A Western blot analysis showing dynamic expression patterns of FBXW7 and c - MYC during the indicated time points of EB induction . α - TUBULIN was used as a loading control .

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A Western blot analysis showing dynamic expression patterns of FBXW7 and c-MYC during the indicated time points of EB induction. α-TUBULIN was used as a loading control.


B Western blot analysis showing FBXW7 and c - MYC expression in control , Mettl5 KO ( KO - 6B ) and KO + HA - MT5WT Day7 EBs , respectively . α - TUBULIN was used as a loading control .

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B Western blot analysis showing FBXW7 and c-MYC expression in control, Mettl5 KO (KO-6B) and KO+HA-MT5WT Day7 EBs, respectively. α-TUBULIN was used as a loading control.


D RT - qPCR analysis of the expression of pluripotency and lineage - specific markers in Day7 FBXW7 - induced EBs at the presence or absence of 10 ng / ml Dox . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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D RT-qPCR analysis of the expression of pluripotency and lineage-specific markers in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.


E Immunostaining analysis of the expression of pluripotency marker POU5F1 , lineage - specific markers FOXA2 , T and NESTIN in Day7 FBXW7 - induced EBs at the presence or absence of 10 ng / ml Dox . Scale bars represent 100 μm .

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E Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 FBXW7-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.


G RT - qPCR analysis the expression of pluripotency and lineage - specific markers in Day7 c - MYC - induced EBs at the presence or absence of 10 ng / ml Dox . Data are represented as mean ± SD from three biological replicates . Student ' s t test , two - tailed . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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G RT-qPCR analysis the expression of pluripotency and lineage-specific markers in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Data are represented as mean ± SD from three biological replicates. Student's t test, two-tailed. *, P < 0.05; **, P < 0.01; ***, P<0.001.


H Immunostaining analysis of the expression of pluripotency marker POU5F1 , lineage - specific markers FOXA2 , T and NESTIN in Day7 c - MYC - induced EBs at the presence or absence of 10 ng / ml Dox . Scale bars represent 100 μm .

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H Immunostaining analysis of the expression of pluripotency marker POU5F1, lineage-specific markers FOXA2, T and NESTIN in Day7 c-MYC-induced EBs at the presence or absence of 10 ng/ml Dox. Scale bars represent 100 μm.


C , Validation of the AR and TRIM33 interaction by co - immunoprecipitation assays . Endogenous AR protein in LNCaP cells ( left ) or VCaP cells ( right ) were immunoprecipitated and detected for TRIM33 by immunoblotting . IgG served as a negative control .

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C, Validation of the AR and TRIM33 interaction by co-immunoprecipitation assays. Endogenous AR protein in LNCaP cells (left) or VCaP cells (right) were immunoprecipitated and detected for TRIM33 by immunoblotting. IgG served as a negative control.


D , In vitro GST pull - down assays were performed with bacterially expressed and purified GST or GST - tagged AR immobilized onto GSH beads . Immobilized GST protein beads were used to pull down purified his - MBP ( left ) or his - MBP - tagged TRIM33 ( right ) .

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

D, In vitro GST pull-down assays were performed with bacterially expressed and purified GST or GST-tagged AR immobilized onto GSH beads. Immobilized GST protein beads were used to pull down purified his-MBP (left) or his-MBP-tagged TRIM33 (right).


AR - dependent luciferase assays were performed on LNCaP cells transfected with siTRIM33 or negative control ( NC ) ( A ) , increasing amounts of TRIM33 plasmid ( B ) Western blot analysis shows the protein level of TRIM33 in LNCaP cells after TRIM33 knockdown ( A ) or overexpression ( B ) . Data information : , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 ; * * P < 0 . 01 ; ns = not significant ( two - way ANOVA ) .

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

AR-dependent luciferase assays were performed on LNCaP cells transfected with siTRIM33 or negative control (NC) (A), increasing amounts of TRIM33 plasmid (B) Western blot analysis shows the protein level of TRIM33 in LNCaP cells after TRIM33 knockdown (A) or overexpression (B). Data information: , data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05; **P < 0.01; ns=not significant (two-way ANOVA).


A - D , AR transcript levels were measured by RT - qPCR on LNCaP cells ( A and C ) or VCaP cells ( B and D ) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT . Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies . Data information : In ( A - D , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ( two - way ANOVA ) .

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

A-D, AR transcript levels were measured by RT-qPCR on LNCaP cells (A and C) or VCaP cells (B and D) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT. Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies. Data information: In (A-D, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).


E and F , LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide ( CHX ) . Protein lysates were harvested at the indicated time points for western blot analysis . AR protein levels were quantified and normalized to β - actin and then plotted relative to 0 h . Data information : In F , data are presented as mean ± SD ( n = 3 independent experiments ) . * P < 0 . 05 , * * P < 0 . 01 , ns = not significant ( two - way ANOVA ) .

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

E and F, LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide (CHX). Protein lysates were harvested at the indicated time points for western blot analysis. AR protein levels were quantified and normalized to β-actin and then plotted relative to 0 h. Data information: In F, data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).


G , LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h . Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies .

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

G, LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h. Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies.


HA - Ub was expressed in LNCaP cells co - transfected with siTRIM33 ( H ) The cells were treated with MG132 for 4 h before harvesting . Endogenous AR was immunoprecipitated from cell lysates , and interacting proteins were detected by immunoblotting with the indicated antibodies .

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HA-Ub was expressed in LNCaP cells co-transfected with siTRIM33 (H) The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.


I , HA - Ub was expressed in LNCaP cells co - transfected with the TRIM33 expression construct ( I ) . The cells were treated with MG132 for 4 h before harvesting . Endogenous AR was immunoprecipitated from cell lysates , and interacting proteins were detected by immunoblotting with the indicated antibodies .

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

I, HA-Ub was expressed in LNCaP cells co-transfected with the TRIM33 expression construct (I). The cells were treated with MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.


LNCaP cells transiently expressing myc - tagged Skp2 with Flag - TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies .

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LNCaP cells transiently expressing myc-tagged Skp2 with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.


K , LNCaP cells transiently expressing myc - tagged SPOP with Flag - TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies .

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K, LNCaP cells transiently expressing myc-tagged SPOP with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.


L , shTRIM33 - or shCtrl - LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies .

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L, shTRIM33- or shCtrl-LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies.


M , HEK293T cells were co - transfected with flag - TRIM33 and various myc - tagged AR constructs . AR - FL and AR truncated mutants were immunoprecipitated with an anti - myc antibody and then subjected to western blot analysis with the indicated antibodies .

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M, HEK293T cells were co-transfected with flag-TRIM33 and various myc-tagged AR constructs. AR-FL and AR truncated mutants were immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.


N , HEK293T cells were co - transfected with myc - AR - N and various flag - tagged TRIM33 constructs . AR - N was immunoprecipitated with an anti - myc antibody and then subjected to western blot analysis with the indicated antibodies .

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N, HEK293T cells were co-transfected with myc-AR-N and various flag-tagged TRIM33 constructs. AR-N was immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.


O , LNCaP cells were transfected with various flag - tagged TRIM33 constructs . AR - associated proteins were immunoprecipitated with an anti - AR antibody and then subjected to western blot analysis with the indicated antibodies .

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O, LNCaP cells were transfected with various flag-tagged TRIM33 constructs. AR-associated proteins were immunoprecipitated with an anti-AR antibody and then subjected to western blot analysis with the indicated antibodies.


P , LNCaP cells were co - transfected with myc - Skp2 and various flag - TRIM33 constructs . AR and other proteins were detected by western blot analysis with the indicated antibodies .

