-incubation with 0.1% DMSO or 100 μM MDC in the bath solution.


A , B . Cm traces with bulk endocytosis ( arrowheads ) in control ( A ) and KD ( B ) neurons induced by 200 - ms depolarization . Expanded bulk events are shown on the right .

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A, B. Cm traces with bulk endocytosis (arrowheads) in control (A) and KD (B) neurons induced by 200-ms depolarization. Expanded bulk events are shown on the right.


C , D . Frequency distribution and bulk endocytic events / cell within 16 s after stimulation .

C, D. Frequency distribution and bulk endocytic events/cell within 16 s after stimulation.


E , F . Size and duration of Cm decay during bulk endocytosis .

E, F. Size and duration of Cm decay during bulk endocytosis.


G , H . Representative z - projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons . Neurons were loaded with 50 μM tetramethylrhodamine - dextran ( 40 kD ) by 2 - min exposure to 100 mM KCl . Scale bars , 10 μm .

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G, H. Representative z-projected fluorescence images and statistics showing the large dextran uptake in Syt11 KD and control DRG neurons. Neurons were loaded with 50 μM tetramethylrhodamine-dextran (40 kD) by 2-min exposure to 100 mM KCl. Scale bars, 10 μm.


A . Representative electron micrographs showing a small / clear vesicle ( SV , arrow ) , dense - core vesicles ( DCVs , arrowheads ) and a clathrin - coated pit ( hatched arrow ) in DRG neurons .

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A. Representative electron micrographs showing a small/clear vesicle (SV, arrow), dense-core vesicles (DCVs, arrowheads) and a clathrin-coated pit (hatched arrow) in DRG neurons.


B , C . Diameter distributions of SVs and DCVs ( statistics from 6 control cells and 9 KD cells ) in DRG neurons . The red and green lines are curves fitted to a Gaussian function .

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B, C. Diameter distributions of SVs and DCVs (statistics from 6 control cells and 9 KD cells) in DRG neurons. The red and green lines are curves fitted to a Gaussian function.


D , E . Diameters of SVs and DCVs in Syt11 KD and control neurons with ( 100K , statistics from 6 control cells and 9 KD cells ) or without 100 mM K + stimulation ( SE , statistics from 6 cells for each group ) .

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D, E. Diameters of SVs and DCVs in Syt11 KD and control neurons with (100K, statistics from 6 control cells and 9 KD cells) or without 100 mM K+ stimulation (SE, statistics from 6 cells for each group).


A . Representative electron micrographs of HRP uptake from standard ( SE ) or 100 mM K + ( 100K ) bath solution . Scale bar , 2 μm .

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A. Representative electron micrographs of HRP uptake from standard (SE) or 100 mM K+ (100K) bath solution. Scale bar, 2 μm.


B - D . HRP - labeled small vesicles ( filled arrows ) and endosomes ( arrowheads ) in DRG neurons . Quantitative data are shown in ( C ) and ( D ) . Scale bar , 200 nm .

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B-D. HRP-labeled small vesicles (filled arrows) and endosomes (arrowheads) in DRG neurons. Quantitative data are shown in (C) and (D). Scale bar, 200 nm.


E . Representative docked vesicles ( arrows ) in a DRG neuron . Quantitative data are on the right . Scale bar , 200 nm .

E. Representative docked vesicles (arrows) in a DRG neuron. Quantitative data are on the right. Scale bar, 200 nm.


F - H . Clathrin - coated pits ( CCPs , hatched arrows ) and bulk - endocytosis - related structures of invaginating membrane ( open arrowhead ) in DRGneurons . Quantitative data are shown in ( G ) and ( H ) . Scale bars , 200 nm .

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F-H. Clathrin-coated pits (CCPs, hatched arrows) and bulk-endocytosis-related structures of invaginating membrane (open arrowhead) in DRGneurons. Quantitative data are shown in (G) and (H). Scale bars, 200 nm.


I , J . Cumulative frequencies of diameter distributions of small vesicles and endosomes ( statistics from 6 control cells and 9 KD cells ) . Kolmogorov - Smirnov test , P = 0 . 304 for ( I ) and P = 0 . 553 for ( J ) .

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I, J. Cumulative frequencies of diameter distributions of small vesicles and endosomes (statistics from 6 control cells and 9 KD cells). Kolmogorov-Smirnov test, P = 0.304 for (I) and P = 0.553 for (J).


Figure 9 . Functional domains of Syt11 in endocytosisA . Diagram of the mutant forms of Syt11 used for rescue experiments . B - D . Statistics of FM4 - 64 ( 5 min ) , Tf , and large dextran ( 40 - KD ) uptake into KD neurons with or without rescue by indicated forms of Syt11 . All data are presented as mean ± s . e . m . of 3 independent experiments . One - way ANOVA , values labeled with different letters ( a , b , and c ) are significantly different from each other , * * * P < 0 . 001 . Student ' s t - test for the comparison between KD and control cells in ( B - D ) , * * P < 0 . 01 , * * * P < 0 . 001 .

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Figure 9. Functional domains of Syt11 in endocytosisA. Diagram of the mutant forms of Syt11 used for rescue experiments.B-D. Statistics of FM4-64 (5 min), Tf, and large dextran (40-KD) uptake into KD neurons with or without rescue by indicated forms of Syt11.All data are presented as mean ± s.e.m. of 3 independent experiments. One-way ANOVA, values labeled with different letters (a, b, and c) are significantly different from each other, ***P < 0.001. Student's t-test for the comparison between KD and control cells in (B-D), **P < 0.01, ***P < 0.001.


Colorectal tumor outcomes in WT ( n = 7 ) , IL - 33KO ( n = 4 ) and ST2KO ( n = 3 ) mice at the completion of the AOM / DSS carcinogenesis protocol . ( A ) Tumor volumes at the endpoint ( each dot represents a tumor , mice with no tumor are marked as zero ) , Skin tumor outcomes in WT ( n = 7 ) , IL - 33KO ( n = 6 ) and ST2KO ( n = 7 ) mice at the completion of the DMBA / DNFB carcinogenesis protocol . ( D ) Tumor volumes at the endpoint ( each dot represents a tumor , mice with no tumor are marked as zero ) ,

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Colorectal tumor outcomes in WT (n=7), IL-33KO (n=4) and ST2KO (n=3) mice at the completion of the AOM/DSS carcinogenesis protocol. (A) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero), Skin tumor outcomes in WT (n=7), IL-33KO (n=6) and ST2KO (n=7) mice at the completion of the DMBA/DNFB carcinogenesis protocol. (D) Tumor volumes at the endpoint (each dot represents a tumor, mice with no tumor are marked as zero),


H ) Quantification of nuclear IL - 33 + epidermal and dermal cells in the DNFB - versus acetone - treated WT skin . Dots represent cell counts from three randomly selected high power field ( HPF ) images per sample ( n = 5 per group ) . J ) Quantification of nuclear IL - 33 + epithelial and stromal cells of colon treated with AOM / DSS versus no treatment . Dots represent cell counts from three randomly selected HPF images per sample ( n = 4 per group ) . Data information : Graphs show mean + SD , unpaired t - test .

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H) Quantification of nuclear IL-33+ epidermal and dermal cells in the DNFB- versus acetone-treated WT skin. Dots represent cell counts from three randomly selected high power field (HPF) images per sample (n=5 per group). J) Quantification of nuclear IL-33+ epithelial and stromal cells of colon treated with AOM/DSS versus no treatment. Dots represent cell counts from three randomly selected HPF images per sample (n=4 per group). Data information: Graphs show mean + SD, unpaired t-test.


( H ) ChIP - qPCR assay for Smad6 in the presence of IL - 33 full length or cytokine domain using an anti - RUNX2 antibody . After Pam212 cells were transfected with IL - 33 full length or cytokine domain for 24 hours , cell lysates were subjected to chromatin - immunoprecipitation with anti - RUNX2 antibody and eluted RUNX2 - bound chromatin was used for qPCR with primers against Smad6 promoter region . Anti - IgG ChIP - qPCR results are shown as negative controls ( n = 4 per group ) .

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(H) ChIP-qPCR assay for Smad6 in the presence of IL-33 full length or cytokine domain using an anti-RUNX2 antibody. After Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours, cell lysates were subjected to chromatin-immunoprecipitation with anti-RUNX2 antibody and eluted RUNX2-bound chromatin was used for qPCR with primers against Smad6 promoter region. Anti-IgG ChIP-qPCR results are shown as negative controls (n=4 per group).


