( c , d ) Control siRNA - or p62 siRNA - silenced HeLa cells were transfected with EGFP - Parkin wild - type . Cells were treated with CCCP for the indicated times and stained with anti - TOM20 ( pseudo coloured ; blue ) to visualize mitochondrial morphology and anti - p62 ( red ) to monitor silencing efficiency . Parkin wild - type was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells with cleared mitochondria are surrounded by a line . (

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

(c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. (


( c - f ) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment ( c - e ) , but completely blocks final clearance after 24 h ( c , d , f ) . ( e , f ) Relative parkin translocation ( e ) and mitochondrial clearance ( f ) after p62 knockdown and CCCP treatment ( n = 4 ) . Data represent the mean ± s . d . , * * * P ≤ 0 . 0005 compared with control siRNA - transfected cells . Representative images of four independent experiments are shown . Scale bars , 10 μm . Immunostainings of control cells are shown in Supplementary Information , Fig . S5d , e .

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(c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e.


( g ) Western blot of siRNA - transfected HeLa cells used in c - f demonstrates the efficiency of p62 silencing . β - Actin probing served as a loading control . WT , wild - type .

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(g) Western blot of siRNA-transfected HeLa cells used in c-f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.


( a ) Time - course experiment of HeLa cells transfected with Flag - Parkin wild - type and treated with CCCP for the indicated periods . Total lysates were prepared , and western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 and anti - VDAC1 antibodies . β - Actin probing served as a loading control . All of the proteins detected are endogenously expressed , except Flag - Parkin .

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(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin.


( b ) Pathogenic Parkin mutants fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . HeLa cells were transfected with empty vector ( - ) , Flag - Parkin wild - type or pathogenic mutants along with 6 × His - tagged ubiquitin wild - type for 24 h . Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer . Denaturing conditions of Ni - NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis - tagged ubiquitin . Western blots of protein lysates and purified Ni - NTA agarose elutions were probed with anti - VDAC1 , anti - TOM20 and anti - Parkin antibodies . Modification of VDAC1 was observed in cells expressing Parkin wild - type and the functional mutant G328E , whereas non - functional mutants did not have this effect . (

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

(b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. (


( c ) In response to CCCP treatment , Parkin catalyses predominantly K27 - linked poly - ubiquitin chains on endogenous VDAC1 and is itself strongly poly - ubiquitylated . HeLa cells were transfected with an empty vector ( - ) or Flag - Parkin wild - type along with 6 × His - tagged ubiquitin variants for 24 h . Treatment of cells , preparation of lysates and Ni - NTA purification were performed as described in c and probed for Parkin , VDAC1 and β - actin . WT , wild - type ; Ub , ubiquitin .

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(c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.


( a ) Western blot analysis of parental SH - SY5Y cells treated with CCCP for the indicated times . Time - course experiments of CCCP - treated neuronal cells reveal increasing modifications of VDAC1 over time . Total lysates were prepared , and western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 and anti - VDAC1 antibodies . All proteins represent endogenous levels .

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 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

(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels.


( b ) Immunoprecipitations of Parkin and VDAC1 from parental SH - SY5Y cells confirm ubiquitylation of both proteins . CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time . Using specific antibodies , Parkin and VDAC1 were immunoprecipitated from lysates of parental SH - SY5Y cells at different time points , western blotted and probed with anti - ubiquitin , anti - Parkin and anti - VDAC1 antibodies .

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

(b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies.


( c ) Pathogenic Parkin mutations fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . Western blot analysis of a time - course experiment with inducible Parkin SH - SY5Y cells after CCCP treatment . Total lysates were prepared , and western blots were probed with specific anti - Parkin and anti - VDAC1 antibodies . β - Actin probing served as a loading control in a - c . WT , wild - type ; Ub , ubiquitin .

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 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

(c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.


VDAC1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , and mitochondrial clearance after 24 h , of CCCP treatment . Both effects can be rescued by re - transfection with wild - type VDAC1 . ( a ) Immunofluorescence and ( b ) western blot of siRNA - transfected HeLa cells used in c - g demonstrate partial silencing of endogenous VDAC1 . ( a ) Silenced cells were stained with anti - HtrA2 / Omi , a mitochondrial marker ( pseudo coloured ; blue ) , and anti - VDAC1 ( red ) antibodies . ( b ) Lysates from silenced cells were subjected to western blot analysis and probed with an anti - VDAC1 antibody , while TOM20 and β - actin probing served as loading controls .

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VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c-g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls.


( c - e ) Control siRNA - or VDAC1 siRNA - silenced HeLa cells were transfected with the indicated combinations of EGFP - Parkin wild - type , VDAC1 - Flag wild - type or appropriate empty vector controls . Cells were treated with CCCP for 2 h and 24 h and stained with anti - HtrA2 / Omi ( pseudo coloured ; blue ) and anti - Flag ( red ) to visualize mitochondrial morphology and re - transfected VDAC1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive cells are surrounded by a line . Representative pictures of three independent experiments are shown . Scale bars , 10 μm .

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(c-e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP-Parkin wild-type, VDAC1-Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm.


( f , g ) Quantification of d ( f ) and e ( g ) . Parkin wild - type colocalization with impaired mitochondria after 2 h ( f ) and uncleared mitochondria after 24 h ( g ) of CCCP treatment ( n = 3 ) . Data represent the mean ± s . d . , * P ≤ 0 . 05 compared with control siRNA - transfected or with VDAC1 - Flag wild - type - transfected cells .

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(f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1-Flag wild-type-transfected cells.


A - C qRT - PCR detection of mRNA levels of IL - 1β , TNFα and IL - 6 mRNA levels in CCDC50 - WT and CCDC50 - KO THP - 1 cells primed with LPS and then treated with ATP and Nigericin ( A ) , or infected with Listeria monocytogenes at a multiplicity of infection ( MOI ) of 10 and Salmonella typhimurium ( MOI of 10 ) for 6 h ( B ) or MSU ( 40 μg / ml ) and SiO2 ( 40 μg / ml ) for 6 h ( C ) ( n = 3 biological replicates ) . Data information : L . m , Listeria monocytogene ; S . t , Salmonella typhimurium ; MSU , Monosodium urate ; mRNA results are normalized to GAPDH and relative to untreated wild - type cells Data are representative of three biological replicates and shown as mean with SEM ( A - C ) ; * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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A-C qRT-PCR detection of mRNA levels of IL-1β, TNFα and IL-6 mRNA levels in CCDC50-WT and CCDC50-KO THP-1 cells primed with LPS and then treated with ATP and Nigericin (A), or infected with Listeria monocytogenes at a multiplicity of infection (MOI) of 10 and Salmonella typhimurium (MOI of 10) for 6 h (B) or MSU (40 μg/ml ) and SiO2 (40 μg/ml) for 6 h (C) (n = 3 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; mRNA results are normalized to GAPDH and relative to untreated wild-type cells Data are representative of three biological replicates and shown as mean with SEM (A-C); **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


D Enzyme - linked immunosorbent assays ( ELISA ) of IL - 1β production in supernatants of THP - 1 cells treated as in ( A - C ) ( n = 2 biological replicates ) . Data information : L . m , Listeria monocytogene ; S . t , Salmonella typhimurium ; MSU , Monosodium urate ; ; data are representative of two biological replicates ( D ) . * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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D Enzyme-linked immunosorbent assays (ELISA) of IL-1β production in supernatants of THP-1 cells treated as in (A-C) (n = 2 biological replicates). Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; ; data are representative of two biological replicates (D). **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


E - G CCDC50 - WT and CCDC50 - KO THP - 1 cells were stimulated as in ( A - C ) . The cells were collected for immunoblot analysis of pro - caspase - 1 , and the supernatants were subjected to cleaved caspase - 1 and IL - 1β analysis . The expression levels of cleaved - caspase - 1 and cleaved - IL - 1β were quantitated using ImageJ software . Data information : L . m , Listeria monocytogene ; S . t , Salmonella typhimurium ; MSU , Monosodium urate ; Casp1 , caspase - 1 ; actin was used as a loading control .