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P, LNCaP cells were co-transfected with myc-Skp2 and various flag-TRIM33 constructs. AR and other proteins were detected by western blot analysis with the indicated antibodies.


Q , AR pull - down was performed on protein extracts from MG132 - treated HEK293T cells overexpressing myc - AR , HA - Ub , myc - Skp2 , and various flag - tagged TRIM33 constructs . AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies .

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

Q, AR pull-down was performed on protein extracts from MG132-treated HEK293T cells overexpressing myc-AR, HA-Ub, myc-Skp2, and various flag-tagged TRIM33 constructs. AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies.


A , TRIM33 mRNA expression levels were analyzed in normal and tumor samples from multiple publicly available patient cohorts . A two - sided Wilcoxon signed - rank test was used for the TCGA Prostate , Ding 2016 , and Jing 2020 datasets , while a two - sided Wilcoxon rank - sum test was used for the other datasets . Measurements shown in box plots are the first and third quartiles and are split by the medians , whiskers extending a 1 . 5 - fold interquartile range beyond the box .

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A, TRIM33 mRNA expression levels were analyzed in normal and tumor samples from multiple publicly available patient cohorts. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.


B and C , A human PCa progression tissue microarray was used for immunohistochemistry staining . Representative staining images are shown in B . TRIM33 staining by Gleason Scores is summarized in the histogram . Scale bar : 100 μm .

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B and C, A human PCa progression tissue microarray was used for immunohistochemistry staining. Representative staining images are shown in B. TRIM33 staining by Gleason Scores is summarized in the histogram. Scale bar: 100 μm.


A , The AR - TRIM33 coactivated gene signature was compared with two PCa patient - derived AR gene signatures in multiple publicly available patient datasets .

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A, The AR-TRIM33 coactivated gene signature was compared with two PCa patient-derived AR gene signatures in multiple publicly available patient datasets.


B , The expression of the gene signature in normal and tumor samples was examined in the same cohorts used in A . A two - sided Wilcoxon signed - rank test was used for the TCGA Prostate , Ding 2016 , and Jing 2020 datasets , while a two - sided Wilcoxon rank - sum test was used for the other datasets . Measurements shown in box plots are the first and third quartiles and are split by the medians , whiskers extending a 1 . 5 - fold interquartile range beyond the box .

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B, The expression of the gene signature in normal and tumor samples was examined in the same cohorts used in A. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.


C , Hierarchical clustering of PCa patients from the TCGA Prostate dataset ( n = 480 ) resulted in two distinct clusters indicated in orange ( high ) and blue ( low ) based on the average expression of our 36 - gene signature .

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C, Hierarchical clustering of PCa patients from the TCGA Prostate dataset (n = 480) resulted in two distinct clusters indicated in orange (high) and blue (low) based on the average expression of our 36-gene signature.


E , Kaplan - Meier analysis showing the recurrence - free survival of patients with low and high expression of the 36 - gene signature ( p - value , log - rank test ) .

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E, Kaplan-Meier analysis showing the recurrence-free survival of patients with low and high expression of the 36-gene signature (p-value, log-rank test).


J , Western blot analysis of AR and TRIM3 protein levels in tumor tissues from H .

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

J, Western blot analysis of AR and TRIM3 protein levels in tumor tissues from H.


( A and B ) FACS - sorted T cell populations and Monocyte - derived Macrophages were treated with the indicated stimuli : Nigericin ( Nig . 4h ) , NeedleTox ( 4h ) , Val - boroPro ( VbP 22h ) , ABT737 / S63845 ( A / S 22h ) . When indicated , cells were primed with LPS for 2 hours prior to stimulation . When indicated , MCC950 was added to the media 30 minutes prior to the addition of Nigericin . LDH - activity ( A ) and IL - 1β and IL - 18 concentration ( B and C , respectively ) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; * * p ≤ 0 . 01 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

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

(A and B) FACS-sorted T cell populations and Monocyte-derived Macrophages were treated with the indicated stimuli: Nigericin (Nig. 4h), NeedleTox (4h), Val-boroPro (VbP 22h), ABT737/S63845 (A/S 22h). When indicated, cells were primed with LPS for 2 hours prior to stimulation. When indicated, MCC950 was added to the media 30 minutes prior to the addition of Nigericin. LDH-activity (A) and IL-1β and IL-18 concentration (B and C, respectively) in the supernatant were determined by LDH cytotoxicity assay and ELISA respectively. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


A ) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI - uptake were monitored by live - cell imaging microscopy using a 25x objective . Representative images from indicated time points are shown . Cyan color coding is used for the fluorescent PI - signal . One donor out of two is shown . ( B ) Representative images were acquired with a 63x objective at the end of the experiment shown in ( A ) at 16 hours . One donor out of two is shown . Scale bars : 25 µm .

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

A) Macrophages and CD4 T cells from the same donor were subjected to the indicated treatments and morphologic changes as well as PI-uptake were monitored by live-cell imaging microscopy using a 25x objective. Representative images from indicated time points are shown. Cyan color coding is used for the fluorescent PI-signal. One donor out of two is shown. (B) Representative images were acquired with a 63x objective at the end of the experiment shown in (A) at 16 hours. One donor out of two is shown. Scale bars: 25 µm.


( C ) CD4 T cells were treated with VbP or ABT737 / S63845 . Lysate and supernatant samples were collected at the indicated time points . Samples were analyzed by immunoblotting . αSS and αLS indicate the use of a small subunit or large subunit - specific caspase - 1 antibody respectively . ( Lys = lysate , SN = supernatant , FL = full length ) . One representative experiment out of three is shown .

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

(C) CD4 T cells were treated with VbP or ABT737/S63845. Lysate and supernatant samples were collected at the indicated time points. Samples were analyzed by immunoblotting. αSS and αLS indicate the use of a small subunit or large subunit-specific caspase-1 antibody respectively. (Lys = lysate, SN = supernatant, FL = full length). One representative experiment out of three is shown.


( A ) Gene expression profile of 15 inflammasome - related genes in human T cell and monocyte subsets . TPM values were retrieved from a public RNASeq dataset and log2 transformed . Both , rows and columns are clustered using euclidean distance and complete linkage . ( Tfh , T follicular helper cells ; Tregs , T regulatory cells ; Th , T helper cells ; CM , central memory T cells ; EM , effector memory T cells ; TE , terminal effector T cells ; CD14 + C16 - , classical monocytes ; CD14 + CD16 + , intermediate monocytes ; CD14 - CD16 + , non - classical monocytes ) .

(A) Gene expression profile of 15 inflammasome-related genes in human T cell and monocyte subsets. TPM values were retrieved from a public RNASeq dataset and log2 transformed. Both, rows and columns are clustered using euclidean distance and complete linkage. (Tfh, T follicular helper cells; Tregs, T regulatory cells; Th, T helper cells; CM, central memory T cells; EM, effector memory T cells; TE, terminal effector T cells; CD14+ C16-, classical monocytes; CD14+ CD16+, intermediate monocytes; CD14- CD16+, non-classical monocytes).


( B ) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours . * indicates an unspecific band . One representative experiment out of three is shown .

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(B) Immunoblotting of CD4 T cells and MDMs treated with Pam3CSK4 for 6 hours. * indicates an unspecific band. One representative experiment out of three is shown.


( C ) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene . One representative experiment out of three is shown .

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(C) Immunoblotting of polyclonal CD4 T cells targeted with two gRNAs for each indicated target gene. One representative experiment out of three is shown.


( D ) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours . Individual data points ± SEM from three independent donors are shown . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Dunnett ) .

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(D) CD4 T cells from the indicated polyclonal gene targeting approach were treated with the indicated stimuli for 18 hours. Individual data points ± SEM from three independent donors are shown. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Dunnett).