( D ) Immunoblot of p - SMAD2 / 3 and SMAD2 / 3 proteins in response to TGF - β and poly ( I : C ) treatment . Pam212 cells were treated with poly ( I : C ) or PBS followed by incubation with and without TGF - β ( 5nM ) . Data represent three independent experiments with similar results . ( E ) Immunoblot of p - SMAD2 / 3 and SMAD2 / 3 proteins in response to TGF - β + poly ( I : C ) after knocking down of IL - 33 . Pam212 cells were transfected with siIl33 knockdown construct or siRNA control ( siCon ) for 30 hours followed by incubation with poly ( I : C ) and TGF - β . Data represent three independent experiments with similar results . ( F ) Immunoblot of p - SMAD2 / 3 and SMAD2 / 3 proteins upon the expression of IL - 33 full length or cytokine domain . Pam212 cells were transfected with IL - 33 full length or cytokine domain for 24 hours followed by incubation with and without TGF - β . Data represent three independent experiments with similar results . Data information : GAPDH is used as the control housekeeping protein in D - F ) . Graphs show mean + SD , NS : not significant , unpaired t - test .

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(D) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β and poly (I:C) treatment. Pam212 cells were treated with poly (I:C) or PBS followed by incubation with and without TGF-β (5nM). Data represent three independent experiments with similar results. (E) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins in response to TGF-β + poly (I:C) after knocking down of IL-33. Pam212 cells were transfected with siIl33 knockdown construct or siRNA control (siCon) for 30 hours followed by incubation with poly (I:C) and TGF-β. Data represent three independent experiments with similar results. (F) Immunoblot of p-SMAD2/3 and SMAD2/3 proteins upon the expression of IL-33 full length or cytokine domain. Pam212 cells were transfected with IL-33 full length or cytokine domain for 24 hours followed by incubation with and without TGF-β. Data represent three independent experiments with similar results. Data information: GAPDH is used as the control housekeeping protein in D-F). Graphs show mean + SD, NS: not significant, unpaired t-test.


( G ) Impact of IL - 33 knockdown on cell proliferation in response to TGF - β . Pam212 cells were treated with siIl33 or siCon followed by TGF - β treatment ( n = 7 in each group ) . ( H ) Impact of IL - 33 knockdown and SB431542 treatment on cell proliferation . Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO ( carrier control , n = 7 in each group ) .

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(G) Impact of IL-33 knockdown on cell proliferation in response to TGF-β. Pam212 cells were treated with siIl33 or siCon followed by TGF-β treatment (n=7 in each group). (H) Impact of IL-33 knockdown and SB431542 treatment on cell proliferation. Pam212 cells were treated with siIl33 or siCon in combination with SB431542 or DMSO (carrier control, n=7 in each group).


( I ) SMAD signaling proteins levels in WT and K14 - IL33tg , ST2KO skin treated with acetone ( control ) at the completion of the DMBA / TPA carcinogenesis protocol . Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p - SMAD2 / 3 , p - SMAD1 / 5 , SMAD6 , SMAD2 / 3 and SMAD1 antibodies . GAPDH is used as the control housekeeping protein ( n = 5 in each group ) .

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(I) SMAD signaling proteins levels in WT and K14-IL33tg,ST2KO skin treated with acetone (control) at the completion of the DMBA/TPA carcinogenesis protocol. Tissue lysates prepared from the whole back skin of the animals were subjected to immunoblot with p-SMAD2/3, p-SMAD1/5, SMAD6, SMAD2/3 and SMAD1 antibodies. GAPDH is used as the control housekeeping protein (n=5 in each group).


( J ) Representative images of IL - 33 and p - SMAD2 / 3 immunostaining on the adjacent sections of human squamous cell carcinoma ( SCC ) and normal skin ( scale bar : 100 μm ) . Data information : Graphs show mean + SD , NS : not significant , unpaired t - test .

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(J) Representative images of IL-33 and p-SMAD2/3 immunostaining on the adjacent sections of human squamous cell carcinoma (SCC) and normal skin (scale bar: 100 μm). Data information: Graphs show mean + SD, NS: not significant, unpaired t-test.


( B ) Il33 and Smad6 mRNA levels in caerulein - treated WT pancreas compared with PBS - treated controls at the completion of chronic pancreatitis protocol ( n = 8 in caerulein and n = 9 in PBS group for Il33 , n = 9 in caerulein and n = 10 in PBS group for Smad6 , unpaired t - test ) .

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(B) Il33 and Smad6 mRNA levels in caerulein-treated WT pancreas compared with PBS-treated controls at the completion of chronic pancreatitis protocol (n=8 in caerulein and n=9 in PBS group for Il33, n=9 in caerulein and n=10 in PBS group for Smad6, unpaired t-test).


( D ) Pancreatic tumor - free survival of WT ( n = 4 ) , IL - 33KO ( n = 5 ) and ST2KO ( n = 6 ) mice at the completion of the DMBA / caerulein carcinogenesis protocol ( log - rank test ) .

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(D) Pancreatic tumor-free survival of WT (n=4), IL-33KO (n=5) and ST2KO (n=6) mice at the completion of the DMBA/caerulein carcinogenesis protocol (log-rank test).


( G , H ) Quantification of nuclear ( G ) IL - 33 + and ( H ) p - SMAD2 / 3 + epithelial cells per HPF in the matched human pancreatic tissues . Each dot represents the average cell counts across three randomly selected HPF images per sample ( n = 18 patients , paired t - test ) .

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(G, H) Quantification of nuclear (G) IL-33+ and (H) p-SMAD2/3+ epithelial cells per HPF in the matched human pancreatic tissues. Each dot represents the average cell counts across three randomly selected HPF images per sample (n=18 patients, paired t-test).


C . Western blot for POT1 and DDR markers upon POT1 deletion induced with 0 . 5 μM 4 - OHT . Cl75 - parental cell line . gRNA1 - POT1 knock - out in the population by transient transfection with pSpCas9 ( BB ) - 2A - puro containing gRNA1 sequence . EV - corresponding empty vector .

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C. Western blot for POT1 and DDR markers upon POT1 deletion induced with 0.5 μM 4-OHT. Cl75 - parental cell line. gRNA1 - POT1 knock-out in the population by transient transfection with pSpCas9(BB)-2A-puro containing gRNA1 sequence. EV - corresponding empty vector.


D . Time course of telomere length changes upon POT1 removal in clone 35 . d ( days ) TP0 - time point 0 , no 4OHT .

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D. Time course of telomere length changes upon POT1 removal in clone 35. d (days) TP0 - time point 0, no 4OHT.


E . Growth curve for POT1 WT and POT1 knockout cells . Population doublings ( PDL ) are represented as mean ± SD ( three biological replicates ) .

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E. Growth curve for POT1 WT and POT1 knockout cells. Population doublings (PDL) are represented as mean ± SD (three biological replicates).


F . Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35 . The bars show percentage of cells containing more than 5 TIFs ± SD . Experiments were performed in triplicate . At least 120 cells were analyzed per condition per replicate . Significance was determined using two - way ANOVA . p - values are indicated on the graph .

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F. Quantification for TIFs in EdU positive and EdU negative cells upon POT1 removal in clone 35. The bars show percentage of cells containing more than 5 TIFs ± SD. Experiments were performed in triplicate. At least 120 cells were analyzed per condition per replicate. Significance was determined using two-way ANOVA. p-values are indicated on the graph.


A . G - overhang assay for clone 35 upon POT1 removal . The bars show relative amounts of single stranded telomeric DNA ± SD . Experiments were performed in triplicate .

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A. G-overhang assay for clone 35 upon POT1 removal. The bars show relative amounts of single stranded telomeric DNA ± SD. Experiments were performed in triplicate.


B . 2D gels for telomeric DNA from clone 35 cells with POT1 ( NT ) and upon POT1 removal for 7 days ( 7d 4OHT ) .

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B. 2D gels for telomeric DNA from clone 35 cells with POT1 (NT) and upon POT1 removal for 7 days (7d 4OHT).