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E-G CCDC50-WT and CCDC50-KO THP-1 cells were stimulated as in (A-C). The cells were collected for immunoblot analysis of pro-caspase-1, and the supernatants were subjected to cleaved caspase-1 and IL-1β analysis. The expression levels of cleaved-caspase-1 and cleaved-IL-1β were quantitated using ImageJ software. Data information: L.m, Listeria monocytogene; S.t, Salmonella typhimurium; MSU, Monosodium urate; Casp1, caspase-1; actin was used as a loading control.


C qRT - PCR detection of Il6 , IL - 1β and Tnfα mRNA levels in Ccdc50 + / + and Ccdc50 - / - BMDMs primed with LPS for 3 h and then induced with ATP and Nigericin ( A ) , L . m and S . t ( B ) , or MSU and SiO2 ( C ) for indicated time points ( n = 3 biological replicates ) . D - F Ccdc50 + / + and Ccdc50 - / - BMDCs treated as BMDMs were collected and subjected to qRT - PCR analysis ( n = 3 biological replicates ) . Data information : mRNA data are normalized to GAPDH and relative to untreated wild - type cells ; Data are representative of three biological replicates and are shown as the mean with SEM * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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C qRT-PCR detection of Il6, IL-1β and Tnfα mRNA levels in Ccdc50+/+ and Ccdc50-/- BMDMs primed with LPS for 3 h and then induced with ATP and Nigericin (A), L.m and S.t (B), or MSU and SiO2 (C) for indicated time points (n = 3 biological replicates). D-F Ccdc50+/+ and Ccdc50-/- BMDCs treated as BMDMs were collected and subjected to qRT-PCR analysis (n = 3 biological replicates). Data information: mRNA data are normalized to GAPDH and relative to untreated wild-type cells; Data are representative of three biological replicates and are shown as the mean with SEM *P <0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


G ELISA analysis of the protein level of secreted IL - 1β in supernatants of Ccdc50 + / + and Ccdc50 - / - BMDMs ( top panel ) and BMDCs ( bottom panel ) treated as in ( A - C ) ( n = 3 biological replicates ) . Data information : Data are representative of three biological replicates and are shown as the mean with SEM * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; two - tailed unpaired Student ' s t - test .

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G ELISA analysis of the protein level of secreted IL-1β in supernatants of Ccdc50+/+ and Ccdc50-/- BMDMs (top panel) and BMDCs (bottom panel) treated as in (A-C) (n = 3 biological replicates). Data information: Data are representative of three biological replicates and are shown as the mean with SEM *P <0.05, **P < 0.01, ***P < 0.001; two-tailed unpaired Student's t-test.


H , I Ccdc50 + / + and Ccdc50 - / - BMDMs ( H ) and BMDCs ( I ) were treated as in ( A - C ) . The cells were lysed for western blotting analysis of pro - caspase - 1 and the supernatant was collected for immunoblots of cleaved - caspase - 1 . The expression levels of cleaved - caspase - 1 were quantitated using ImageJ software .

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H,I Ccdc50+/+ and Ccdc50-/- BMDMs (H) and BMDCs (I) were treated as in (A-C). The cells were lysed for western blotting analysis of pro-caspase-1 and the supernatant was collected for immunoblots of cleaved-caspase-1. The expression levels of cleaved-caspase-1 were quantitated using ImageJ software.


A , B Co - Immunoprecipitation ( Co - IP ) with anti - HA ( A ) or anti - Flag ( B ) was performed for the analysis of the association between HA - CCDC50 and Flag - AIM2 , - NLRP3 , and - ASC in cotransfected HEK293 cells . Data information : L . C , light - chain ; actin was used as a loading control . All the experiments were repeated at least once .

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A,B Co-Immunoprecipitation (Co-IP) with anti-HA (A) or anti-Flag (B) was performed for the analysis of the association between HA-CCDC50 and Flag-AIM2, -NLRP3, and -ASC in cotransfected HEK293 cells. Data information: L.C, light-chain; actin was used as a loading control. All the experiments were repeated at least once.


C Immunoprecipitation ( IP ) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP - 1 cells activated by LPS plus ATP . IgG was used as a negative control . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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C Immunoprecipitation (IP) analysis of the interaction of NLRP3 with endogenous CCDC50 and ASC in THP-1 cells activated by LPS plus ATP. IgG was used as a negative control. Data information: actin was used as a loading control. All the experiments were repeated at least once.


D Representative endogenous colocalization analysis of CCDC50 and NLRP3 in PMA - induced THP - 1 cells treated with LPS and ATP . Scale bar , 10 μm .

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D Representative endogenous colocalization analysis of CCDC50 and NLRP3 in PMA-induced THP-1 cells treated with LPS and ATP. Scale bar, 10 μm.


E Representative confocal microscopy image of HCT116 cells overexpressing GFP - CCDC50 and stimulated with LPS as well as ATP . Scale bar , 10 μm .

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E Representative confocal microscopy image of HCT116 cells overexpressing GFP-CCDC50 and stimulated with LPS as well as ATP. Scale bar, 10 μm.


F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag - AIM2 , - NLRP3 , - ASC and HA - CCDC50 or empty control vector . The expression levels of Flag - tagged proteins and Actin were quantitated using ImageJ software . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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F Immunoblot analysis of lysates from HEK293 cells cotransfected with Flag- AIM2, -NLRP3, -ASC and HA-CCDC50 or empty control vector. The expression levels of Flag-tagged proteins and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.


G Immunoblot analysis of HEK293 cells transfected with FLAG - NLRP3 and an increasing amount of HA - CCDC50 plasmids . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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G Immunoblot analysis of HEK293 cells transfected with FLAG-NLRP3 and an increasing amount of HA-CCDC50 plasmids. Data information: actin was used as a loading control. All the experiments were repeated at least once.


H Immunoblot analysis of lysates from CCDC50 - KO and CCDC50 - WT THP - 1 cells induced with LPS plus ATP and then treated with cycloheximide ( CHX ) for indicated time points . The expression levels of NLRP3 and Actin were quantitated using ImageJ software . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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H Immunoblot analysis of lysates from CCDC50-KO and CCDC50-WT THP-1 cells induced with LPS plus ATP and then treated with cycloheximide (CHX) for indicated time points. The expression levels of NLRP3 and Actin were quantitated using ImageJ software. Data information: actin was used as a loading control. All the experiments were repeated at least once.


I HEK293 cells were co - transfected with Flag - NLRP3 and HA - CCDC50 or empty control vector for 24 h and then treated with MG132 , NH4Cl , 3 - MA , CQ or DMSO for another 6 h . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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I HEK293 cells were co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h and then treated with MG132, NH4Cl, 3-MA, CQ or DMSO for another 6 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.


J Immunoblot analysis of NLRP3 in CCDC50 - WT and CCDC50 - KO THP - 1 cells stimulated with LPS in combination with ATP . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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J Immunoblot analysis of NLRP3 in CCDC50-WT and CCDC50-KO THP-1 cells stimulated with LPS in combination with ATP. Data information: actin was used as a loading control. All the experiments were repeated at least once.


K Western blotting analysis of ATG5 wild - type or knockout HEK293T cells co - transfected with Flag - NLRP3 and HA - CCDC50 or empty control vector for 24 h . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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K Western blotting analysis of ATG5 wild-type or knockout HEK293T cells co-transfected with Flag-NLRP3 and HA-CCDC50 or empty control vector for 24 h. Data information: actin was used as a loading control. All the experiments were repeated at least once.


L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs . Data information : actin was used as a loading control . All the experiments were repeated at least once .

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L Immunoblot analyses of NLRP3 in HEK293 cells in the presence of CCDC50 or its mutant CCDC50 with the deletion of MIUs. Data information: actin was used as a loading control. All the experiments were repeated at least once.