( B ) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing . DPP9 and DPP8 were targeted with one gRNA / target gene , CARD8 was targeted with two gRNAs . On the day of nucleofection , T cells were activated with human T activator CD3 / CD28 beads and expanded in the presence of IL - 2 . On day 10 , cells were switched to IL - 7 / IL - 15 containing medium . ( C ) Fraction of wildtype and in - frame reads are depicted . One representative experiment out of three is shown .

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(B) MiSeq analysis of the indicated gene targeting approaches from samples collected at the indicated time points post editing. DPP9 and DPP8 were targeted with one gRNA/target gene, CARD8 was targeted with two gRNAs. On the day of nucleofection, T cells were activated with human T activator CD3/CD28 beads and expanded in the presence of IL-2. On day 10, cells were switched to IL-7/IL-15 containing medium. (C) Fraction of wildtype and in-frame reads are depicted. One representative experiment out of three is shown.


( A ) CD4 T cells were activated with human T activator CD3 / CD28 beads ( bead : cell ratio 1 : 5 ) for 4 days . Controls were kept resting in the presence of IL - 7 and IL - 15 . On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH - activity in the supernatant . Individual data points ± SEM from four independent donors . Statistics indicate significance by two - way ANOVA : * * * p ≤ 0 . 001 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) .

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

(A) CD4 T cells were activated with human T activator CD3/CD28 beads (bead:cell ratio 1:5) for 4 days. Controls were kept resting in the presence of IL-7 and IL-15. On day 4 cells were stimulated as indicated for 18 hours and cytotoxicity was assessed by LDH-activity in the supernatant. Individual data points ± SEM from four independent donors. Statistics indicate significance by two-way ANOVA: ***p ≤ 0.001; ns, not significant. P-values were corrected for multiple comparisons (Sidak).


( B ) Immunoblotting of CD4 T cell lysates treated as in ( A ) for 3 to 4 days . * indicates an unspecific band . ( C ) Quantification of immunoblots shown in ( B ) and additional donors ( n = 9 for all but GSDMD , GSDMD n = 8 , for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution ) . Individual data points ± SEM are shown . Statistics indicate significance by one - way ANOVA of log2 - transformed fold change values of band intensities ( activated / non - activated T cells ) normalized to β - Actin : * * * p ≤ 0 . 001 ; * p ≤ 0 . 05 ; ns , not significant . P - values were corrected for multiple comparisons ( Sidak ) . All band intensity ratios were tested against β - Actin . CARD8 full length was additionally tested against CARD8 cleaved .

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

(B) Immunoblotting of CD4 T cell lysates treated as in (A) for 3 to 4 days. * indicates an unspecific band. (C) Quantification of immunoblots shown in (B) and additional donors (n= 9 for all but GSDMD, GSDMD n=8, for one donor quantification of the GSDMD band was considered unreliable due to uneven substrate distribution). Individual data points ± SEM are shown. Statistics indicate significance by one-way ANOVA of log2-transformed fold change values of band intensities (activated/non-activated T cells) normalized to β-Actin: ***p ≤ 0.001; *p ≤ 0.05; ns, not significant. P-values were corrected for multiple comparisons (Sidak). All band intensity ratios were tested against β-Actin. CARD8 full length was additionally tested against CARD8 cleaved.


A , B Data were normalised using the control group and are represented as fold - change ( FC ) of the mean fluorescence intensity ( MFI ) . Macrophage cells were obtained from human donors ( n = 4 biological replicas ) and incubated with medium ( CON ) or recombinant human IL - 10 ( hIL - 10r ) , MPN wild - type ( WT ) or MPN expressing IL - 10 ORF ( WT _ IL10 ) . Data are represented as mean ± Standard Error of the Mean ( SEM ) . Statistical analysis was performed using one - way ANOVA + post - hoc Tukey multiple comparison test ( * P < 0 . 05 ; * * P < 0 . 001 , * * * P < 0 . 0001 ) .

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

A, B Data were normalised using the control group and are represented as fold-change (FC) of the mean fluorescence intensity (MFI). Macrophage cells were obtained from human donors (n = 4 biological replicas) and incubated with medium (CON) or recombinant human IL-10 (hIL-10r), MPN wild-type (WT) or MPN expressing IL-10 ORF (WT_IL10). Data are represented as mean ± Standard Error of the Mean (SEM). Statistical analysis was performed using one-way ANOVA + post-hoc Tukey multiple comparison test (*P < 0.05; **P < 0.001, ***P < 0.0001).


( A ) EC - 50 ( molar , M ) for human IL - 10 recombinant protein ( hIL - 10r ) and IL - 10 WT ( IL - 10 ORF ) and different variants ( MutM , hIL - 10r , Mut1 , Mut2 , Mut3 , MutSC1 , and MutSC2 ) expressed by M . pneumoniae . Data are represented as mean ± SD . Statistical comparison was done by One - way ANOVA + post - hoc Bonferroni multiple comparison test , using the IL - 10 ORF condition as a reference ( * P < 0 . 05 ) .

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

(A) EC-50 (molar, M) for human IL-10 recombinant protein (hIL-10r) and IL-10 WT (IL-10 ORF) and different variants (MutM, hIL-10r, Mut1, Mut2, Mut3, MutSC1, and MutSC2) expressed by M. pneumoniae. Data are represented as mean ± SD. Statistical comparison was done by One-way ANOVA + post-hoc Bonferroni multiple comparison test, using the IL-10 ORF condition as a reference (*P < 0.05).


( B ) Fold - change ( FC ) in expression levels ( fg / CFU ) of IL - 10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL - 10 ORF ( ORF ) . Statistical comparison of mean ± SD was performed by One - way ANOVA + Tukey post - hoc test ( * P < 0 . 05 ) .

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(B) Fold-change (FC) in expression levels (fg/CFU) of IL-10 variants Mut3 and MutSC1 secreted to the medium normalised by expression level of IL-10 ORF (ORF). Statistical comparison of mean ± SD was performed by One-way ANOVA + Tukey post-hoc test (*P < 0.05).


( C , D ) Average and SD values for the FC in the relative EC - 50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20 - min exposure of the BlaER1 ( C ) or HAFTL ( D ) cell lines to IL - 10 ORF ( reference ) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD .

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

(C, D) Average and SD values for the FC in the relative EC-50 values determined by flow cytometry analysis of phosphorylated STAT3 after a 20-min exposure of the BlaER1 (C) or HAFTL (D) cell lines to IL-10 ORF (reference) or the mutant MutSC1 or MutSC2 Numbers indicate the average FC ± SD.


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( A ) Average ± SD bacterial loads recovered of the WT ( circle ) and CV8 ( square ) strains in mouse lungs at 2 - dpi or 4 - dpi . Data are shown as log10 CFU / lung homogenate .

O O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-ORGANISM O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (A) Average ± SD bacterial loads recovered of the WT (circle) and CV8 (square) strains in mouse lungs at 2-dpi or 4-dpi. Data are shown as log10 CFU/ lung homogenate.


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( B ) Inflammatory profile of mouse lungs inoculated with WT ( dark blue ) , CV8 ( light blue ) , or PBS ( grey ) . Gene expression was analysed by RT - qPCR using gapdh as an endogenous control ( see Materials and Methods ) . Data are shown as average ± SD of fold - change ( FC ) in mRNA expression ( One - way ANOVA + Tukey post - hoc test ; * P < 0 . 05 ) .

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

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (B) Inflammatory profile of mouse lungs inoculated with WT (dark blue), CV8 (light blue), or PBS (grey). Gene expression was analysed by RT-qPCR using gapdh as an endogenous control (see Materials and Methods). Data are shown as average ± SD of fold-change (FC) in mRNA expression (One-way ANOVA + Tukey post-hoc test; *P < 0.05).