C . Examples of metaphase spreads for non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . White arrowheads indicate fragile telomeres , the red arrowhead indicates a sister - chromatid fusion . Scale bar equals 6 µm . D . Quantification of fragile telomeres ( left graph ) , sister chromatid fusions ( middle graph ) and chromosome fusions ( right graph ) as a percentage of events per metaphase . Experiments were performed in triplicate . At least 25 metaphases were examined per condition per replicate . The mean is displayed and error bars represent ± SD . Significance was determined using one - way ANOVA . p - values are indicated on the graph .

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C. Examples of metaphase spreads for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. White arrowheads indicate fragile telomeres, the red arrowhead indicates a sister-chromatid fusion. Scale bar equals 6 µm. D. Quantification of fragile telomeres (left graph), sister chromatid fusions (middle graph) and chromosome fusions (right graph) as a percentage of events per metaphase. Experiments were performed in triplicate. At least 25 metaphases were examined per condition per replicate. The mean is displayed and error bars represent ± SD. Significance was determined using one-way ANOVA. p-values are indicated on the graph.


E . Quantification and representative images of anaphases with bridges and micronuclei for non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . Scale bar equals 6 µm . Experiments were performed in triplicate . The bars show percentage of cells containing anaphases , anaphases with bridges or micronuclei + SD . At least 650 cells were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on the graph . In all experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT .

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E. Quantification and representative images of anaphases with bridges and micronuclei for non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Scale bar equals 6 µm. Experiments were performed in triplicate. The bars show percentage of cells containing anaphases, anaphases with bridges or micronuclei + SD. At least 650 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT.


A . Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1 .

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A. Western blot for POT1 and DDR markers upon POT1 removal in clone 35 and overexpression of ectopic WT POT1.


B . Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 ( constant - field gel electrophoresis ) .

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B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic WT POT1 (constant-field gel electrophoresis).


D , E Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease - associated mutations ( constant - field gel electrophoresis ) .

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D, E Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and overexpression of ectopic POT1 variants carrying disease-associated mutations (constant-field gel electrophoresis).


B . Quantification of precipitated telomeric DNA in 2 - step QTIP ( % of input ) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA ( based on dot blot analyses ) . Bars represent data from three independent experiments ± SD .

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B. Quantification of precipitated telomeric DNA in 2-step QTIP (% of input) and fold enrichment of precipitated telomeric DNA compared with precipitated Alu repeat DNA (based on dot blot analyses). Bars represent data from three independent experiments ± SD.


C . Scatter plots representing immunoprecipitation specificity ( 2 - step QTIP / IgG ratio ) versus difference between non treated cells ( NT ) and cells at day 4 upon 4 - OHT induction ( 4d 4OHT / NT ratio ) or difference between non treated cells ( NT ) and cells at day 7 upon 4 - OHT induction ( 7d 4OHT / NT ratio ) respectively ( data from Table EV1 ) . MRN complex in red , 9 - 1 - 1 complex in blue , shelterin complex in orange except for POT1 in black . Values are the average of the 3 biological replicates .

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C. Scatter plots representing immunoprecipitation specificity (2-step QTIP/IgG ratio) versus difference between non treated cells (NT) and cells at day 4 upon 4-OHT induction (4d 4OHT/NT ratio) or difference between non treated cells (NT) and cells at day 7 upon 4-OHT induction (7d 4OHT/NT ratio) respectively (data from Table EV1). MRN complex in red, 9-1-1 complex in blue, shelterin complex in orange except for POT1 in black. Values are the average of the 3 biological replicates.


D . Heat map representing changes in levels of selected proteins involved in DNA damage ( MRN complex in red , 9 - 1 - 1 complex in blue ) and HDR proteins ( purple ) after POT1 knockout induction . Proteins marked with * pass t - test , but not t - test with Benjamini - Hochberg correction ( p < 0 . 05 , q < 0 . 09 ) . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated cells .

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D. Heat map representing changes in levels of selected proteins involved in DNA damage (MRN complex in red, 9-1-1 complex in blue) and HDR proteins (purple) after POT1 knockout induction. Proteins marked with * pass t-test, but not t-test with Benjamini-Hochberg correction (p < 0.05, q < 0.09). Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells.


A . Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA ( pulsed - field gel electrophoresis ) .

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A. Telomere length for clone 35 upon POT1 removal and depletion of selected recombination proteins with siRNA (pulsed-field gel electrophoresis).


B . Telomere length and G - overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L ( pulsed - field gel electrophoresis ) .

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B. Telomere length and G-overhang length analysis for clone 35 upon POT1 removal and inhibition of telomerase with 1 µM GRN163L (pulsed-field gel electrophoresis).


C . DRIP assay for DNA - RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9 . 6 antibody . Dot blots represent two replicates . Bars represent data from three independent experiments ± SD . The IgG signal was subtracted . Significance was determined using one - way ANOVA , p - values are indicated on the graph .

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C. DRIP assay for DNA-RNA hybrid detection at telomeres in clone 35 upon POT1 removal with S9.6 antibody. Dot blots represent two replicates. Bars represent data from three independent experiments ± SD. The IgG signal was subtracted. Significance was determined using one-way ANOVA, p-values are indicated on the graph.


D . C - circle ( CC ) assay for clone 35 upon POT1 removal . Bars represent data from three independent experiments ± SD . DNA from ALT positive cell line U2OS was used as positive control . Significance was determined using one - way ANOVA , p - values are indicated on the graph . In all experiments , POT1 deletions was induced with 0 . 5 μM 4 - OHT for 4 and 7 days .

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D. C-circle (CC) assay for clone 35 upon POT1 removal. Bars represent data from three independent experiments ± SD. DNA from ALT positive cell line U2OS was used as positive control. Significance was determined using one-way ANOVA, p-values are indicated on the graph. In all experiments, POT1 deletions was induced with 0.5 μM 4-OHT for 4 and 7 days.


E . C - overhang length analysis for clone 35 upon POT1 removal ( pulsed - field gel electrophoresis , hybridization with Telo - G probe ) .

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E. C-overhang length analysis for clone 35 upon POT1 removal (pulsed-field gel electrophoresis, hybridization with Telo-G probe).


F . Quantification and representative pictures for visualization of the PML protein ( IF , green ) and telomeres ( FISH , red ) in non - treated ( NT ) clone 35 cells and clone 35 treated with 4 - OHT for 4 and 7 days . Arrows indicate co - localizations . Scale bar equals 10 µm . Bars represent data from three independent experiments ± SD . At least 150 cells were examined per condition per replicate . Significance was determined using one - way ANOVA . p - values are indicated on the graph . In all experiments , POT1 deletion was induced with 0 . 5 μM 4 - OHT . Data information : 4 - OHT - 4 - hydroxytamoxifen , NT - non - treated cells , GRN163L - telomerase inhibitor Imitelstat , siNT - non - tagreting siRNA , negative control .

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F. Quantification and representative pictures for visualization of the PML protein (IF, green) and telomeres (FISH, red) in non-treated (NT) clone 35 cells and clone 35 treated with 4-OHT for 4 and 7 days. Arrows indicate co-localizations. Scale bar equals 10 µm. Bars represent data from three independent experiments ± SD. At least 150 cells were examined per condition per replicate. Significance was determined using one-way ANOVA. p-values are indicated on the graph. In all experiments, POT1 deletion was induced with 0.5 μM 4-OHT. Data information: 4-OHT - 4-hydroxytamoxifen, NT - non-treated cells, GRN163L - telomerase inhibitor Imitelstat, siNT -non-tagreting siRNA, negative control.


Differences in clinical variables including ( B ) liver fat , plasma levels of ( C ) ALT , ( D ) AST , ( E ) uric acid and ( F ) creatinine are presented in the CMA and placebo groups on Days 0 , 14 and 70 after weight loss adjustment . Adj . P indicates p value after weight loss adjustment . Statistical significance is defined based on paired Student ' s t test . p < 0 . 05 . The boxes show the distribution of the clinical parameters in different groups . The bottom and top of the boxes represent the 25th and 75th percentiles . The central band represents the median value . The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values . The sample sizes on Days 0 , 14 or 70 were marked in each boxplot .