A Denature - IP ( with anti - Flag ) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag - NLRP3 , HA - Ub ( ubiquitin ) as well as HA - CCDC50 or empty vector for 24 h .

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A Denature-IP (with anti-Flag) and immunoblot analysis of polyubiquitinated NLRP3 in HEK293 cells transfected with Flag-NLRP3, HA-Ub (ubiquitin) as well as HA- CCDC50 or empty vector for 24 h.


B Denature - IP analysis ( with anti - Flag ) of K63 - linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h .

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B Denature-IP analysis (with anti-Flag) of K63-linked polyubiquitination of NLRP3 in cotransfected HEK293 cells left untreated or treated with CQ for 6 h.


C The colocalization analysis of K63 - Ub ( Red ) , Flag - NLRP3 ( Purple ) and GFP - CCDC50 in HeLa cells ; scale bars , 5 μM .

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C The colocalization analysis of K63-Ub (Red), Flag-NLRP3 (Purple) and GFP- CCDC50 in HeLa cells; scale bars, 5 μM.


D , E Images of HeLa cells transfected with CCDC50 ( red ) ( D ) or K63 - Ub ( red ) ( E ) and Flag - NLRP3 ( Purple ) and GFP - LC3B for 24 h and then induced with LPS plus ATP ; scale bars , 5 μM .

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D,E Images of HeLa cells transfected with CCDC50 (red) (D) or K63-Ub (red) (E) and Flag-NLRP3 (Purple) and GFP-LC3B for 24 h and then induced with LPS plus ATP; scale bars, 5 μM.


F The colocalization analysis of NLRP3 and LC3B in CCDC50 - WT and CCDC50 - KO HeLa cells stimulated with LPS plus ATP ; scale bars , 5 μM . Data information : The nuclei were stained with DAPI ; data are representative of three individual experiments .

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F The colocalization analysis of NLRP3 and LC3B in CCDC50-WT and CCDC50- KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.


G The oligomerization analysis of NLRP3 in CCDC50 wild - type and knockout THP - 1 cells stimulated with LPS as well as ATP . LPS - primed ( 1 μg / ml , 3 h ) THP - 1 cells were stimulated with ATP ( 5 mM ) for another 30 min and then were collected and lysed . The cell lysates were centrifuged and separated into soluble and insoluble fractions . The soluble fraction was analyzed by SDS - PAGE . The insoluble fraction was analyzed by native - PAGE .

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G The oligomerization analysis of NLRP3 in CCDC50 wild-type and knockout THP-1 cells stimulated with LPS as well as ATP. LPS-primed (1 μg/ml, 3 h) THP-1 cells were stimulated with ATP (5 mM) for another 30 min and then were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.


H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag - NLRP3 , HA - CCDC50 or empty vector for 24 h , and then treated with DMSO or CQ for 6 h . After treatment , the cells were collected and lysed . The cell lysates were centrifuged and separated into soluble and insoluble fractions . The soluble fraction was analyzed by SDS - PAGE . The insoluble fraction was analyzed by native - PAGE .

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H The oligomerization analysis of NLRP3 in HEK293 cells transfected with plasmids expressing Flag-NLRP3, HA-CCDC50 or empty vector for 24 h, and then treated with DMSO or CQ for 6 h. After treatment, the cells were collected and lysed. The cell lysates were centrifuged and separated into soluble and insoluble fractions. The soluble fraction was analyzed by SDS-PAGE. The insoluble fraction was analyzed by native-PAGE.


I Co - IP ( with anti - HA ) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h , and treated with or without CQ for 6 h .

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I Co-IP (with anti-HA) and immunoblot analysis of the interaction between NLRP3 and ASC in HEK293 cells transfected with indicated plasmids for 24 h, and treated with or without CQ for 6 h.


J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50 - WT and CCDC50 - KO HeLa cells stimulated with LPS plus ATP ; scale bars , 5 μM . Data information : The nuclei were stained with DAPI ; data are representative of three individual experiments .

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J Confocal microscopy analysis of colocalization between NLRP3 and ASC in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.


K Confocal microscopy analysis of ASC speck formation in CCDC50 - WT and CCDC50 - KO HeLa cells stimulated with LPS plus ATP ; scale bars , 5 μM . Data information : The nuclei were stained with DAPI ; data are representative of three individual experiments .

K Confocal microscopy analysis of ASC speck formation in CCDC50-WT and CCDC50-KO HeLa cells stimulated with LPS plus ATP; scale bars, 5 μM. Data information: The nuclei were stained with DAPI; data are representative of three individual experiments.


( A ) HeLa cells were incubated for 4 h in either EBSS or media without glucose / pyruvate . Lysates were then analysed by western blot for the presence of MCL ‐ 1 , as well as actin as a loading control .

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(A) HeLa cells were incubated for 4 h in either EBSS or media without glucose/pyruvate. Lysates were then analysed by western blot for the presence of MCL‐1, as well as actin as a loading control.


( B ) CGNs were subjected to 17 h hypoxia after 7 daysin vitro . Protein lysates were collected at indicated times after reoxygenation and blotted for MCL ‐ 1 expression and for actin as a loading control .

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(B) CGNs were subjected to 17 h hypoxia after 7 daysin vitro. Protein lysates were collected at indicated times after reoxygenation and blotted for MCL‐1 expression and for actin as a loading control.


( C ) H1299 cells stably expressing GFP - LC3 were incubated for 2 h in EBSS , and expression of various BCL ‐ 2 homologues was analysed by western blot . Alternatively , primary cortical neurons were incubated for 4 h in media without glucose / pyruvate .

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(C) H1299 cells stably expressing GFP-LC3 were incubated for 2 h in EBSS, and expression of various BCL‐2 homologues was analysed by western blot. Alternatively, primary cortical neurons were incubated for 4 h in media without glucose/pyruvate.


( D ) H1299 cells stably expressing GFP - LC3 were incubated for 3 h in media without glucose / pyruvate , followed by re ‐ addition of glucose and pyruvate for the indicated times .

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(D) H1299 cells stably expressing GFP-LC3 were incubated for 3 h in media without glucose/pyruvate, followed by re‐addition of glucose and pyruvate for the indicated times.


( E ) HeLa cells were infected with 5 p . f . u . per cell of either Ad HA ‐ tBID or Ad LacZ for 7 h , the media being changed to EBSS for the last 3 h where indicated . Alternatively , cells were treated with 50 μM camptothecin for 3 h .

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(E) HeLa cells were infected with 5 p.f.u. per cell of either Ad HA‐tBID or Ad LacZ for 7 h, the media being changed to EBSS for the last 3 h where indicated. Alternatively, cells were treated with 50 μM camptothecin for 3 h.


( F , G ) HeLa cells were treated as in E in the presence of 50 μM zVAD ‐ FMK and analysed by immunofluorescence microscopy for active BAX ( 6A7 epitope ; F ) or cyt c ( G ) . Data are expressed as the average of three experiments ± s . d . * P0 . 01 .

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(F, G) HeLa cells were treated as in E in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence microscopy for active BAX (6A7 epitope; F) or cyt c (G). Data are expressed as the average of three experiments±s.d. *P0.01.


( A ) H1299 cells stably expressing GFP - LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD ‐ FMK and analysed by immunofluorescence for cyt c or active BAX ( 6A7 epitope ) . As a positive control , cells were infected with 5 p . f . u . per cell Ad HA ‐ tBID for 7 h . Data are expressed as the average of three experiments ± s . d .

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(A) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD‐FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA‐tBID for 7 h. Data are expressed as the average of three experiments±s.d.


( B ) H1299 cells stably expressing GFP - LC3 were incubated for 4 h in either EBSS or media without glucose / pyruvate . Cells were then fixed , stained with an antibody against cyt c and analysed by immunofluorescence . Scale bars = 50 μm .

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(B) H1299 cells stably expressing GFP-LC3 were incubated for 4 h in either EBSS or media without glucose/pyruvate. Cells were then fixed, stained with an antibody against cyt c and analysed by immunofluorescence. Scale bars=50 μm.