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post - infection ( dpi ) or 4 - dpi ( n ≥ 3 biological replicas / group ) . ( C ) Histological findings of mouse lung samples at the analysed time points . Left , plots representing the quantitative evaluation ( score : 0 - 5 ) of alveolar infiltrate ( top panel ) and peribronquial / olar infiltrate ( FC ) ( bottom panel ) ( see Materials and Methods ) . Parameters were normalised using the average of the PBS group ( FC = sample value / average PBS ; FC control group , ~ 1 ) . Data are shown as average ± SD of FC ( one - way ANOVA + Tukey post - hoc test ) ( * P < 0 . 05 ) . Right , representative images of lungs stained with hematoxylin - eosin ( the line represents 250 μm ) .

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 B-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE 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 B-SMALL_MOLECULE 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 B-TISSUE B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and sacrificed at 2 days post-infection (dpi) or 4-dpi (n ≥ 3 biological replicas/group). (C) Histological findings of mouse lung samples at the analysed time points. Left, plots representing the quantitative evaluation (score: 0-5) of alveolar infiltrate (top panel) and peribronquial/olar infiltrate (FC) (bottom panel) (see Materials and Methods). Parameters were normalised using the average of the PBS group (FC = sample value/average PBS; FC control group, ~1). Data are shown as average ± SD of FC (one-way ANOVA + Tukey post-hoc test) (*P < 0.05). Right, representative images of lungs stained with hematoxylin-eosin (the line represents 250 μm).


C57Bl / 6 were infected with 107 CFUs of WT or CV8 strains and ( D ) IL - 10 secretion by the MPN WT or CV8 strain coding for hIL - 10 WT ( ORF ) , MutSC1 , or MutSC2 . Data are expressed as the mean ± SD of IL - 10 supernatant concentration ( μg / mL ) by biomass ( protein content in μg / mL ) ( n = 3 biological replicas ) .

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C57Bl/6 were infected with 107 CFUs of WT or CV8 strains and (D) IL-10 secretion by the MPN WT or CV8 strain coding for hIL-10 WT (ORF), MutSC1, or MutSC2. Data are expressed as the mean ± SD of IL-10 supernatant concentration (μg/mL) by biomass (protein content in μg/mL) (n=3 biological replicas).


( B ) CFUs of PAO1 ( left ) or Mycoplasma strains ( right ) recovered from lung samples . Data are shown as mean ± SD of Log10 CFU / lung homogenate . Two replicates were performed and individual symbols are shown ( experiment 1 , circle ; experiment 2 , square ) .

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(B) CFUs of PAO1 (left) or Mycoplasma strains (right) recovered from lung samples. Data are shown as mean ± SD of Log10 CFU/lung homogenate. Two replicates were performed and individual symbols are shown (experiment 1, circle; experiment 2, square).


( C ) Fold - change ( FC ) of mRNA gene expression of inflammatory markers determined by qPCR . The 2 - ΔΔCt method was used to normalise the values , using gapdh as endogenous control Data are shown as average ± SD of FC . Statistical analyses were performed using unpaired t - test ( * P < 0 . 05 ; * * P < 0 . 01 * * * ; P < 0 . 001 , * * * * P < 0 . 0001 ) . Two replicates were performed and individual symbols are shown ( Experiment 1 , circle ; Experiment 2 , square ) .

O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY 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 O O O O O O O O O O O O O O O O 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) Fold-change (FC) of mRNA gene expression of inflammatory markers determined by qPCR. The 2-ΔΔCt method was used to normalise the values, using gapdh as endogenous control Data are shown as average ± SD of FC. Statistical analyses were performed using unpaired t-test (* P < 0.05; ** P <0.01***; P <0.001, **** P <0.0001). Two replicates were performed and individual symbols are shown (Experiment 1, circle; Experiment 2, square).


( D ) Quantitative analysis of immunochemistry ( IHC ) against neutrophil elastase ( NE ) of lung samples . Data are represented as the mean ± SD of percentage of positive cells , calculated as follows : % = 100 × ( positive cells / positive cells + negative cells ) ( One - Way ANOVA + Tukey post - hoc test , * P < 0 . 05 ) . ( E ) Representative images of IHC staining of lung samples against NE are shown . Arrows indicate NE - positive cells . Scale bar size : 50 µm .

O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-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 O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O

(D) Quantitative analysis of immunochemistry (IHC) against neutrophil elastase (NE) of lung samples. Data are represented as the mean ± SD of percentage of positive cells, calculated as follows: % = 100 × (positive cells/positive cells + negative cells) (One-Way ANOVA + Tukey post-hoc test, * P <0.05). (E) Representative images of IHC staining of lung samples against NE are shown. Arrows indicate NE-positive cells. Scale bar size: 50 µm.


( a ) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT - α in HCT116 cells .

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(a) Ultrastructural evidence of autophagy induced by depletion of p53 with a specific siRNA or pharmacological inhibition of p53 with PFT-α in HCT116 cells.


( b ) Autophagy induced by PFT - α , p53 knockdown , or p53 knockout in HCT116 cells . The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments ( mean ± s . e . m . ; * P 0 . 05 ) . Culture in nutrient - free ( NF ) conditions was used as a positive control .

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(b) Autophagy induced by PFT-α, p53 knockdown, or p53 knockout in HCT116 cells. The number of autophagosomes and autolysosomes was determined for at least 50 cells in 3 independent experiments (mean ± s.e.m.; *P 0.05). Culture in nutrient-free (NF) conditions was used as a positive control.


( c ) Effect of p53 on the maturation of LC3 . Immunoblots are shown for wild - type ( WT ) or p53 − / − HCT116 ( n = 3 ) .

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(c) Effect of p53 on the maturation of LC3. Immunoblots are shown for wild-type (WT) or p53−/− HCT116 (n = 3).


( d ) GFP - LC3 puncta induced by PFT - α , p53 knockdown or knockout . WT and p53 − / − HCT116 cells were transfected with control or p53 - specific siRNAs , re - transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT - α . The percentage of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) is reported ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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

(d) GFP-LC3 puncta induced by PFT-α, p53 knockdown or knockout. WT and p53−/− HCT116 cells were transfected with control or p53-specific siRNAs, re-transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h and kept for 6 h in the presence or absence of 30 μM PFT-α. The percentage of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac) is reported (mean ± s.d., n = 3; *P 0.05).


( e ) GFP - LC3 puncta in p53 − / − MEF , compared with WT MEF transfected with GFP - LC3 ( mean ± s . d . , n = 3 ; * P 0 . 05 ) .

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(e) GFP-LC3 puncta in p53−/− MEF, compared with WT MEF transfected with GFP-LC3 (mean ± s.d., n = 3; *P 0.05).


( f ) GFP - LC3 dots in tissues from p53 + / + or p53 − / − mice expressing a GFP - LC3 transgene ( mean ± s . d . ; * P 0 . 05 ) , without or after 24 h of starvation . The number of GFP - LC3 dots per area was determined for a minimum of 4 fields ( at × 400 magnification for 4 slides per animal , 3 animals per condition ) .

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(f) GFP-LC3 dots in tissues from p53+/+ or p53−/− mice expressing a GFP-LC3 transgene (mean ± s.d.; *P 0.05), without or after 24 h of starvation. The number of GFP-LC3 dots per area was determined for a minimum of 4 fields (at ×400 magnification for 4 slides per animal, 3 animals per condition).


( g ) TEM pictures of p53 − / − and p53 + / − mouse livers . p53 − / − hepatocytes carry approximately 3 autophagosomes , p53 + / − controls approximately 1 per cell .