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Differences in clinical variables including (B) liver fat, plasma levels of (C) ALT, (D) AST, (E) uric acid and (F) creatinine are presented in the CMA and placebo groups on Days 0, 14 and 70 after weight loss adjustment. Adj.P indicates p value after weight loss adjustment. Statistical significance is defined based on paired Student's t test. p< 0.05. The boxes show the distribution of the clinical parameters in different groups. The bottom and top of the boxes represent the 25th and 75th percentiles. The central band represents the median value. The whiskers represent the minimum and maximum values that are not outliers and dots represent outlier values. The sample sizes on Days 0, 14 or 70 were marked in each boxplot.


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Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B ) in both ( n = 4 ) ;

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Association between the plasma level of significantly different metabolites on Day 70 vs Day 0 B) in both (n=4);


A ) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups . Asterisks indicate statistical significance based on paired Student ' s t test . p < 0 . 05 ;

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A) Heatmap shows log2FC based alterations between the significantly different inflammation related proteins on Day 70 vs Day 0 in the CMA and placebo groups. Asterisks indicate statistical significance based on paired Student's t test. p < 0.05;


B ) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine , cysteine , carnitine and nicotinamide . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM )

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B) Heatmap shows the correlation between the plasma levels of all inflammation related proteins and plasma levels of the individual metabolic activators including serine, cysteine, carnitine and nicotinamide. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM)


Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Heatmap shows the associations between the significantly different inflammation related proteins ( CD8A , CSF - 1 , CCL23 , FGF - 21 and OSM ) C ) with the 10 most significantly correlated plasma metabolites ,

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Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Heatmap shows the associations between the significantly different inflammation related proteins (CD8A, CSF-1, CCL23, FGF-21 and OSM) C) with the 10 most significantly correlated plasma metabolites,


Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A ) gut microbiome between CMA and placebo groups on Day 70 vs Day 0 ; B ) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups ; C ) oral microbiome between CMA and placebo groups on Day 70 vs Day 0 ; D ) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups . Each dot represents a species and it has been colored according to its corresponding phylum . Asterisks indicate statistical significance based on paired Wilcoxon signed - rank test . p < 0 . 05 . Log2FC : log2 ( fold change ) .

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Scatter plot with linear regression line and heatmap show log2FC based alterations of the significantly different species in the A) gut microbiome between CMA and placebo groups on Day 70 vs Day 0; B) gut microbiome between Day 70 vs Day 0 in the CMA and placebo groups; C) oral microbiome between CMA and placebo groups on Day 70 vs Day 0; D) oral microbiome between Day 70 vs Day 0 in the CMA and placebo groups. Each dot represents a species and it has been colored according to its corresponding phylum. Asterisks indicate statistical significance based on paired Wilcoxon signed-rank test. p< 0.05. Log2FC: log2(fold change).


Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , B ) plasma level of 10 most significant inflammation related

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

Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , B) plasma level of 10 most significant inflammation related proteins


Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with , C ) the abundance of the species in gut

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Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with , C) the abundance of the species in gut microbiome


Heatmap shows the association between the plasma level of clinical variables including liver fat , ALT , AST , uric acid , and creatinine with D ) the abundance of the species in oral microbiome . AST , Aspartate aminotransferase ; ALT , Alanine transaminase . Asterisks indicate statistical significance based on Spearman correlation analysis . p < 0 . 05 ; Cor . Coeff : Correlation coefficient .

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Heatmap shows the association between the plasma level of clinical variables including liver fat, ALT, AST, uric acid, and creatinine with D) the abundance of the species in oral microbiome. AST, Aspartate aminotransferase; ALT, Alanine transaminase. Asterisks indicate statistical significance based on Spearman correlation analysis. p < 0.05; Cor.Coeff: Correlation coefficient.


Low magnification views of a CD1 Otof - / - organ of Corti ( P23 ) transduced with otoferlin dual ‑ AAV ‑ TS vectors . IHCs : inner hair cells , OHCs : outer hair cells maximum intensity projections of confocal optical

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Low magnification views of a CD1 Otof-/- organ of Corti (P23) transduced with otoferlin dual‑AAV‑TS vectors. IHCs: inner hair cells, OHCs: outer hair cells maximum intensity projections of confocal optical sections


High magnification views of CD1B6F1 Otof - / - IHCs transduced with otoferlin dual ‑ AAV ‑ TS ( P26 ) ( B ) and dual ‑ AAV ‑ Hyb ( P26 ) ( C ) vectors . Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in ( A - C ) with warmer colors representing higher pixel intensities ) , maximum intensity projections of confocal optical

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High magnification views of CD1B6F1 Otof-/- IHCs transduced with otoferlin dual‑AAV‑TS (P26) (B) and dual‑AAV‑Hyb (P26) (C) vectors. Individual eGFP and otoferlin immunostainings are depicted as color lookup tables in (A-C) with warmer colors representing higher pixel intensities ), maximum intensity projections of confocal optical sections


Percentage of N ‑ and C ‑ terminal otoferlin labelled IHCs in dual ‑ AAV ‑ TS ( n = 10 mice ) and dual ‑ AAV ‑ Hyb ( n = 9 mice ) injected CD1B6F1 Otof - / - mice ( P18 ‑ 30 )

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Percentage of N‑ and C‑terminal otoferlin labelled IHCs in dual‑AAV‑TS (n=10 mice) and dual‑AAV‑Hyb (n=9 mice) injected CD1B6F1 Otof-/- mice (P18‑30)


Average N - terminal and C ‑ terminal otoferlin immunofluorescence levels in dual ‑ AAV transduced Otof - / - and wild - type IHCs ( P23 - 30 ) . Otoferlin levels were normalized to immunofluorescence levels in non - transduced B6 wild - type IHCs for each antibody

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Average N-terminal and C‑terminal otoferlin immunofluorescence levels in dual‑AAV transduced Otof-/- and wild-type IHCs (P23-30). Otoferlin levels were normalized to immunofluorescence levels in non-transduced B6 wild-type IHCs for each antibody separately


High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons ( CtBP2 ) from wild ‑ type ( B6 : P27 , CD1B6F1 : P29 ) , dual - AAV injected CD1B6F1 Otof - / - ( dual - AAV - TS : P26 , dual - AAV - Hyb : P28 ) , and their contralateral non - injected ears . ( * ) Transduced cells . Maximum intensity projections of optical confocal sections . Scale bars : 5

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High magnification views of IHCs immunolabeled for otoferlin and synaptic ribbons (CtBP2) from wild‑type (B6: P27, CD1B6F1: P29), dual-AAV injected CD1B6F1 Otof-/- (dual-AAV-TS: P26, dual-AAV-Hyb: P28), and their contralateral non-injected ears. (*) Transduced cells. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm


Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild ‑ type ( B6 : n = 48 IHCs , CD1B6F1 : n = 108 IHCs ) , transduced Otof - / - ( dual ‑ AAV ‑ TS : n = 59 IHCs , dual - AAV - Hyb : n = 37 IHCs ) , and non - transduced Otof - / - IHCs from injected ( - AAV injected ear , n = 65 IHCs ) and contralateral non - injected ( - AAV non - injected ear , n = 46 IHCs ) ears ( P25 - 29 )

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Synaptic ribbon numbers quantified from IHCs in apical cochlear turns of wild‑type (B6: n=48 IHCs, CD1B6F1: n=108 IHCs), transduced Otof-/- (dual‑AAV‑TS: n=59 IHCs, dual-AAV-Hyb: n=37 IHCs), and non-transduced Otof-/- IHCs from injected (-AAV injected ear, n=65 IHCs) and contralateral non-injected (-AAV non-injected ear, n=46 IHCs) ears (P25-29)


IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild - type and Otof - / - P6 and P14 organs of Corti . Maximum intensity projections of optical confocal sections . Scale bars : 5 µm . D Synapse numbers quantified from IHCs in apical cochlear turns ( C ) of B6 wild - type ( P6 : n = 53 IHCs ; P14 : n = 73 IHCs ) and B6 Otof - / - ( P6 : n = 62 IHCs ; P14 : n = 65 IHCs ) mice at two different developmental stages ( P6 and P14 )

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IHC synapses labelled with CtBP2 and the postsynaptic marker Shank1a in B6 wild-type and Otof-/- P6 and P14 organs of Corti. Maximum intensity projections of optical confocal sections. Scale bars: 5 µm. D Synapse numbers quantified from IHCs in apical cochlear turns (C) of B6 wild-type (P6: n= 53 IHCs; P14: n=73 IHCs) and B6 Otof-/- (P6: n=62 IHCs; P14: n=65 IHCs) mice at two different developmental stages (P6 and P14)