( Alternatively ( C ) , the cells were analysed by western blot for the presence of MCL ‐ 1 and the autophagic markers GFP - LC3 ( anti ‐ GFP antibody ) and p62 .

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( Alternatively (C), the cells were analysed by western blot for the presence of MCL‐1 and the autophagic markers GFP-LC3 (anti‐GFP antibody) and p62.


( D ) H1299 cells stably expressing GFP - LC3 were incubated for the indicated times in either EBSS or media without glucose / pyruvate and analysed by western blot for the presence of MCL ‐ 1 and autophagic markers .

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(D) H1299 cells stably expressing GFP-LC3 were incubated for the indicated times in either EBSS or media without glucose/pyruvate and analysed by western blot for the presence of MCL‐1 and autophagic markers.


( E , F ) H1299 cells stably expressing GFP - LC3 were pre ‐ incubated for 1 h with 5 mM 3MA ( E ) or 200 nM bafilomycin ( F ) , and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin . Lysates were then analysed for the presence of MCL ‐ 1 , the autophagy marker p62 and actin as a loading control .

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(E, F) H1299 cells stably expressing GFP-LC3 were pre‐incubated for 1 h with 5 mM 3MA (E) or 200 nM bafilomycin (F), and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin. Lysates were then analysed for the presence of MCL‐1, the autophagy marker p62 and actin as a loading control.


( A ) H1299 cells stably expressing GFP - LC3 were infected with 10 p . f . u . per cell of either Ad LacZ or Ad Myc ‐ MCL ‐ 1 for 24 h then incubated in EBSS for 4 h . Alternatively , cells were transfected with control or MCL ‐ 1 siRNA 24 h before EBSS treatment . Cells were stained with an antibody against cyt c and analysed by immunofluorescence . Representative images are shown . Scale bars = 50 μm .

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(A) H1299 cells stably expressing GFP-LC3 were infected with 10 p.f.u. per cell of either Ad LacZ or Ad Myc‐MCL‐1 for 24 h then incubated in EBSS for 4 h. Alternatively, cells were transfected with control or MCL‐1 siRNA 24 h before EBSS treatment. Cells were stained with an antibody against cyt c and analysed by immunofluorescence. Representative images are shown. Scale bars=50 μm.


( B ) The percent of the cell surface area covered by GFP - LC3 vesicles was quantified using the Image J software . n > 70 cells in three experiment , * P0 . 01 .

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(B) The percent of the cell surface area covered by GFP-LC3 vesicles was quantified using the Image J software. n>70 cells in three experiment, *P0.01.


( C , D ) H1299 cells stably expressing GFP - LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated . The levels of endogenous LC3 ‐ II relative to actin were quantified by western blot in C . Data are expressed as the average of three experiments ± s . d , * P0 . 05 .

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(C, D) H1299 cells stably expressing GFP-LC3 were transfected as in A and incubated with 200 nM bafilomycin for 1 h where indicated. The levels of endogenous LC3‐II relative to actin were quantified by western blot in C. Data are expressed as the average of three experiments±s.d, *P0.05.


( E , F ) H1299 cells stably expressing GFP - LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP - LC3 .

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(E, F) H1299 cells stably expressing GFP-LC3 were treated as in A and analysed by western blot for the presence of p62 and GFP-LC3.


( G ) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL ‐ 1 and p62 by western blot 3 days later .

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(G) Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of MCL‐1 and p62 by western blot 3 days later.


( H ) Transformed wild ‐ type ( WT ) and MCL ‐ 1Δ / Δ ( KO ) MEFs were analysed by western blot for the expression of endogenous LC3 .

(H) Transformed wild‐type (WT) and MCL‐1Δ/Δ (KO) MEFs were analysed by western blot for the expression of endogenous LC3.


( I , J ) Colocalization of GFP - LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A . The number of LC3 ‐ positive vesicles that colocalised with LAMP1 was quantified in I . Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1 ‐ positive ± s . e . m . , with at least 14 cells per condition . Representative images are shown in J . Arrows indicate LC3 ‐ positive vesicles colocalizing with LAMP1 . Scale bars = 10 μm .

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(I, J) Colocalization of GFP-LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunofluorescence in cells treated as in A. The number of LC3‐positive vesicles that colocalised with LAMP1 was quantified in I. Data are expressed as the percentage of LC3 vesicles per cell that are LAMP1‐positive±s.e.m., with at least 14 cells per condition. Representative images are shown in J. Arrows indicate LC3‐positive vesicles colocalizing with LAMP1. Scale bars=10 μm.


( A ) MCL ‐ 1 interacts with Beclin ‐ 1 . Lysates from H1299 stably expressing GFP - LC3 were immunoprecipitated using control or MCL ‐ 1 ‐ specific antibodies and analysed for the presence of Beclin ‐ 1 by western blot .

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(A) MCL‐1 interacts with Beclin‐1. Lysates from H1299 stably expressing GFP-LC3 were immunoprecipitated using control or MCL‐1‐specific antibodies and analysed for the presence of Beclin‐1 by western blot.


( B ) H1299 cells stably expressing GFP - LC3 were fractionated into HM ( containing mitochondria ) , and LM ( containing ER ) . Fractions were analysed for the presence of MCL ‐ 1 , BCL ‐ 2 , Beclin ‐ 1 , the mitochondrial marker mtHSP70 and the ER marker calreticulin .

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(B) H1299 cells stably expressing GFP-LC3 were fractionated into HM (containing mitochondria), and LM (containing ER). Fractions were analysed for the presence of MCL‐1, BCL‐2, Beclin‐1, the mitochondrial marker mtHSP70 and the ER marker calreticulin.


( C ) MCL ‐ 1 was immunoprecipitated from HM and LM before analysing the presence of MCL ‐ 1 and Beclin ‐ 1 .

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(C) MCL‐1 was immunoprecipitated from HM and LM before analysing the presence of MCL‐1 and Beclin‐1.


( B ) Expression levels of various BCL ‐ 2 homologues in the cortices of animals of the indicated genotypes .

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(B) Expression levels of various BCL‐2 homologues in the cortices of animals of the indicated genotypes.


( C ) Brain morphology of MCL ‐ 1Δ / Δ . Brain sections were stained with cresyl violet and imaged using a × 1 objective .

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(C) Brain morphology of MCL‐1Δ/Δ. Brain sections were stained with cresyl violet and imaged using a × 1 objective.


( D - G ) Cortical neurons around the lesion in MCL ‐ 1Δ / Δ animals are positive for the autophagic marker LC3 . Brain sections from animals with the indicated genotypes ( P14 for E , G ; P7 for H ) were stained for LC3 ( Green ) and the neuronal marker NeuN ( in H , red ) , along with the nuclear stain Hoechst ( Blue ) . Confocal images were taken using × 10 ( E ) or × 63 ( G , H ) objectives . Scale bars = 200 μm ( E ) ; 50 μm ( G , H ) . Quantification of the LC3 staining is shown in D . The total number of cells was determined by counting the Hoechst ‐ positive nuclei . Data are expressed as percent of LC3 ‐ positive cells in at least three animals per genotype ± s . d . * P0 . 01 . ( F ) Quantification of the total surface area occupied by autophagosomes . Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype ± s . d . P0 . 005 ( I ) Representative EM images from MCL ‐ 1 + / Δ and MCL ‐ 1Δ / ΔP14mice . N , nucleus ; * , autophagosomal structures ; arrowheads , double membrane ; arrow , mitochondrion inside a vesicle .