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(g) TEM pictures of p53−/− and p53+/− mouse livers. p53−/− hepatocytes carry approximately 3 autophagosomes, p53+/− controls approximately 1 per cell.


( h ) Modulation of DsRed : : LGG - 1 levels by the p53 orthologue CEP - 1 in C . elegans . Representative pictures are shown for WT animals and for nematodes carrying a cep - 1 deletion allele ( gk138 ) . The count of puncta per cell was 6 . 9 ± 3 . 3 for WT , 52 . 2 ± 9 . 4 for cep - 1 ( gk138 ) embryos ( mean ± s . e . m . for 20 individuals , 5 cells per embryo ) . The quantification of DsRed : : LGG - 1 puncta is shown for the indicated genetic backgrounds and conditions ( dots represent pixel intensity for individual embryos ) .

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(h) Modulation of DsRed::LGG-1 levels by the p53 orthologue CEP-1 in C. elegans. Representative pictures are shown for WT animals and for nematodes carrying a cep-1 deletion allele (gk138). The count of puncta per cell was 6.9 ± 3.3 for WT, 52.2 ± 9.4 for cep-1 (gk138) embryos (mean ± s.e.m. for 20 individuals, 5 cells per embryo). The quantification of DsRed::LGG-1 puncta is shown for the indicated genetic backgrounds and conditions (dots represent pixel intensity for individual embryos).


( a ) PFT - α or p53 siRNA - induced GFP - LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5 , Atg10 , Atg12 , Beclin 1 or hVps34 . Culture in nutrient - free ( NF ) conditions was used as a control of autophagy induction .

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(a) PFT-α or p53 siRNA-induced GFP-LC3 puncta in WT HCT116 cells transfected with siRNAs to deplete the essential autophagy gene products Atg5, Atg10, Atg12, Beclin 1 or hVps34. Culture in nutrient-free (NF) conditions was used as a control of autophagy induction.


( b ) PFT - α or p53 siRNA - mediated GFP - LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1 .

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(b) PFT-α or p53 siRNA-mediated GFP-LC3 puncta in MEFs transfected with siRNAs specific for mouse Atg5 or Beclin 1.


( c ) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells . MCF7 cells stably transfected with a tetracycline - repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression , transfected with GFP - LC3 and subjected to p53 knockdown before quantification of GFP - LC3 puncta . Data in a - c are mean ± s . d . of 3 independent experiments .

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(c) Requirement of Beclin 1 expression for autophagy induction by p53 depletion in MCF7 cells. MCF7 cells stably transfected with a tetracycline-repressible Beclin 1 construct were maintained in conditions that prevent or allow Beclin 1 expression, transfected with GFP-LC3 and subjected to p53 knockdown before quantification of GFP-LC3 puncta. Data in a-c are mean ± s.d. of 3 independent experiments.


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - α and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

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(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-α and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( d - f ) Effect of bafilomycin A1 ( BafA1 ) on PFT - α - induced GFP - LC3 puncta . GFP - LC3 - expressing HeLa cells were treated with PFT - alpha and / or BafA1 and then immunostained for Lamp - 2 to observe the colocalization between GFP - LC3 and Lamp - 2 . Representative confocal microphotographs are shown together with the profiles of colocalization ( d ) within the area of interest , indicated by the orientation of the arrow . Percentage overlap was plotted for control cells and cells cultured in the presence of PFT - α and / or BafA1 after 6 h ( d , e ) . Columns represent the percentage of colocalization of GFP - LC3 and Lamp - 2 ( mean ± s . d . ; * P 0 . 05 ) , quantified for at least 50 cells for each condition . The kinetics of GFP - LC3 redistribution was quantified by conventional fluorescence microscopy ( mean ± s . d . , 3 experiments , f ) .

(d-f) Effect of bafilomycin A1 (BafA1) on PFT-α-induced GFP-LC3 puncta. GFP-LC3-expressing HeLa cells were treated with PFT-alpha and/or BafA1 and then immunostained for Lamp-2 to observe the colocalization between GFP-LC3 and Lamp-2. Representative confocal microphotographs are shown together with the profiles of colocalization (d) within the area of interest, indicated by the orientation of the arrow. Percentage overlap was plotted for control cells and cells cultured in the presence of PFT-α and/or BafA1 after 6 h (d, e). Columns represent the percentage of colocalization of GFP-LC3 and Lamp-2 (mean ± s.d.; *P 0.05), quantified for at least 50 cells for each condition. The kinetics of GFP-LC3 redistribution was quantified by conventional fluorescence microscopy (mean ± s.d., 3 experiments, f).


( g ) Immunoblot detection of LC3 - I / II in cells treated with PFT - α and / or leupeptin in WT HCT116 cells ( n = 3 ) .

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(g) Immunoblot detection of LC3-I/II in cells treated with PFT-α and/or leupeptin in WT HCT116 cells (n = 3).


( a ) Immunoblot detection of the phosphorylation status of AMPKα , ACCα , TSC2 and p70S6K in HCT116 cells . Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα , ACCα and TSC2 was detected in p53 − / − HCT116 , compared with WT HCT116 cells after culture in complete medium ( n = 5 ) .

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(a) Immunoblot detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in HCT116 cells. Hypophosphorylation of p70S6K and hyperphosphorylation of AMPKα, ACCα and TSC2 was detected in p53−/− HCT116, compared with WT HCT116 cells after culture in complete medium (n = 5).


( b ) Quantification of GFP - LC3vac cells in WT and p53 − / − HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK ( mean ± s . d . of 3 independent experiments ; * P 0 . 05 ) . The inset in b demonstrates the efficiency of siRNA - mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis ( n = 3 ) . Note that the antibody recognizes both AMPKα1 and AMPKα2 .

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(b) Quantification of GFP-LC3vac cells in WT and p53−/− HCT116 cells transfected with siRNAs specific for the catalytic α1 and α2 subunits of AMPK (mean ± s.d. of 3 independent experiments; *P 0.05). The inset in b demonstrates the efficiency of siRNA-mediated downregulation of AMPKα1 and AMPKα2 , as assessed by immunoblot analysis (n = 3). Note that the antibody recognizes both AMPKα1 and AMPKα2.


( c ) Immunoblot detection of ACCα phosphorylation in WT and p53 − / − HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 ( n = 5 ) ; Co , control .

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(c) Immunoblot detection of ACCα phosphorylation in WT and p53−/− HCT116 cells subjected to knockdown of AMPKα1 and AMPKα2 (n = 5); Co, control.


( d ) Quantification of GFP - LC3vac cells ( means ± s . d . , n = 3 separate experiments ) in WT HCT116 and p53 − / − HCT116 treated with PFT - α and / or rapamycin .

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(d) Quantification of GFP-LC3vac cells (means ± s.d., n = 3 separate experiments) in WT HCT116 and p53−/− HCT116 treated with PFT-α and/or rapamycin.


( a ) Cytosolic ATP levels in WT and p53 − / − HCT116 cells in the absence of glucose . Measurements were performed on luciferase - expressing HCT116 , after perfusion of the cells with medium first containing then missing glucose .

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(a) Cytosolic ATP levels in WT and p53−/− HCT116 cells in the absence of glucose. Measurements were performed on luciferase-expressing HCT116, after perfusion of the cells with medium first containing then missing glucose.


( b ) Effect of methylpyruvate ( MetPyr ) on ATP levels in WT p53 − / − HCT116 cells depleted of glucose . ATP levels were measured as in a , in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr .

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(b) Effect of methylpyruvate (MetPyr) on ATP levels in WT p53−/− HCT116 cells depleted of glucose. ATP levels were measured as in a, in the presence of glucose and 15 min after its withdrawal or replacement by MetPyr.