Ca2 + ‑ current - voltage relationship of control CD1B6F1 wild ‑ type ( n = 6 IHCs ) , dual - AAV - TS transduced ( n = 8 IHCs ) , and non ‑ transduced CD1B6F1 Otof - / - ( n = 10 IHCs ) IHCs ( P14 ‑ 18 )

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Ca2+‑current-voltage relationship of control CD1B6F1 wild‑type (n=6 IHCs), dual-AAV-TS transduced (n=8 IHCs), and non‑transduced CD1B6F1 Otof-/- (n=10 IHCs) IHCs (P14‑18)


Representative Ca2 + ‑ currents ( Ica ) and IHC plasma membrane capacitance increments ( ΔCm ) of a wild ‑ type control , transduced , and non ‑ transduced Otof - / - IHC in response to a 20 ms depolarization pulse at maximum Ca2 + ‑ current potentials ( typically - 14 mV )

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Representative Ca2+‑currents (Ica) and IHC plasma membrane capacitance increments (ΔCm) of a wild‑type control, transduced, and non‑transduced Otof-/- IHC in response to a 20 ms depolarization pulse at maximum Ca2+‑current potentials (typically -14 mV)


Average exocytosis level measured as ΔCm ( G ) and corresponding Ca2 + ‑ current integrals ( QCa2 + ) ( H ) in wild ‑ type ( CD1B6F1 : n = 6 IHCs ; B6 : n = 11 IHCs ( B6 data replotted from Strenzke et al , 2016 ) ) , dual - AAV - TS transduced Otof - / - ( n = 8 IHCs ) , and non ‑ transduced ( n = 11 IHCs ) Otof - / - IHCs . Transduced IHCs that showed eGFP expression , but had almost no exocytosis are depicted as dashed lines ( not included into the average )

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

Average exocytosis level measured as ΔCm (G) and corresponding Ca2+‑current integrals (QCa2+) (H) in wild‑type (CD1B6F1: n=6 IHCs; B6: n=11 IHCs (B6 data replotted from Strenzke et al, 2016)), dual-AAV-TS transduced Otof-/- (n=8 IHCs), and non‑transduced (n=11 IHCs) Otof-/- IHCs. Transduced IHCs that showed eGFP expression, but had almost no exocytosis are depicted as dashed lines (not included into the average)


Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual ‑ AAV ‑ TS and dual ‑ AAV ‑ Hyb injected CD1B6F1 Otof - / - animals . AAV2 / 6 . eGFP ( + AAV . eGFP ) and dual ‑ AAV ‑ TS injected CD1B6F1 wild ‑ type , and non ‑ injected control Otof - / - littermate ( - AAV ) mice served as controls . SP : summating potential ; ABR waves are indicated from I -

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Representative ABR wave traces in response to broadband click sound stimuli from otoferlin dual‑AAV‑TS and dual‑AAV‑Hyb injected CD1B6F1 Otof-/- animals. AAV2/6.eGFP (+AAV.eGFP) and dual‑AAV‑TS injected CD1B6F1 wild‑type, and non‑injected control Otof-/- littermate (-AAV) mice served as controls. SP: summating potential; ABR waves are indicated from I-V


Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks /

Average ABRs evoked by 80 dB SPL click sound stimuli for 20 clicks/s


ABR click sound ( C thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control

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ABR click sound (C thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals


ABR tone burst ( D ) thresholds in otoferlin dual ‑ AAV treated Otof - / - mice compared to wild ‑ type and non ‑ treated Otof - / - control animals . In D , the two best animals are depicted with open circles . Animals with thresholds exceeding the maximum loudspeaker output ( arrows ) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL , respectively . Apical and basal cochlear turns are indicated as Apex and Base ,

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ABR tone burst (D) thresholds in otoferlin dual‑AAV treated Otof-/- mice compared to wild‑type and non‑treated Otof-/- control animals. In D, the two best animals are depicted with open circles. Animals with thresholds exceeding the maximum loudspeaker output (arrows) of 100 dB SPL for clicks and 90 dB SPL for tone bursts were set to 110 dB SPL and 100 dB SPL, respectively. Apical and basal cochlear turns are indicated as Apex and Base, respectively


Summed ABR wave I - V amplitudes at different click sound intensities in otoferlin dual ‑ AAV injected , non ‑ injected Otof - / - , and wild ‑ type control

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Summed ABR wave I-V amplitudes at different click sound intensities in otoferlin dual‑AAV injected, non‑injected Otof-/-, and wild‑type control mice


Summed ABR wave I ‑ V amplitudes of individual dual ‑ AAV ‑ TS treated CD1B6F1 Otof - / - animals ( n = 8 animals plotted against their full - length otoferlin IHC transduction

Summed ABR wave I‑V amplitudes of individual dual‑AAV‑TS treated CD1B6F1 Otof-/- animals (n=8 animals plotted against their full-length otoferlin IHC transduction rate


B . Rev3l + / + and Rev3l - / - MEFs were pulse - labelled with EdU for 15min , permeabilized , fixed and stained for EdU incorporation ( green ) . S phase sub - stages from I to IV were evaluated by visual inspection of the cycling population ( > 300 EdU + cells , top panel ) . Scale bar = 5 μm . Dot plots and pie charts show the relative proportion ( percentage of total S ) of each sub - stage from I to IV ( middle and bottom panel , respectively ) . Each dot represents the mean of two technical replicates .

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B. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with EdU for 15min, permeabilized, fixed and stained for EdU incorporation (green). S phase sub-stages from I to IV were evaluated by visual inspection of the cycling population (>300 EdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of each sub-stage from I to IV (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.


C . Analysis of the active marks H3K27ac and H3K4me3 and facultative heterochromatin mark H3K27me3 content in early , mid , late and TTR compared to disturbed replicating regions found in Rev3l - / - MEFs . Bar in boxplot represents the median , and red points represents the mean . The limit of the boxes corresponds to the 0 . 25 to 0 . 75 quartiles with whiskers extending to the maximum value of 1 . 5 times the interquartile range . Graphs show data from two biological experiments .

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C. Analysis of the active marks H3K27ac and H3K4me3 and facultative heterochromatin mark H3K27me3 content in early, mid, late and TTR compared to disturbed replicating regions found in Rev3l-/- MEFs. Bar in boxplot represents the median, and red points represents the mean. The limit of the boxes corresponds to the 0.25 to 0.75 quartiles with whiskers extending to the maximum value of 1.5 times the interquartile range. Graphs show data from two biological experiments.


D . Relative mRNA levels of four genes involved in development ( Hoxb2 , 8 , 9 and WT1 ) were validated through qRT - PCR from Rev3l + / + and Rev3l - / - MEFs samples . The data were normalized to the amount of GAPDH mRNA . Error bars indicate standard error of the mean for three independent experiments . * * * , P < 0 . 001 and * * , P < 0 . 005 by Student ' s t - test . E . Relative mRNA levels of genes involved in imprinting ( paternal expressed genes : Dlk1and Slc18a4 and maternal expressed genes : Dcn1 and H19 ) were validated through qRT - PCR from Rev3l + / + and Rev3l - / - MEFs samples . The data were normalized to the amount of GAPDH mRNA . Error bars indicate standard error of the mean for three independent experiments . * * * P < 0 . 001 by Student ' s t - test .

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D. Relative mRNA levels of four genes involved in development (Hoxb2, 8, 9 and WT1) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. ***, P<0.001 and **, P<0.005 by Student's t-test. E. Relative mRNA levels of genes involved in imprinting (paternal expressed genes: Dlk1and Slc18a4 and maternal expressed genes: Dcn1 and H19) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. *** P<0.001 by Student's t-test.


A . Total histones were acid extracted from Rev3l + / + and Rev3l - / - MEFs . Samples were analyzed by western - blot using indicated antibodies specific to histone marks ( right panel ) . An anti - pan H4 or H3 was used as loading control ( left panel ) . Histograms show WT / KO Rev3l fold change relative to the immunoblot shown on the top panel ( using 1X sample intensity ) .

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A. Total histones were acid extracted from Rev3l+/+ and Rev3l-/- MEFs. Samples were analyzed by western-blot using indicated antibodies specific to histone marks (right panel). An anti-pan H4 or H3 was used as loading control (left panel). Histograms show WT/KO Rev3l fold change relative to the immunoblot shown on the top panel (using 1X sample intensity).