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(D-G) Cortical neurons around the lesion in MCL‐1Δ/Δ animals are positive for the autophagic marker LC3. Brain sections from animals with the indicated genotypes (P14 for E, G; P7 for H) were stained for LC3 (Green) and the neuronal marker NeuN (in H, red), along with the nuclear stain Hoechst (Blue). Confocal images were taken using × 10 (E) or × 63 (G, H) objectives. Scale bars=200 μm (E); 50 μm (G, H). Quantification of the LC3 staining is shown in D. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of LC3‐positive cells in at least three animals per genotype±s.d. *P0.01. (F) Quantification of the total surface area occupied by autophagosomes. Data are expressed as percent of total cortical area covered by autophagosomes in at least 16 EM images per genotype±s.d. P0.005 (I) Representative EM images from MCL‐1+/Δ and MCL‐1Δ/ΔP14mice. N, nucleus; *, autophagosomal structures; arrowheads, double membrane; arrow, mitochondrion inside a vesicle.


( A ) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase ‐ 3 , p62 and MCL ‐ 1 . Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase ‐ 3 .

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(A) Cortices from the indicated genotypes and ages were analysed by western blot for the presence of active caspase‐3, p62 and MCL‐1. Primary cortical neurons treated for 12 h with camptothecin were used as a positive control for active caspase‐3.


( B ) Active BAX ( 6A7 ) and active caspase ‐ 3 were also quantified by staining the cortex of 1 ‐ to 14 ‐ day ‐ old MCL ‐ 1Δ / Δ mice . Representative images from at least three sections from each animal were counted . The total number of cells was determined by counting the Hoechst ‐ positive nuclei . Data are expressed as percent of positive cells for at least three animals per genotype ± s . d . * P0 . 05 .

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(B) Active BAX (6A7) and active caspase‐3 were also quantified by staining the cortex of 1‐ to 14‐day‐old MCL‐1Δ/Δ mice. Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for at least three animals per genotype±s.d. *P0.05.


( C ) Active BAX is present in a subset of LC3 ‐ positive cells . Sections from P7 and P14 MCL ‐ 1Δ / Δ brains were stained for LC3 ( Green ) and active BAX ( 6A7 ; Red ) . Confocal images were taken using × 63 objective . Arrows point to BAX ‐ positive , LC3 ‐ negative cells . Representative images are shown . Scale bars = 50 μm .

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(C) Active BAX is present in a subset of LC3‐positive cells. Sections from P7 and P14 MCL‐1Δ/Δ brains were stained for LC3 (Green) and active BAX (6A7; Red). Confocal images were taken using × 63 objective. Arrows point to BAX‐positive, LC3‐negative cells. Representative images are shown. Scale bars=50 μm.


Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase ‐ cleaved fragment of the caspase ‐ 3 substrate CAS5 days later . Alternatively ( E ) , the neurons were analysed for cytochrome c release by immunofluorescence . Data are expressed as percent of positive cells for at least three embryos per genotype ± s . d .

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Primary cortical neurons of the indicated genotypes were infected with a lentivirus expressing Cre at the time of plating and analysed for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate CAS5 days later. Alternatively (E), the neurons were analysed for cytochrome c release by immunofluorescence. Data are expressed as percent of positive cells for at least three embryos per genotype±s.d.


( A ) Brains from E12 . 5 Foxg1 Cre MCL ‐ 1 animals were analysed by western blot for the presence of the caspase ‐ cleaved fragment of the caspase ‐ 3 substrate p130CAS and the autophagic markers LC3 and p62 .

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(A) Brains from E12.5 Foxg1 Cre MCL‐1 animals were analysed by western blot for the presence of the caspase‐cleaved fragment of the caspase‐3 substrate p130CAS and the autophagic markers LC3 and p62.


( B ) Autophagy in Foxg1 Cre MCL ‐ 1Δ / Δ mice . Brain sections from E15 . 5 animals with the indicated genotypes were stained for LC3 ( green ) . Confocal images were taken using × 20 ( bottom panel ) or × 63 ( top and middle panels ) objectives . Scale bars = 50 μm .

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(B) Autophagy in Foxg1 Cre MCL‐1Δ/Δ mice. Brain sections from E15.5 animals with the indicated genotypes were stained for LC3 (green). Confocal images were taken using × 20 (bottom panel) or × 63 (top and middle panels) objectives. Scale bars=50 μm.


( C ) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15 . 5 wild ‐ type embryos . Data are expressed as the percentage of LC3 vesicles that are LAMP1 ‐ positive ± s . d . About 100 LC3 vesicles were counted per section , with four sections per embryo . VZ , ventricular zone .

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(C) Colocalization of LC3 vesicles with the lysosomal marker LAMP1 was analysed by immunohistochemistry in E15.5 wild‐type embryos. Data are expressed as the percentage of LC3 vesicles that are LAMP1‐positive±s.d. About 100 LC3 vesicles were counted per section, with four sections per embryo. VZ, ventricular zone.


( D ) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis ( BIM , Puma ) and autophagy ( ATG7 , ATG5 ‐ ATG12 ) related proteins as well as Tuj ‐ 1 ( neuronal marker ) , Nestin ( neural progenitor marker ) and HSC70 ( loading control ) .

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(D) Primary cortical neurons and neuronal progenitor cells were analysed for the presence of the indicated apoptosis (BIM, Puma) and autophagy (ATG7, ATG5‐ATG12) related proteins as well as Tuj‐1 (neuronal marker), Nestin (neural progenitor marker) and HSC70 (loading control).


( E ) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL ‐ 1 and actin as a loading control .

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(E) Primary neurospheres were treated for 8 h with EBSS and analysed by western blot for the presence of MCL‐1 and actin as a loading control.


( F ) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130CAS , LC3 and actin as a loading control .

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(F) Primary neurons and neurospheres were treated as in E and analysed by western blot for p130CAS, LC3 and actin as a loading control.


Alternatively , they were analysed by immunofluorescence for c release ( G ) in the presence of 50 μM zVAD ‐ FMK to prevent apoptotic cells from detaching from the coverslip . Data are expressed as the average of 3 - 7 embryos ± s . d . * P0 . 001 .

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Alternatively, they were analysed by immunofluorescence for c release (G) in the presence of 50 μM zVAD‐FMK to prevent apoptotic cells from detaching from the coverslip. Data are expressed as the average of 3-7 embryos±s.d. *P0.001.


( A ) Brain morphology of P14 MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − mice . Brain sections were stained with cresyl violet and imaged using a × 1 objective .

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(A) Brain morphology of P14 MCL‐1Δ/Δ Beclin‐1+/− mice. Brain sections were stained with cresyl violet and imaged using a × 1 objective.


( B - D ) Active BAX ( 6A7 ; B ) and active caspase ‐ 3 ( C ) were quantified by staining the cortex of 14 ‐ day ‐ old MCL ‐ 1Δ / Δ and MCL ‐ 1Δ / Δ Beclin - 1 + / − mice . The number of BAX ‐ positive LC3 ‐ negative cells was also quantified . ( D ) Representative images from at least three sections from each animal were counted . The total number of cells was determined by counting the Hoechst ‐ positive nuclei . Data are expressed as percent of positive cells for least three animals per genotype ± s . d . * P0 . 05 .

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(B-D) Active BAX (6A7; B) and active caspase‐3 (C) were quantified by staining the cortex of 14‐day‐old MCL‐1Δ/Δ and MCL‐1Δ/Δ Beclin-1+/− mice. The number of BAX‐positive LC3‐negative cells was also quantified. (D)Representative images from at least three sections from each animal were counted. The total number of cells was determined by counting the Hoechst‐positive nuclei. Data are expressed as percent of positive cells for least three animals per genotype±s.d. *P0.05.


( E ) Survival data from MCL ‐ 1Δ / Δ ( n = 14 ) and their MCL ‐ 1Δ / Δ Beclin ‐ 1 + / − littermates ( n = 13 ) .

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(E) Survival data from MCL‐1Δ/Δ (n=14) and their MCL‐1Δ/Δ Beclin‐1+/− littermates (n=13).


( A ) Upper panels - Immunofluorescence imaging of PRR7 with Shank ( synaptic ) and MAP2 ( dendritic ) markers in DIV21 hippocampal neurons . Lower panels - Single plane apotome images of nuclei ( DAPI ) show PRR7 can be present ( arrow ) or absent ( arrowhead ) from nuclei .