( c ) Effect of autophagy on the ATP levels of WT and p53 − / − HCT116 cells depleted of glucose . Cells were transfected with siRNAs specific for Beclin 1 and AMPKα , or they were treated with rapamycin for 6 h , followed by measurement of ATP levels as in a , before or after glucose withdrawal ( means ± s . d . of triplicates , 3 independent experiments ) .

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(c) Effect of autophagy on the ATP levels of WT and p53−/− HCT116 cells depleted of glucose. Cells were transfected with siRNAs specific for Beclin 1 and AMPKα, or they were treated with rapamycin for 6 h, followed by measurement of ATP levels as in a, before or after glucose withdrawal (means ± s.d. of triplicates, 3 independent experiments).


( d ) Metabolic stress - induced cell death is attenuated in the absence of p53 . HCT116 cells were transfected with control , Atg5 - , Atg10 - , or AMPKα - specific siRNAs and were subjected 48 h later to metabolic stress ( cultured for 48 h in nutrient - free , hypoxic conditions ) and stained with DiOC6 ( 3 ) and PI . The black and white portions of the columns refer to the DiOC6 ( 3 ) low PI − ( dying ) and DiOC6 ( 3 ) low PI + ( dead ) population , respectively .

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(d) Metabolic stress-induced cell death is attenuated in the absence of p53. HCT116 cells were transfected with control, Atg5-, Atg10-, or AMPKα-specific siRNAs and were subjected 48 h later to metabolic stress (cultured for 48 h in nutrient-free, hypoxic conditions) and stained with DiOC6(3) and PI. The black and white portions of the columns refer to the DiOC6(3)low PI− (dying) and DiOC6(3)low PI+(dead) population, respectively.


( e ) Metabolic stress - induced decrease of clonogenic survival was attenuated in the absence of p53 . HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival .

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(e) Metabolic stress-induced decrease of clonogenic survival was attenuated in the absence of p53. HCT116 cells subjected to metabolic stress as in d were monitored for clonogenic survival.


( f ) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion . MCF7 cells that carry a tetracycline - repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it , transfected with a control siRNA or a p53 - specific siRNA , subjected to metabolic stress , and finally stained with DiOC6 ( 3 ) / PI .

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(f) Expression of Beclin 1 in MCF7 cells restores the survival advantage conferred by p53 depletion. MCF7 cells that carry a tetracycline-repressible Beclin 1 expression construct were cultured to avoid Beclin 1 expression or to induce it, transfected with a control siRNA or a p53-specific siRNA, subjected to metabolic stress, and finally stained with DiOC6(3)/PI.


( g ) Effect of p53 and autophagy on the survival of metabolically stressed MEFs . WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays . Results in d - g are mean ± s . d . of 3 separate experiments ( * P 0 . 05 ) ; Co , control .

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(g) Effect of p53 and autophagy on the survival of metabolically stressed MEFs. WT MEFs were transfected with the indicated siRNAs and then subjected to metabolic stress before performing clonogenic assays. Results in d-g are mean ± s.d. of 3 separate experiments (*P 0.05); Co, control.


( a , b ) PFT - α - mediated stimulation of autophagy in cytoplasts . GFP - LC3 - transfected HeLa cells were enucleated by density - gradient centrifugation after cytochalasin B treatment and cultured on polylysine - coated coverslips . Mixtures of cells and cytoplasts ( arrows , identified by the lack of Hoechst 33342 staining ) were then exposed to three different autophagy inducers : PFT - α , nutrient starvation and rapamycin . Results are means ± s . d . n = 3 experiments ) .

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(a, b) PFT-α-mediated stimulation of autophagy in cytoplasts. GFP-LC3-transfected HeLa cells were enucleated by density-gradient centrifugation after cytochalasin B treatment and cultured on polylysine-coated coverslips. Mixtures of cells and cytoplasts (arrows, identified by the lack of Hoechst 33342 staining) were then exposed to three different autophagy inducers: PFT-α, nutrient starvation and rapamycin. Results are means ± s.d. n = 3 experiments).


( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 cells . Representative micrographs of cells transfected with plasmids expressing WT , nuclear and cytoplasmic p53 are shown in

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(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells.Representative micrographs of cells transfected with plasmids expressing WT, nuclear and cytoplasmic p53 are shown in d


( c - e ) Effect of p53 mutants on autophagy in p53 − / − HCT116 cells . Quantification of GFP - LC3 puncta of p53 − / − HCT116 cells transiently transfected with p53 mutants is shown in e ( mean ± s . d . , n = 3 experiments , * P 0 . 05 )

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(c-e) Effect of p53 mutants on autophagy in p53−/− HCT116 cells. Quantification of GFP-LC3 puncta of p53−/− HCT116 cells transiently transfected with p53 mutants is shown in e (mean ± s.d., n = 3 experiments, *P 0.05)


( f ) Detection of the phosphorylation status of AMPKα , ACCα , TSC2 and p70S6K in p53 − / − HCT116 cells transiently expressing WT or mutant p53 ( n = 5 ) .

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(f) Detection of the phosphorylation status of AMPKα, ACCα, TSC2 and p70S6K in p53−/− HCT116 cells transiently expressing WT or mutant p53 (n = 5).


p53 inhibition induces autophagy by ER stress ( a - c ) WT HCT116 cells were transiently transfected with GFP - LC3 , treated with PFT - α for 2 , 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP - LC3 + puncta with the ER marker ERp57 ( a ) or a mitochondrial marker Complex III ( b ) . Note that cells with GFP - LC3 + puncta often contain scarce amounts of ERp57 ( white arrows ) after prolonged incubation with PFT - α . The percentage of colocalization ( means ± s . d . , n = 3 experiments with 50 images per experiment ) was determined by confocal microscopy ( c ) .

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p53 inhibition induces autophagy by ER stress (a-c) WT HCT116 cells were transiently transfected with GFP-LC3, treated with PFT-α for 2, 4 or 6 h and subjected to immunofluorescence staining to observe the colocalization of GFP-LC3+ puncta with the ER marker ERp57 (a) or a mitochondrial marker Complex III (b). Note that cells with GFP-LC3+ puncta often contain scarce amounts of ERp57 (white arrows) after prolonged incubation with PFT-α. The percentage of colocalization (means ± s.d., n = 3 experiments with 50 images per experiment) was determined by confocal microscopy (c).


( d ) eIF2α phosphorylation in WT or p53 − / − HCT116 cells treated with PFT - α ( n = 5 ) .

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(d) eIF2α phosphorylation in WT or p53−/− HCT116 cells treated with PFT-α (n = 5).


( e , f ) WT HCT116 cells ( e , f ) or p53 − / − HCT116 cells ( f ) were transfected with GFP - LC3 , plus a control siRNA or the indicated combination of IRE1α - and / or p53 - specific siRNA , and treated with PFT - α , thapsigargin , tunicamycin or brefeldin A .

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(e, f) WT HCT116 cells (e, f) or p53−/− HCT116 cells (f) were transfected with GFP-LC3, plus a control siRNA or the indicated combination of IRE1α- and/or p53-specific siRNA, and treated with PFT-α, thapsigargin, tunicamycin or brefeldin A.


( g ) GFP - LC3 - transfected WT or ire1α − / − MEF were treated with PFT - α . Columns in e - g show means ± s . d . of 3 independent experiments ( * P 0 . 05 ) .

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(g) GFP-LC3-transfected WT or ire1α−/− MEF were treated with PFT-α. Columns in e-g show means ± s.d. of 3 independent experiments (*P 0.05).