B . Histone mark levels were examined at selected genes repressed in Rev3l - / - MEFs ( Hoxb2 , Hoxb8 , WT1 ) . Histograms represent enrichment of H3K9me3 and H3K27me3 at indicated loci assessed by ChIP - qPCR in Rev3l + / + and Rev3l - / - MEFs . Error bars indicate standard error of the mean for three independent experiments .

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B. Histone mark levels were examined at selected genes repressed in Rev3l-/- MEFs (Hoxb2, Hoxb8, WT1). Histograms represent enrichment of H3K9me3 and H3K27me3 at indicated loci assessed by ChIP-qPCR in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.


C . H3K9me3 and H3K27me3 mark levels were evaluated at selected genes that localized in genomic loci displaying replication timing delay and downregulated in Rev3l - / - MEFs ( FoxG1 , GATA6 ) . Histograms represent enrichment of H3K9me3 and H3K27me3 assessed by ChIP - qPCR at indicated loci in Rev3l + / + and Rev3l - / - MEFs . Error bars indicate standard error of the mean for three independent experiments .

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C. H3K9me3 and H3K27me3 mark levels were evaluated at selected genes that localized in genomic loci displaying replication timing delay and downregulated in Rev3l-/- MEFs (FoxG1, GATA6). Histograms represent enrichment of H3K9me3 and H3K27me3 assessed by ChIP-qPCR at indicated loci in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.


G . MEFs cells were transfected with GFP - REV3L761 - 1029 WT PSVVL or ASAVA mutant and fixed with 4 % formaldehyde . The distribution of GFP - REV3L constructs was detected by autofluorescence ( green ) , chromocenters were visualized by HP1α immunostaining ( red ) and DNA was counterstained with DAPI . Scale bar = 10

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G. MEFs cells were transfected with GFP-REV3L761-1029 WT PSVVL or ASAVA mutant and fixed with 4% formaldehyde. The distribution of GFP-REV3L constructs was detected by autofluorescence (green), chromocenters were visualized by HP1α immunostaining (red) and DNA was counterstained with DAPI. Scale bar= 10 μm


H . Human 293T cells were transfected with either FH - REV3L700 - 900 , mutant V802D F - H - REV3L700 - 900 or empty vector . Forty - eight hours after transfection cell lysates were made and used for immunoprecipitation with HP1 antibody . Western blot was processed and membranes were immunoblotted with the indicated antibodies .

H. Human 293T cells were transfected with either FH-REV3L700-900, mutant V802D F-H-REV3L700- 900 or empty vector. Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with HP1 antibody. Western blot was processed and membranes were immunoblotted with the indicated antibodies.


I . 293T cells were co - transfected with F - H - REV3L700 - 900 or empty vector and V5 - HP1γ , mutant IY / EE V5 - HP1γ or empty vector . Forty - eight hours after transfection cell lysates were made and used for immunoprecipitation with α - V5 antibody . After electrophoresis , samples were immunoblotted with anti - HA , anti - HP1γ or anti - α - Tubulin as indicated .

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I. 293T cells were co-transfected with F-H-REV3L700-900 or empty vector and V5-HP1γ, mutant IY/EE V5-HP1γ or empty vector. Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with α-V5 antibody. After electrophoresis, samples were immunoblotted with anti-HA, anti-HP1γ or anti-α-Tubulin as indicated.


B . Asynchronous Rev3l + / + and Rev3l - / - MEFs were fixed with PFA then subjected to in situ proximity ligation assay ( PLA ) using 53BP1 and HP1α antibodies then PLA foci were counted in both cell lines ( more than 150 nuclei for each condition were counted ) . p - values were calculated by Mann - Whitney test ( * * * * : p < 0 . 0001 ) . Red lines indicate the mean values . Error bars : SEM . Controls with a single antibody are also shown . Experiments were repeated three times . Representative images are shown . Scale bar = 5 μm .

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B. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were fixed with PFA then subjected to in situ proximity ligation assay (PLA) using 53BP1 and HP1α antibodies then PLA foci were counted in both cell lines (more than 150 nuclei for each condition were counted). p-values were calculated by Mann-Whitney test ( ****: p<0.0001). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Experiments were repeated three times. Representative images are shown. Scale bar= 5 μm.


C . Asynchronous Rev3l + / + and Rev3l - / - MEFs were subjected to PLA as by using γH2AX and HP1β antibodies and processed as in ( B ) . p - values were calculated by Mann - Whitney test ( * : p < 0 . 05 ) . Red lines indicate the mean values . Error bars : SEM . Controls with a single antibody are also shown . Three independent experiments were performed .

C. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were subjected to PLA as by using γH2AX and HP1β antibodies and processed as in (B). p-values were calculated by Mann-Whitney test ( *: p<0.05). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Three independent experiments were performed.


Rev3l + / + and Rev3l - / - MEFs were incubated with or without 0 . 23 μM aphidicolin for 24h before metaphase spreading . FISH was performed using major satellite probe to quantify breaks in pericentromeric regions ( F ) . Representative images of chromosomes showing abnormalities ( see arrows ) in pericentromeric regions from Rev3l - / - MEFs . Chromosomes were labelled with DAPI Error bars indicate standard error of the mean for three independent experiments . Mann - Whitney test ( ns : not significant , * p < 0 . 05 ; * * * p < 0 . 001 ) .

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

Rev3l+/+ and Rev3l-/- MEFs were incubated with or without 0.23 μM aphidicolin for 24h before metaphase spreading. FISH was performed using major satellite probe to quantify breaks in pericentromeric regions (F). Representative images of chromosomes showing abnormalities (see arrows) in pericentromeric regions from Rev3l-/- MEFs. Chromosomes were labelled with DAPI Error bars indicate standard error of the mean for three independent experiments. Mann-Whitney test (ns: not significant, * p<0.05; *** p< 0.001).


A . Asynchronous Rev3l + / + and Rev3l - / - MEFs were UV - irradiated at the indicated doses and harvested 6 h or 24 h later . Cell lysates were analyzed by immunoblotting with indicated antibodies . B . Asynchronous Rev3l - / - MEFs complemented with empty vector ( EV ) or REV3L were UV - irradiated at the indicated doses and harvested 6 h or 24 h later . Cell lysates were analyzed by immunoblotting with indicated antibodies . C

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A. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. B. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. C


Asynchronous Rev3l + / + and Rev3l - / - MEFs were pulse labelled for 15 min using 10 μM EdU then UV - irradiated at 4 J / m2 . Cells pre - treated with CSK buffer were fixed and 53BP1 , RPA and RAD51 were detected by immunofluorescence . Representative images in non - irradiated cells ( NI ) and quantification of 53BP1 foci in EdU positive cells at different time points are shown Scale bar = 10 μm The number of cells analyzed is indicated in ( n ) on each graph . * * * p < 0 . 001 ( Kruskal - Wallis test ) . The presented data are representative of 2 repeats ) . Bars represent the median ± interquartile

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Asynchronous Rev3l+/+ and Rev3l-/- MEFs were pulse labelled for 15 min using 10 μM EdU then UV-irradiated at 4 J/m2. Cells pre-treated with CSK buffer were fixed and 53BP1, RPA and RAD51 were detected by immunofluorescence. Representative images in non-irradiated cells (NI) and quantification of 53BP1 foci in EdU positive cells at different time points are shown Scale bar= 10 μm The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile range


F - H . Asynchronous Rev3l - / - MEFs complemented with empty vector ( EV ) or REV3L were processed as in ( C ) . Quantification of 53BP1 foci in EdU positive cells at different time points is shown ( F ) . Quantification of the intensity of chromatin - bound RPA2 ( G ) and RAD51 foci ( H ) 24 hours after UV - irradiation was performed . The number of cells analyzed is indicated in ( n ) on each graph . * * * p < 0 . 001 ( Kruskal - Wallis test ) . The presented data are representative of 2 repeats . Bars represent the median ± interquartile range .

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F-H. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were processed as in (C). Quantification of 53BP1 foci in EdU positive cells at different time points is shown (F). Quantification of the intensity of chromatin-bound RPA2 (G) and RAD51 foci (H) 24 hours after UV-irradiation was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats. Bars represent the median ± interquartile range.