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(A) Upper panels- Immunofluorescence imaging of PRR7 with Shank (synaptic) and MAP2 (dendritic) markers in DIV21 hippocampal neurons. Lower panels- Single plane apotome images of nuclei (DAPI) show PRR7 can be present (arrow) or absent (arrowhead) from nuclei.


( B ) Synaptic and dendritic distribution of PRR7 and pre ( Bassoon ) and postsynaptic ( PSD95 ) markers ( arrowheads - triple colocalization ) .

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(B) Synaptic and dendritic distribution of PRR7 and pre (Bassoon) and postsynaptic (PSD95) markers (arrowheads-triple colocalization).


( C ) PRR7 - GFP expressed in 17DIV hippocampal neurons is present in dendritic spines ( arrows ) . Scale bar is 15 µm .

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(C) PRR7-GFP expressed in 17DIV hippocampal neurons is present in dendritic spines (arrows). Scale bar is 15 µm.


( D ) PRR7 distribution across subcellular fractions ( TOT = whole lysate , Syn = synaptosomes , Nuc = nuclei ) isolated from rat hippocampal tissue . Fib ( Fibrillarin ) , SNAP25 and PSD95 were used as nuclear , presynaptic and postsynaptic markers , respectively .

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(D) PRR7 distribution across subcellular fractions (TOT = whole lysate, Syn = synaptosomes, Nuc = nuclei) isolated from rat hippocampal tissue. Fib (Fibrillarin), SNAP25 and PSD95 were used as nuclear, presynaptic and postsynaptic markers, respectively.


( A ) Immunocytochemistry analyses show that glutamate stimulation ( Glut , 100 µM 5 min + 25 min washout ) increased PRR7 in the nucleus . Cycloheximide ( 100 µM ) added 30 min prior to glutamate stimulation did not affect this increase ( Cont = 100 ± 12 . 5 % , Glut = 202 . 1 ± 15 . 2 % ; + cycloheximide Cont = 104 . 9 ± 3 . 6 , Glut = 231 . 4 ± 10 . 9 ) ( n = ~ 30 - 50 cells ) .

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(A) Immunocytochemistry analyses show that glutamate stimulation (Glut, 100 µM 5 min + 25 min washout) increased PRR7 in the nucleus. Cycloheximide (100 µM) added 30 min prior to glutamate stimulation did not affect this increase (Cont=100±12.5%, Glut=202.1±15.2%; +cycloheximide Cont=104.9±3.6, Glut=231.4±10.9) (n=~30-50 cells).


( B ) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons . Epoxomicin ( 50 nM ) added 20 min prior to stimulation did not affect this decrease ( Cont = 100 ± 3 . 6 % , Glut = 32 . 3 ± 1 . 3 % ; + epoxomicin Cont = 99 . 8 ± 4 . 6 , Glut = 42 . 9 ± 1 . 1 ) . ( n = 14 - 17 dendrites , > 1200 puncta for each group ) . Statistical analyses for ( A ) and ( B ) were performed using the Mann Whitney test .

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(B) Decreased synaptic PRR7 immunostaining following glutamate stimulation in DIV 21 hippocampal neurons. Epoxomicin (50 nM) added 20 min prior to stimulation did not affect this decrease (Cont=100±3.6%, Glut=32.3±1.3%; +epoxomicin Cont=99.8±4.6, Glut=42.9±1.1). (n=14-17 dendrites, >1200 puncta for each group). Statistical analyses for (A) and (B) were performed using the Mann Whitney test.


( C ) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons . Treatments ( APV [ 50 µM , APV - ( 2R ) - amino - 5 - phosphonovaleric acid ] , CNQX [ 40 µM , 6 - cyano - 7 nitroquinoxaline - 2 , 3 - dione ] , TTX [ 1 µM , tetrodotoxin ] , NMDA [ 50 µM , N - Methyl - D - Aspartate ] ) as indicated . Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 ( 108 . 1 ± 19 % ) . Statistical significance was determined using one - way ANOVA in conjugation with Sidak post - hoc test .

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(C) Quantification of nuclear PRR7 immunostaining in cultured hippocampal neurons. Treatments (APV [50 µM, APV-(2R)-amino-5-phosphonovaleric acid], CNQX [40 µM, 6-cyano-7 nitroquinoxaline-2,3-dione], TTX [1 µM, tetrodotoxin], NMDA [50 µM, N-Methyl-D-Aspartate]) as indicated. Only APV treatment blocked the glutamate dependent increase in nuclear PRR7 (108.1 ± 19%). Statistical significance was determined using one-way ANOVA in conjugation with Sidak post-hoc test.


( D ) PRR7 - GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment . Depicted are representative averaged confocal images ( 30x30 µm2 ) from optical sections ( 300 nm step size ) of PRR7 - GFP expressing neurons ( DIV17 ) before and after bleaching . Prior to stimulation there was substantial PRR7 - GFP in the nucleus due to basal neuronal activity ( upper panel ) . This was reduced by incubation overnight with MK801 ( 10 µM ) and TTX ( 1 µM ) ( lower panel ) . NMDA ( 50 µM ) stimulation , but not MK - 801 , led to recovery of nuclear PRR7 - GFP fluorescence following bleaching of nuclear ROIs at t0 ` ( blue arrow in E ) . ( E ) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0 ` and corrected for bleaching . No significant differences in PRR7 - GFPFRAP were found in control ( MK801 ) conditions . GFP intensities are displayed via look - up - table ( LUT ) . Data are represented as mean ± SEM ; n = 9 cells ( NMDA ) and n = 5 cells ( MK801 ) . Analysis was done using one - way ANOVA using Bonferroni post - hoc correction .

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(D) PRR7-GFP accumulates in the nucleus of hippocampal neurons upon NMDA treatment. Depicted are representative averaged confocal images (30x30 µm2) from optical sections (300 nm step size) of PRR7-GFP expressing neurons (DIV17) before and after bleaching. Prior to stimulation there was substantial PRR7-GFP in the nucleus due to basal neuronal activity (upper panel). This was reduced by incubation overnight with MK801 (10 µM) and TTX (1 µM) (lower panel). NMDA (50 µM) stimulation, but not MK-801, led to recovery of nuclear PRR7-GFP fluorescence following bleaching of nuclear ROIs at t0`(blue arrow in E).(E) Quantification of changes in nuclear GFP fluorescent intensities were normalized to time point t0` and corrected for bleaching. No significant differences in PRR7-GFPFRAP were found in control (MK801) conditions. GFP intensities are displayed via look-up-table (LUT). Data are represented as mean±SEM; n=9 cells (NMDA) and n=5 cells (MK801). Analysis was done using one-way ANOVA using Bonferroni post-hoc correction.


( F ) PRR7 - mEos translocates from distal dendrites to the nucleus upon NMDA treatment . ( Left panels ) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7 - mEos before and after photoconversion of distal dendrites ( ROIs ) through the image z - stack from from green ( 488nm ) to red ( 568nm ) using a UV laser ( 405 nm ) . ( Right panels ) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7 - mEos ( 568nm ) in the nucleus following NMDA stimulation ( 50 µM - 30min ) . ( G ) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7 - mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment . ( Average increase ; NMDA = 617 . 8 ± 47 . 7 % , n = 11 cells , p < 0 . 0005 ; MK801 = 206 . 1 ± 13 . 6 % , n = 5 cells , n . s . ) . Statistical significance was determined by two - way ANOVA using Sidak ' s correction for multiple comparisons .