Kinetics of p53 degradation ( a ) and induction of GFP - LC3 puncta ( b ) in WT HCT116 cells cultured in complete medium in the presence of rapamycin ( Rapa ) , tunicamycin ( Tn ) , lithium ( Li ) or in nutrient - free medium ( NF ) .

O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O

Kinetics of p53 degradation (a) and induction of GFP-LC3 puncta (b) in WT HCT116 cells cultured in complete medium in the presence of rapamycin (Rapa), tunicamycin (Tn), lithium (Li) or in nutrient-free medium (NF).


( c ) WT HCT116 cells transfected with control or HDM2 - targeted siRNAs were cultured in the presence or absence of MG132 for 3 h , followed by the treatment with autophagy inducers for 6 h ( Tp , thapsigargin ; other abbreviations as in b ) . The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP - LC3 in vacuoles ( GFP - LC3vac ) was determined .

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

(c) WT HCT116 cells transfected with control or HDM2-targeted siRNAs were cultured in the presence or absence of MG132 for 3 h, followed by the treatment with autophagy inducers for 6 h (Tp, thapsigargin; other abbreviations as in b). The abundance of p53 and HDM2 was determined by immunoblotting analysis and the percentage of cells showing accumulation of GFP-LC3 in vacuoles (GFP-LC3vac) was determined.


( d ) Effects of HDM2 inhibitors on p53 protein levels and autophagy . WT HCT116 were left untreated or treated with Nutlin - 3 , RITA and / or the indicated autophagy inducers , followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP - LC3 redistribution .

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

(d) Effects of HDM2 inhibitors on p53 protein levels and autophagy. WT HCT116 were left untreated or treated with Nutlin-3, RITA and/or the indicated autophagy inducers, followed by the p53 immunoblot or immunofluorescence microscopy to visualize GFP-LC3 redistribution.


( e ) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53 − / − HCT116 cells .

O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_LINE O O

(e) Effect of MG132 and HDM2 siRNA on autophagy of WT and p53−/− HCT116 cells.


( f ) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53 . Nutlin - 3 and RITA were added to p53 − / − HCT116 cells , followed by quantification of GFP - LC3 puncta .

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

(f) Failure of HDM2 inhibitors to prevent autophagy induced by the absence of p53. Nutlin-3 and RITA were added to p53−/− HCT116 cells, followed by quantification of GFP-LC3 puncta.


( g ) Effect of WT and ubiquitination - resistant p53 on autophagy . p53 − / − HCT116 cells were transfected with GFP - LC3 alone or in combination with different concentrations of pcDNA3 . 1 , WT p53 or p53 lacking the ubiquitination site at amino acids 13 - 19 ( p53ΔI ) , then treated with rapamycin , tunicamycin or thapsigargin . Thereafter , p53 expression and GFP - LC3 puncta were quantified .

O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD 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 B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O

(g) Effect of WT and ubiquitination-resistant p53 on autophagy. p53−/− HCT116 cells were transfected with GFP-LC3 alone or in combination with different concentrations of pcDNA3.1, WT p53 or p53 lacking the ubiquitination site at amino acids 13-19 (p53ΔI), then treated with rapamycin, tunicamycin or thapsigargin. Thereafter, p53 expression and GFP-LC3 puncta were quantified.


( h ) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors . In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1 , p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin , tunicamycin or thapsigargin .

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

(h) Depletion of Atg5 or Beclin 1 does not prevent p53 degradation triggered by rapamycin or ER stressors. In WT HCT116 cells transfected with siRNA targeting Atg5 or Beclin 1, p53 levels were determined by immunoblot analysis after 6 h of treatment with rapamycin, tunicamycin or thapsigargin.


B . RIBEYE mutations do not alter mouse survival . Data show surviving post - weaning mice ( at > P21 ) derived from matings between mice heterozygous for the RBEKI allele ( dark red ) or the RBEKO allele ( bright red ) normalized for the total number of mice ( absolute numbers per genotype / absolute number of all mice per respective breeding strategy ) The expected Mendelian ratio is indicated by the dashed line . Statistical analysis using the 2 - test revealed no statistical difference between wild - type and mutant mice ( RBEKI allele p = 0 . 33 ; RBEKO allele p = 0 . 11 ) .

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

B. RIBEYE mutations do not alter mouse survival. Data show surviving post-weaning mice (at >P21) derived from matings between mice heterozygous for the RBEKI allele (dark red) or the RBEKO allele (bright red) normalized for the total number of mice (absolute numbers per genotype/absolute number of all mice per respective breeding strategy) The expected Mendelian ratio is indicated by the dashed line. Statistical analysis using the 2-test revealed no statistical difference between wild-type and mutant mice (RBEKI allele p=0.33; RBEKO allele p=0.11).


C . Representative immunoblot of retina homogenates from homozygous RBEWT / WT , RBEKI / KI and RBEKO / KO mice probed with antibodies to CtBP2 ( to detect the B - domain of RIBEYE and CtBP2 ) and to EGFP ( to detect GCamP3 in the RIBEYE A - domain GCamP3 fusion protein referred to as RIB - G3 ) .

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

C. Representative immunoblot of retina homogenates from homozygous RBEWT/WT, RBEKI/KI and RBEKO/KO mice probed with antibodies to CtBP2 (to detect the B-domain of RIBEYE and CtBP2) and to EGFP (to detect GCamP3 in the RIBEYE A-domain GCamP3 fusion protein referred to as RIB-G3).


D . Measurements by RT - PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild - type mice ( RBEWT / WT ) , KI - mice expressing the RIBEYE - A - domain fusion protein with GCamP3 ( RBEKI / KI ) , and KO mice ( RBEKO / KO ) . These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina , and to examine whether the RIBEYE A - domain KO impairs CtBP2 expression in brain , which expresses little RIBEYE . Assays are based on a qRT - PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms ( RIBEYE and CtBP2 ) . Statistical analyses were performed using Student ' s t - test ; data are presented as mean ± SEM ( * * * , p < 0 . 001 ; n = 4 mice per genotype ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-TISSUE O O O O O O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD 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 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 O O O O O O O O B-ORGANISM O O O O

D. Measurements by RT-PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild-type mice (RBEWT/WT), KI-mice expressing the RIBEYE-A-domain fusion protein with GCamP3 (RBEKI/KI), and KO mice (RBEKO/KO). These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina, and to examine whether the RIBEYE A-domain KO impairs CtBP2 expression in brain, which expresses little RIBEYE. Assays are based on a qRT-PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms (RIBEYE and CtBP2). Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (***, p<0.001; n=4 mice per genotype).


E . Representative immunofluorescence labeling of cryostat sections from littermate wild - type and RBEKI / KI mice for EGFP ( green , to detect GCamP3 in RIB - G3 ) and SV2 ( red , to label synapses ) demonstrates expression of the A - domain / GCamP3 fusion protein in knockin mice ( OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , granule cell layer ) . Scale bar = 5 µm .

O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O

E. Representative immunofluorescence labeling of cryostat sections from littermate wild-type and RBEKI/KI mice for EGFP (green, to detect GCamP3 in RIB-G3) and SV2 (red, to label synapses) demonstrates expression of the A-domain/GCamP3 fusion protein in knockin mice (OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, granule cell layer). Scale bar = 5 µm.


F . Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT / WT and RBEKO / KO mice . Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies ; results are normalized to loading controls depending on molecular weights ( actin , fodrin , VCP , and GDI ) and to wild - type controls . Statistical analyses were performed using Student ' s t - test ; data are presented as mean ± SEM ( * , p < 0 . 05 ; n = 4 mice per genotype ) .