Agar plugs containing actively growing cultures of wild type S . sclerotiorum ( strain 1980 ) and the OA deficient A2 mutant were inoculated onto Col - 0 and ced - 9 expressing Arabidopsis leaves . ( A ) Wild type inoculations onto Col - 0 plants resulted in typical lesions for this pathogen including a rapid , spreading cell death ; however , infection was completely suppressed in ced - 9 expressing plants . The expression of this gene had no effect on the A2 phenotype .

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Agar plugs containing actively growing cultures of wild type S. sclerotiorum (strain 1980) and the OA deficient A2 mutant were inoculated onto Col-0 and ced-9 expressing Arabidopsis leaves. (A) Wild type inoculations onto Col-0 plants resulted in typical lesions for this pathogen including a rapid, spreading cell death; however, infection was completely suppressed in ced-9 expressing plants. The expression of this gene had no effect on the A2 phenotype.


( B ) Trypan blue staining indicates the extent of cell death for each genotype / strain combination , including an agar plug control . All images were recorded 48 hours post inoculation .

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(B) Trypan blue staining indicates the extent of cell death for each genotype/strain combination, including an agar plug control. All images were recorded 48 hours post inoculation.


Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation . S . sclerotiorum A2 ( A ) and wild type ( B ) strains were inoculated onto tomato leaves using colonized agar plugs . 24 hours post inoculation ; leaves were post - fixed in osmium tetroxide , and embedded in Spurr ' s epoxy resin . A microtome was used to cut 400 nm sections . Toluidine blue stain was used to reveal fungal hyphae . H = hyphae . The dotted line represents the leading edge of the visible lesion . Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software , version 2 . 2 . 1 . 227 .

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Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.S. sclerotiorum A2 (A) and wild type (B) strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were post-fixed in osmium tetroxide, and embedded in Spurr's epoxy resin. A microtome was used to cut 400 nm sections. Toluidine blue stain was used to reveal fungal hyphae. H = hyphae. The dotted line represents the leading edge of the visible lesion. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227.


S . sclerotiorum A2 strain induces autophagic structures in plants . S . sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs . 24 hours post inoculation ; leaves were stained with 100 µM final concentration of MDC ( Sigma ) in PBS for 30 min . Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope ( Olympus systems , Germany ) , with an excitation wavelength of 335 nm and an emission wavelength of 508 nm . Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software , version 2 . 2 . 1 . 227 . Scale bar = 10 µm .

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S. sclerotiorum A2 strain induces autophagic structures in plants.S. sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were stained with 100 µM final concentration of MDC (Sigma) in PBS for 30 min. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope (Olympus systems, Germany), with an excitation wavelength of 335 nm and an emission wavelength of 508 nm. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227. Scale bar = 10 µm.


Transmission Electron Microscopy ( TEM ) fungal inoculated tomato leaves . Representative TEM images from four independent experiments . ( A , G ) ; Healthy non - inoculated leaf tissue . ( B - F ) Tomato leaves inoculated with the OA deficient A2 strain . ( H , I ) Tomato leaves inoculated with wild type S . sclerotiorum . Arrows , autolysosomal / autophagosomal - like structures ; C , chloroplast ; V , vacuole ; N , nucleus ; Circle , active dismantlement of chloroplast ; Rectangle , chromatin condensation within the nucleus . Black scale bars = 2 µm , white scale bars = 1 µm . Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV . Digital images were recorded with a MegaViewIII digital camera operated with iTEM software .

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Transmission Electron Microscopy (TEM) fungal inoculated tomato leaves.Representative TEM images from four independent experiments. (A, G); Healthy non-inoculated leaf tissue. (B-F) Tomato leaves inoculated with the OA deficient A2 strain. (H,I) Tomato leaves inoculated with wild type S. sclerotiorum. Arrows, autolysosomal/autophagosomal-like structures; C, chloroplast; V, vacuole; N, nucleus; Circle, active dismantlement of chloroplast; Rectangle, chromatin condensation within the nucleus. Black scale bars = 2 µm, white scale bars = 1 µm. Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were recorded with a MegaViewIII digital camera operated with iTEM software.


( A , B ) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col - 0 and select Arabidopsis autophagy mutant plants . These mutants showed enhanced susceptibility to the normally non - pathogenic A2 strain . Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation .

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(A,B) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col-0 and select Arabidopsis autophagy mutant plants. These mutants showed enhanced susceptibility to the normally non-pathogenic A2 strain. Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation.


( C ) Tomato leaves were either pre - infiltrated with water ( control ) or autophagy inhibitors Wortmannin , LY294002 , Chloroquine ( CQ ) , and 3 - methyladenine ( 3 - MA ) . Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection . ( B ) 48 hours post inoculation ; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol ( 1 : 3 , v / v ) . Images were taken 48 hours post inoculation .

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(C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.


( A ) NBT treated Arabidopsis ( Col - 0 and two independent atg8a mutant lines ) following agar plug inoculation with the A2 mutant . Images were collected 48 hours post inoculation . Dotted lines represent the edge of the observable legion .

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(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion.


( B ) RT - PCR was used to evaluate the transcript levels of three catalases ( CAT1 , 2 , and 3 ) and three superoxide dismutases in Col - 0 plants following inoculation with wild type S . sclerotiorum ( black bars ) and the A2 mutant strain ( grey bars ) . * > 2 fold change , * * > 5 fold change .

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(B) RT-PCR was used to evaluate the transcript levels of three catalases (CAT1, 2, and 3) and three superoxide dismutases in Col-0 plants following inoculation with wild type S. sclerotiorum (black bars) and the A2 mutant strain (grey bars). *>2 fold change, **>5 fold change.


( C ) TNFemARE / ARE mice show significantly elevated serum levels of TNF ( wild - type n = 5 , TNFemARE / + n = 5 , TNFemARE / ARE n = 5 ) . Data information : data are represented as Mean + / - SEM , n = biological replicates . one - way ANOVA test was used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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(C) TNFemARE/ARE mice show significantly elevated serum levels of TNF (wild-type n=5, TNFemARE/+ n=5, TNFemARE/ARE n=5). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( D ) Histologic H & E sections of ileum and hind paw indicate severe ileitis and arthritis in 20 - 30 w / o SPF TNFemARE / ARE mice ( Scale bars upper panel : 100 μm , scale bars lower panel : 500 μm ) .

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(D) Histologic H&E sections of ileum and hind paw indicate severe ileitis and arthritis in 20-30 w/o SPF TNFemARE/ARE mice (Scale bars upper panel: 100 μm, scale bars lower panel: 500 μm).


( G ) tSNE analysis performed on CD45 + lineage ( excluding CD3 + , CD19 + , NK1 . 1 + fractions ) of lamina propria of ileum samples ( n = 5 / genotype ) .

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(G) tSNE analysis performed on CD45+ lineage (excluding CD3+, CD19+, NK1.1+ fractions) of lamina propria of ileum samples (n=5/genotype).


( H ) Ileal flow cytometry data of 25 w / o TNFemARE mice show a highly activated innate immune system ( n = 5 / genotype ) . Data information : data are represented as Mean + / - SEM , n = biological replicates . one - way ANOVA test was used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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(H) Ileal flow cytometry data of 25 w/o TNFemARE mice show a highly activated innate immune system (n=5/genotype). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( J ) Ileal flow cytometry data show an increase of CD4 + cells in transgenic mice , which can be designated to the enrichment of the CD4 + RORgt + ( Th17 ) cell population ( n = 5 / genotype ) . Data information : data are represented as Mean + / - SEM , n = biological replicates . one - way ANOVA test was used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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(J) Ileal flow cytometry data show an increase of CD4+ cells in transgenic mice, which can be designated to the enrichment of the CD4+RORgt+ (Th17) cell population (n=5/genotype). Data information: data are represented as Mean +/- SEM, n=biological replicates. one-way ANOVA test was used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( C ) Serum levels of TNF are elevated in hetero - and homozygous transgenic mice , in both SPF and GF conditions ( n = 5 mice / group , except for GF wild - type n = 4 ) . Data information : data are represented as Mean + / - SEM , n = biological replicates , two - way ANOVA test used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

(C) Serum levels of TNF are elevated in hetero- and homozygous transgenic mice, in both SPF and GF conditions (n=5 mice/group, except for GF wild-type n=4). Data information: data are represented as Mean +/- SEM, n=biological replicates, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( D ) Quantitative real - time PCR reveals increased expression of the tnf gene in the ileum in homozygous SPF and GF mice ( SPF wild - type n = 10 , SPF TNFemARE / + n = 10 , SPF TNFemARE / ARE n = 13 ; GF wild - type n = 3 , GF TNFemARE / + n = 4 , GF TNFemARE / ARE n = 4 ) . Data information : data are represented as Mean + / - SEM , n = biological replicates , two - way ANOVA test used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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(D) Quantitative real-time PCR reveals increased expression of the tnf gene in the ileum in homozygous SPF and GF mice (SPF wild-type n=10, SPF TNFemARE/+ n=10, SPF TNFemARE/ARE n=13; GF wild-type n=3, GF TNFemARE/+ n=4, GF TNFemARE/ARE n=4). Data information: data are represented as Mean +/- SEM, n=biological replicates, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( F ) Comparing SPF versus GF H & E sections of the ileum shows complete rescue of gut inflammation under axenic conditions in 20 - 30 w / o TNFemARE / ARE mice ( Scale bars : 100 μm ) .