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(F) PRR7-mEos translocates from distal dendrites to the nucleus upon NMDA treatment. (Left panels) Confocal maximal intensity projection images of hippocampal neurons expressing PRR7-mEos before and after photoconversion of distal dendrites (ROIs) through the image z-stack from from green (488nm) to red (568nm) using a UV laser (405 nm). (Right panels) Averaged intensity projection images from nuclear planes showing accumulation of photoconverted PRR7-mEos (568nm) in the nucleus following NMDA stimulation (50 µM- 30min).(G) Paired quantification of changes in background corrected fluorescent intensities of nuclear PRR7-mEos at 0 min and 30 min following NMDA stimulation or MK801 treatment. (Average increase; NMDA=617.8±47.7%, n=11 cells, p<0.0005; MK801=206.1±13.6%, n=5 cells, n.s.). Statistical significance was determined by two-way ANOVA using Sidak's correction for multiple comparisons.


( A ) Co - Immunoprecipitations ( Co - IPs ) of PRR7 with GluN1 , PSD95 and GluN2B from rat brain lysates . FT = flowthrough , IgG = normal IgGs .

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(A) Co-Immunoprecipitations (Co-IPs) of PRR7 with GluN1, PSD95 and GluN2B from rat brain lysates. FT = flowthrough, IgG = normal IgGs.


( B ) Co - IPs from HEK293 cells of c - terminal myc - tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95 .

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(B) Co-IPs from HEK293 cells of c-terminal myc-tagged PRR7 with GluN1 in the absence of other NMDAR subunits or PSD95.


( C ) Co - IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 ( myc - tagged GluN2B and GluA2 and flag - tagged PRR7 ) .

(C) Co-IPs from HEK293 cells transfected with tagged NMDA or AMPA receptor subunits and PRR7 (myc-tagged GluN2B and GluA2 and flag-tagged PRR7).


( D ) ( top ) Schematics of the different PRR7 constructs used for co - IP ( FL = full length ) . ( bottom ) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co - IPs performed as indicated .

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(D) (top) Schematics of the different PRR7 constructs used for co-IP (FL= full length). (bottom) PRR7 deletion constructs and NMDAR subunits were expressed in HEK293 cells and Co-IPs performed as indicated.


( E ) Co - IPs of PRR7 with GluN1 from high - density primary neuronal cultures following treatment with NMDA ( 100µM , 30 min , representative of n = 4 ) .

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(E) Co-IPs of PRR7 with GluN1 from high-density primary neuronal cultures following treatment with NMDA (100µM, 30 min, representative of n=4).


( A ) Western blots of lysates of primary neurons transduced with control lentiviruses ( NT ) or lentiviruses to knockdown ( KD ) or overexpress ( OE ) PRR7 . PRR7 knockdown reduced ( 75 . 6 ± 6 . 9 % ) while PRR7 overexpression ( OE ) increased ( 157 . 6 ± 22 . 8 % ) c - Jun levels compared to controls . No change was detected in c - Fos levels . One - way ANOVA test was used to analyze results with Dunett ' s post hoc test ( n = 16 gels for c - Jun and 4 gels for c - Fos ) .

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(A) Western blots of lysates of primary neurons transduced with control lentiviruses (NT) or lentiviruses to knockdown (KD) or overexpress (OE) PRR7. PRR7 knockdown reduced (75.6 ± 6.9%) while PRR7 overexpression (OE) increased (157.6 ± 22.8%) c-Jun levels compared to controls. No change was detected in c-Fos levels. One-way ANOVA test was used to analyze results with Dunett's post hoc test (n=16 gels for c-Jun and 4 gels for c-Fos).


( B ) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c - Jun protein levels were measured by Western blotting . PRR7 expression significantly stabilized c - Jun levels at 120 min ( 110 . 0 ± 7 . 5 % vs control 68 . 7 ± 7 . 9 % ) and 240 minutes ( 83 . 6 ± 5 . 2 % vs control 43 . 7 ± 3 . 4 % ) . Values for c - Jun were normalized to their respective 0 min time point ( n = 4 gels for each group performed blind ) . T - tests between similar time points were used to analyze results .

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(B) Control or PRR7 transfected HEK293 cells were treated with cycloheximide for the indicated time points and c-Jun protein levels were measured by Western blotting. PRR7 expression significantly stabilized c-Jun levels at 120 min (110.0 ± 7.5% vs control 68.7 ± 7.9%) and 240 minutes (83.6 ± 5.2% vs control 43.7 ± 3.4%). Values for c-Jun were normalized to their respective 0 min time point (n=4 gels for each group performed blind). T-tests between similar time points were used to analyze results.


( C ) PRR7 , FBW7 and / or a control protein ( PSD95 ) were transfected into HEK293 cells and c - Jun ubiquitination was measured by immunoprecipitating c - Jun and blotting for ubiquitin . Ubiquitination of c - Jun in the presence of FBW7 ( 178 . 3 ± 26 . 9 % ) was significantly reduced in the presence of PRR7 ( 103 . 7 ± 20 . 6 % ) but not a control protein ( PSD95 ) ( n = 5 gels for all groups ) .

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(C) PRR7, FBW7 and/or a control protein (PSD95) were transfected into HEK293 cells and c-Jun ubiquitination was measured by immunoprecipitating c-Jun and blotting for ubiquitin. Ubiquitination of c-Jun in the presence of FBW7 (178.3 ± 26.9%) was significantly reduced in the presence of PRR7 (103.7 ± 20.6%) but not a control protein (PSD95) (n=5 gels for all groups).


( D ) Western blots of c - Junubiquitination in hippocampal primary neuronal cultures . PRR7 knockdown ( KD ) elevated ( 149 ± 10 . 62 % , n = 6 gels ) c - Junubiquitination level in comparison to control neurons ( NT ) . Statistical significance was determined by t - test .

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(D) Western blots of c-Junubiquitination in hippocampal primary neuronal cultures. PRR7 knockdown (KD) elevated (149±10.62%, n=6 gels) c-Junubiquitination level in comparison to control neurons (NT). Statistical significance was determined by t-test.


( E ) Western blots showing knockdown ( KD ) or overexpression ( OE ) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc .

(E) Western blots showing knockdown (KD) or overexpression (OE) of PRR7 in primary hippocampal neurons do not alter levels of other FBW7 targets mTOR or Myc.


( A ) Co - IPs of PRR7 with c - Jun from lysates of HEK293 cells overexpressing PRR7 .

(A) Co-IPs of PRR7 with c-Jun from lysates of HEK293 cells overexpressing PRR7.


( B ) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c - Jun .

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(B) Pulldown assays in vitro using purified proteins showed a direct interaction between PRR7 and c-Jun.


( C ) Co - IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 .

(C) Co-IPs of PRR7 with FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7.


( D ) Pulldown assays in vitro using purified PRR7 and FBW7 .

(D) Pulldown assays in vitro using purified PRR7 and FBW7.


( E ) Co - IPs of endogenous PRR7 and FBW7 from lysates of ratbrain .

(E) Co-IPs of endogenous PRR7 and FBW7 from lysates of ratbrain.


( F ) Co - IPs of PRR7 with c - Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7 . FBW7 expression increased the amount of c - Jun pulled down with PRR7 ( n = 4 gels for each panel , representative blots ) . Lower bar graphs - quantitation for Western blots show c - Jun / FBW7 binding is 41 . 4 ± 13 . 9 % greater in the presence of PRR7 ( normalized for FBW7 expression ) . Similarly , c - Jun / PRR7 binding is 97 . 4 ± 38 . 1 % greater in the presence of FBW7 ( normalized for PRR7 expression ) . Statistical significance was calculated using unpaired t - tests .

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(F) Co-IPs of PRR7 with c-Jun and FBW7 from lysates of HEK293 cells overexpressing PRR7 and FBW7. FBW7 expression increased the amount of c-Jun pulled down with PRR7 (n=4 gels for each panel, representative blots). Lower bar graphs- quantitation for Western blots show c-Jun/FBW7 binding is 41.4±13.9% greater in the presence of PRR7 (normalized for FBW7 expression). Similarly, c-Jun/PRR7 binding is 97.4±38.1% greater in the presence of FBW7 (normalized for PRR7 expression). Statistical significance was calculated using unpaired t-tests.