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

F. Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT/WT and RBEKO/KO mice. Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies; results are normalized to loading controls depending on molecular weights (actin, fodrin, VCP, and GDI) and to wild-type controls. Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (*, p<0.05; n=4 mice per genotype).


G . Immunofluoresence labeling for CtBP2 ( green , to label RIBEYE ) and mGluR6 ( red , to label photoreceptorsynapses ) in cryostat sections from littermate RBEWT / WT and RBEKO / KOmouseretinas demonstrates that the KO abolishes RIBEYE expression ( OPL , outer plexiform layer ) . Scale bars : 5 µm .

O O B-EXP_ASSAY O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O

G. Immunofluoresence labeling for CtBP2 (green, to label RIBEYE) and mGluR6 (red, to label photoreceptorsynapses) in cryostat sections from littermate RBEWT/WT and RBEKO/KOmouseretinas demonstrates that the KO abolishes RIBEYE expression (OPL, outer plexiform layer). Scale bars: 5 µm.


A . Overview of the synaptic organization of the retina in RBEWT / WT and RBEKO / KOmice . Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B - domain ( CtBP2 ; green ) and SV2 . Scale bar : 20 µm ; abbreviations : ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer .

O O O O O O O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O

A. Overview of the synaptic organization of the retina in RBEWT/WT and RBEKO/KOmice. Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B-domain (CtBP2; green) and SV2. Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


B . Double immunofluorescence staining of retinas from RBEWT / WT and RBEKO / KO mice for PSD95 ( green , to label postsynaptic specializations ) and mGluR6 ( red , to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses ) . Abbreviations are as in A . Scale bars : 20 µm ( left and right overview ) , 5 µm ( left panel magnification ) , 2 µm ( right panel magnification ) . C . Same as B , except that retinas were stained for PKCα ( green , to label bipolar neurons ) and PSD95 ( red , to label amacrine cells ) .

O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE 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 O O O O O O O O O O O B-TISSUE O O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O B-CELL_TYPE I-CELL_TYPE O O

B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice for PSD95 (green, to label postsynaptic specializations) and mGluR6 (red, to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses). Abbreviations are as in A. Scale bars: 20 µm (left and right overview), 5 µm (left panel magnification), 2 µm (right panel magnification).C. Same as B, except that retinas were stained for PKCα (green, to label bipolar neurons) and PSD95 (red, to label amacrine cells).


A and B . Double immunofluorescence staining of retinas from RBEWT / WT and RBEKO / KO mice , respectively , for PSD95 ( green , to label presynaptic photoreceptor terminals ) and Cav1 . 4 ( red , to label presynaptic Ca2 + - channel ) . Scale bar : 20 µm ; abbreviations : ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer . ( A1 to A4 ) Magnification of Cav1 . 4 staining in the outer plexiform layer ( OPL ) in the retina of RBEWT / WT and ( B1 to B4 ) RBEKO / KO mice . White arrow heads indicate Ca2 + - channel distribution at selected synapses . Images represent 2 µm z - stack projections . Scale bar : 2 µm .

O O O O O B-EXP_ASSAY O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O B-TISSUE O B-GENEPROD O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O

A and B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice, respectively, for PSD95 (green, to label presynaptic photoreceptor terminals) and Cav1.4 (red, to label presynaptic Ca2+-channel). Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (A1 to A4) Magnification of Cav1.4 staining in the outer plexiform layer (OPL) in the retina of RBEWT/WT and (B1 to B4) RBEKO/KO mice. White arrow heads indicate Ca2+-channel distribution at selected synapses. Images represent 2 µm z-stack projections. Scale bar: 2 µm.


A . Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in the terminals of RBEWT / WTmice . Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO / KOmice in which the synaptic ribbon is absent . Except for the absence of synaptic ribbons , the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT / WTmice . Abbreviations : sr , synaptic ribbon ; pre , presynaptic ; post , postsynaptic . Scale bars : 500 nm .

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

A. Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO/KOmice in which the synaptic ribbon is absent. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT/WTmice. Abbreviations: sr, synaptic ribbon; pre, presynaptic; post, postsynaptic. Scale bars: 500 nm.


B . Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in the terminals of RBEWT / WTmice . Except for the absence of synaptic ribbons , the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT / WTmice . Abbreviations are the same as in A . Scale bars : 1 µm ( B1 , B2 , B4 , B5 ) ; 300 nm ( B3 , B6 ) .

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

B. Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT/WTmice. Abbreviations are the same as in A. Scale bars: 1 µm (B1, B2, B4, B5); 300 nm (B3, B6).


A and B . Representative transmission EM pictures of RBEWT / WT ( A ) and RBEKO / KO ( B ) photoreceptor ribbon synapses ( blue rectangles , area adjacent to synaptic junction used for measuring junction - associated vesicle density [ size = 200 nm x 300 nm ] ; black squares , are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [ size = 200 nm x 200 nm ] ) . C . Average vesicle ratio ( expressed as vesicle numbers in the area adjacent to synaptic junctions ( blue rectangle ) or the cytosol ( black square ) normalized to RBEWT / WT . D . Average number of docked vesicles within a 150 nm range in the vicinity of the active zone .

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

A and B. Representative transmission EM pictures of RBEWT/WT (A) and RBEKO/KO (B) photoreceptor ribbon synapses (blue rectangles, area adjacent to synaptic junction used for measuring junction-associated vesicle density [size = 200 nm x 300 nm]; black squares, are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [size = 200 nm x 200 nm]).C. Average vesicle ratio (expressed as vesicle numbers in the area adjacent to synaptic junctions (blue rectangle) or the cytosol (black square) normalized to RBEWT/WT.D. Average number of docked vesicles within a 150 nm range in the vicinity of the active zone.


A . Experimental design and representative traces for analysis of synaptic transmission in retinas from wild - type ( black ) and RIBEYE KO mice ( red ) . Presynaptic bipolar neurons were depolarized in voltage - clamp mode from - 70 mV to - 10 mV for 0 . 5 s as indicated on top to open Ca2 + - channels and trigger release . Presynaptic Ca2 + - currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces ; dotted box displays an expansion of the initial Ca2 + - currents and EPSCs . B . RIBEYE KO does not alter presynaptic Ca2 + - currents . Summary graphs show average peak Ca2 + - currents and Ca2 + - current . C . RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization . Plot shows integrated EPSC charge as a function of time ; both initial and sustained neurotransmitter release are impaired . D . RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude . E . RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization . Summary graphs display integrated EPSC charges for the initial phase ( 0 - 50 ms ) and the sustained phase ( 50 - 500 ms ) . F . RIBEYE KO does not alter the kinetics of EPSCs . Summary graphs depict the mean synaptic delays ( left ) , rise times ( center ) and decay time constants ( right ) of EPSCs .

O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 O O O O O O O O O O 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 O

A. Experimental design and representative traces for analysis of synaptic transmission in retinas from wild-type (black) and RIBEYE KO mice (red). Presynaptic bipolar neurons were depolarized in voltage-clamp mode from -70 mV to -10 mV for 0.5 s as indicated on top to open Ca2+-channels and trigger release. Presynaptic Ca2+-currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces; dotted box displays an expansion of the initial Ca2+-currents and EPSCs.B. RIBEYE KO does not alter presynaptic Ca2+-currents. Summary graphs show average peak Ca2+-currents and Ca2+-current.C. RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization. Plot shows integrated EPSC charge as a function of time; both initial and sustained neurotransmitter release are impaired.D. RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude.E. RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization. Summary graphs display integrated EPSC charges for the initial phase (0-50 ms) and the sustained phase (50-500 ms).F. RIBEYE KO does not alter the kinetics of EPSCs. Summary graphs depict the mean synaptic delays (left), rise times (center) and decay time constants (right) of EPSCs.