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(F) Comparing SPF versus GF H&E sections of the ileum shows complete rescue of gut inflammation under axenic conditions in 20-30 w/o TNFemARE/ARE mice (Scale bars: 100 μm).


( C ) 20 - 30 w / o SPF TNFemARE / ARE mice clearly suffer from inflammation in the synovio - entheseal complex , which is not rescued in age - matched GF mice . ( Scale bars : 200 μm )

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(C) 20-30 w/o SPF TNFemARE/ARE mice clearly suffer from inflammation in the synovio-entheseal complex, which is not rescued in age-matched GF mice. (Scale bars: 200 μm)


( F ) < em > μ < / em > CT images of tails of wild - type versus SPF and GF TNFemARE / ARE mouse display fusion of sacral vertebrae in homozygous SPF and GF mice , as indicated by yellow arrows .

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(F) μCT images of tails of wild-type versus SPF and GF TNFemARE/ARE mouse display fusion of sacral vertebrae in homozygous SPF and GF mice, as indicated by yellow arrows.


( B ) Gait analysis of TNFemARE mice reveals distinct moving patterns of SPF and GF TNFemARE / ARE mice . Homozygous mice tend to have smaller footprints , place their feet with lower mean intensity and generally have a smaller stride length . ( SPF wild - type n = 12 , SPF TNFemARE / + n = 12 , SPF TNFemARE / ARE n = 12 ; GF wild - type n = 8 , GF TNFemARE / + n = 20 , GF TNFemARE / ARE n = 12 ) Data information : For panel ( B ) , boxplots represent the 25 - and 75 - percentile with median values as central band , whiskers span min to max value range , n = biological replicates with every datapoint representing 1 paw , two - way ANOVA test used with Tukey ' s multiple comparisons test , ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

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(B) Gait analysis of TNFemARE mice reveals distinct moving patterns of SPF and GF TNFemARE/ARE mice. Homozygous mice tend to have smaller footprints, place their feet with lower mean intensity and generally have a smaller stride length. (SPF wild-type n=12, SPF TNFemARE/+ n=12, SPF TNFemARE/ARE n=12; GF wild-type n=8, GF TNFemARE/+ n=20, GF TNFemARE/ARE n=12) Data information: For panel (B), boxplots represent the 25- and 75- percentile with median values as central band, whiskers span min to max value range, n=biological replicates with every datapoint representing 1 paw, two-way ANOVA test used with Tukey's multiple comparisons test, ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( C ) H & E sections of the calcaneo - cuboid joint of 5 - 15 w / o A20myel - KO mice indicate immune cell infiltrates in the synovium and the fat pad and severe bone marrow edema . ( Scale bars : 100 μm )

(C) H&E sections of the calcaneo-cuboid joint of 5-15 w/o A20myel-KO mice indicate immune cell infiltrates in the synovium and the fat pad and severe bone marrow edema. (Scale bars: 100 μm)


( E ) Histologic images of hind paws of old ( 44 w / o ) GF wild - type ( left ) versus A20myel - KO ( right ) mice . The morphology of the paw is completely lost in the transgenic mouse due to severe inflammation . ( Scale bars : 200 μm )

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(E) Histologic images of hind paws of old (44 w/o) GF wild-type (left) versus A20myel-KO (right) mice. The morphology of the paw is completely lost in the transgenic mouse due to severe inflammation. (Scale bars: 200 μm)


( F ) Splenomegaly in GF A20myel - KO mice of 15 w / o indicates systemic inflammation in this mouse model ( wild - type n = 7 , A20myel - KO n = 8 ) . Data information : For panels F ) , data are represented as Mean + / - SEM , n = biological replicates . For graph ( F ) , two - tailed unpaired t - test was used . ns = p - value > 0 . 05 , * = p - value ≤ 0 . 05 , * * = p - value ≤ 0 . 01 , * * * = p - value ≤ 0 . 001 , * * * * = p - value ≤ 0 . 0001 .

(F) Splenomegaly in GF A20myel-KO mice of 15 w/o indicates systemic inflammation in this mouse model (wild-type n=7, A20myel-KO n=8). Data information: For panels F), data are represented as Mean +/- SEM, n=biological replicates. For graph (F), two-tailed unpaired t-test was used. ns= p-value > 0.05, * = p-value ≤ 0.05, ** = p-value ≤ 0.01, *** = p-value ≤ 0.001, **** = p-value ≤ 0.0001.


( D , E ) representative confocal micrographs and quantification of LC3 - II accumulation in CTNSWT and CTNS - / - cells in presence and absence of 25 nM bafilomycin ( BafA1 ) for 4 hrs , respectively ( n = 3 ) . Scale bars are 20 µm .

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(D, E) representative confocal micrographs and quantification of LC3-II accumulation in CTNSWT and CTNS-/- cells in presence and absence of 25 nM bafilomycin (BafA1) for 4 hrs, respectively (n = 3). Scale bars are 20 µm.


( F , G , H ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio and SQSTM1 protein levels in CTNSWT , CTNS - / - , and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h , respectively ( n = 3 ) .

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(F, G, H) Western blotting and densitometric analyses for LC3-II/LC3-I ratio and SQSTM1 protein levels in CTNSWT, CTNS-/-, and CTNSPatient cells cultured in the presence or in the absence of 25 nM BafA1 for 4 h, respectively (n = 3).


( I , J ) Representative confocal micrographs and quantification of DQ - BSA and BSA in CTNSWT , CTNS - / - , and CTNSPatient cells , respectively ( n = 7 - 14 quantified images ) . Scale bars are 20 µm .

(I, J) Representative confocal micrographs and quantification of DQ-BSA and BSA in CTNSWT, CTNS-/-, and CTNSPatient cells, respectively (n = 7-14 quantified images). Scale bars are 20 µm.


( D ) List of metabolites that were shared and significantly altered in both the CTNS - / - and CTNSPatient cells compared to control cells ( n = 6 ) .

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(D) List of metabolites that were shared and significantly altered in both the CTNS-/- and CTNSPatient cells compared to control cells (n = 6).


( I ) List of proteins that were significantly upregulated and downregulated in CTNS - / - cells compared to control cells ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , LIPA ; Lysosomal acid lipase , ACP2 ; Lysosomal acid phosphatase , CTSS ; Cathepsin S , CTSC ; Dipeptidyl peptidase , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , SORT1 ; Sortilin , CAT ; Catalase , CTSA ; Lysosomal protective protein , and SOD ; Superoxide dismutase .

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(I) List of proteins that were significantly upregulated and downregulated in CTNS-/- cells compared to control cells (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, LIPA; Lysosomal acid lipase, ACP2; Lysosomal acid phosphatase, CTSS; Cathepsin S, CTSC; Dipeptidyl peptidase, IGF2R; Cation-independent mannose-6-phosphate receptor, SORT1; Sortilin, CAT; Catalase, CTSA; Lysosomal protective protein, and SOD; Superoxide dismutase.


( B , C , D ) ROS production ( ROS / mg protein ) in CTNSWT , CTNS ‑ / - , and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG ( 4 hrs ) , respectively ( n = 3 ) .

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(B, C, D) ROS production (ROS/mg protein) in CTNSWT, CTNS‑/-, and CTNSPatient cells upon starvation for 4 hrs in the presence and absence of DMKG (4 hrs), respectively (n = 3).