( A ) Western blots of phospho - c - Jun in primary neuronal cultures . Quantification of phospho - c - Jun represents a ratio of phospho - c - Jun to total c - Jun and all ratios were normalized to NT . PRR7 knockdown ( KD ) decreased , while PRR7 overexpression ( OE ) increased phospho - c - Jun levels ( black bars ) . Glutamate stimulation ( gray bars ) increased phospho - c - Jun / c - Jun ratio in NT but not in KD or OE neurons ( n = 7 gels for all groups ) . Significance was determined using two - way ANOVA with Sidak post - hoc test .

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(A) Western blots of phospho-c-Jun in primary neuronal cultures. Quantification of phospho-c-Jun represents a ratio of phospho-c-Jun to total c-Jun and all ratios were normalized to NT. PRR7 knockdown (KD) decreased, while PRR7 overexpression (OE) increased phospho-c-Jun levels (black bars). Glutamate stimulation (gray bars) increased phospho-c-Jun/c-Jun ratio in NT but not in KD or OE neurons (n=7 gels for all groups). Significance was determined using two-way ANOVA with Sidak post-hoc test.


( B ) AP - 1 driven luciferase luminescence assays . PRR7 expression in HEK293 cells increased AP - 1 activity ( 190 . 6 ± 13 . 9 % , n = 12 ; RLU = relative light units ) . Statistical significance was determined using t - tests .

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(B) AP-1 driven luciferase luminescence assays. PRR7 expression in HEK293 cells increased AP-1 activity (190.6 ± 13.9%, n=12; RLU= relative light units). Statistical significance was determined using t-tests.


( D ) Quantitation of cell death using 30 minutes of PI in - situ staining following 48h of WT - PRR7 - GFP or mNLS - PRR7 - GFP overexpression in primary hippocampal neuronal cultures ( n = 24 cells per group ) . Images represent a max projection of five z - stack sections across the cell body .

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(D) Quantitation of cell death using 30 minutes of PI in-situ staining following 48h of WT-PRR7-GFP or mNLS-PRR7-GFP overexpression in primary hippocampal neuronal cultures (n=24 cells per group). Images represent a max projection of five z-stack sections across the cell body.


( E ) NMDA treatment ( 25 min , 100 µM followed by 14h chase ) caused 83 . 6 ± 3 . 5 % cell death in control neurons compared to 60 . 3 ± 4 . 6 % cell death in neurons with PRR7 expression knocked down . A c - Jun inhibitory peptide reduced NMDA - mediated cell death to 66 . 2 ± 6 . 0 % in control neurons and to 55 . 6 ± 5 . 3 % in neurons knocked down for PRR7 , but this difference was not statistically significant ( n = 5 independent experiments ) . Two - way ANOVA was used in conjugation with Tukey post - hoc test . ( Left ) Representative images showing transduced neurons ( green - GFP ) and PI staining ( red ) .

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(E) NMDA treatment (25 min, 100 µM followed by 14h chase) caused 83.6±3.5% cell death in control neurons compared to 60.3±4.6% cell death in neurons with PRR7 expression knocked down. A c-Jun inhibitory peptide reduced NMDA-mediated cell death to 66.2±6.0% in control neurons and to 55.6±5.3% in neurons knocked down for PRR7, but this difference was not statistically significant (n=5 independent experiments). Two-way ANOVA was used in conjugation with Tukey post-hoc test. (Left) Representative images showing transduced neurons (green- GFP) and PI staining (red).


( F ) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c - Jun or a dominant negative c - Jun4A ( 4A ) . Overexpression of WT c - Jun caused extensive neuronal cell death ( 84 . 8 ± 2 . 4 % ) that was reduced in neurons knocked down for PRR7 ( 43 . 8 ± 9 . 2 ) . Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity ( 50 . 8 ± 7 . 9 % ) but was not additive with PRR7 knockdown ( 42 . 3 ± 8 . 1 % ) ( n = 3 independent experiments ) . Significance was determined using two - way ANOVA in conjugation with Tukey post - hoc test . Images are representative 10X widefield micrographs showing transduced neurons ( green - GFP ) and PI staining ( red ) . Data is presented as mean ± sem . All % values represent comparisons to control .

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

(F) Quantification of cell death in response to NMDA stimulation in the presence or absence of PRR7 and either WT c-Jun or a dominant negative c-Jun4A (4A). Overexpression of WT c-Jun caused extensive neuronal cell death (84.8±2.4%) that was reduced in neurons knocked down for PRR7 (43.8±9.2). Overexpressing 4A conferred neuroprotection to NMDA excitotoxicity (50.8±7.9%) but was not additive with PRR7 knockdown (42.3±8.1%) (n=3 independent experiments). Significance was determined using two-way ANOVA in conjugation with Tukey post-hoc test. Images are representative 10X widefield micrographs showing transduced neurons (green- GFP) and PI staining (red). Data is presented as mean±sem. All % values represent comparisons to control.


Cell count of Ronin fl / del and Ronin del / del ESC . Data represent mean ± SD , n = three independent experiments .

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Cell count of Ronin fl/del and Ronin del/del ESC. Data represent mean ± SD, n = three independent experiments.


Annexin V cell death assay of Ronin fl / del and Ronin del / del cultured for 6 days . Two - tailed unpaired Student ' s t test , data represent mean ± SD , n = three independent experiments .

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Annexin V cell death assay of Ronin fl/del and Ronin del/del cultured for 6 days. Two-tailed unpaired Student's t test, data represent mean ± SD, n = three independent experiments.


Ronin fl / del and Ronin del / del ESC stained for Ronin ( left panel ) or Oct4 ( right panel ) . The nuclei are counterstained with DAPI .

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Ronin fl/del and Ronin del/del ESC stained for Ronin (left panel) or Oct4 (right panel). The nuclei are counterstained with DAPI.


Haematoxylin / eosin staining of paraffin sections of Ronin fl / del and ecto - Ronin teratomas .

Haematoxylin/eosin staining of paraffin sections of Ronin fl/del and ecto-Ronin teratomas.


In vitro differentiation of Ronin fl / del , ecto - Ronin ( grown in the presence of Dox ) and Ronin del / del ESC .

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In vitro differentiation of Ronin fl/del, ecto-Ronin (grown in the presence of Dox) and Ronin del/del ESC.


Volcano plot of gene expression in Ronin del / del day 4 vs . Ronin fl / del ( left panel ) and Ronin del / del day 8 vs . Ronin fl / del ESC ( right panel ) with annotated naïve and primed pluripotency markers .

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Volcano plot of gene expression in Ronin del/del day 4 vs. Ronin fl/del (left panel) and Ronin del/del day 8 vs. Ronin fl/del ESC (right panel) with annotated naïve and primed pluripotency markers.


Volcano plot of gene expression in Ronin del / del day 8 vs . Ronin del / del day 4 ESC .

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Volcano plot of gene expression in Ronin del/del day 8 vs. Ronin del/del day 4 ESC.


Analysis of nascent protein synthesis using OP - puro assay . Ronin fl / del cells treated with protein synthesis inhibitor ( cycloheximide ) are used as negative control . The nuclei are counterstained with DAPI . Quantification of the OP - puro signal , normalised to DAPI . Ordinary one - way Anova data represent mean ± SD ; n = three independent experiments for each genotype .

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Analysis of nascent protein synthesis using OP-puro assay. Ronin fl/del cells treated with protein synthesis inhibitor (cycloheximide) are used as negative control. The nuclei are counterstained with DAPI.   Quantification of the OP-puro signal, normalised to DAPI. Ordinary one-way Anova data represent mean ± SD; n = three independent experiments for each genotype.


Transmission electron microscopy analysis of mitochondrial structure in Ronin fl / del and Ronin del / del ESC . Mitochondria analysed , n > 150 for each condition .

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Transmission electron microscopy analysis of mitochondrial structure in Ronin fl/del and Ronin del/del ESC. Mitochondria analysed, n > 150 for each condition.


Quantification of the OCR analysis . Kruskal - Wallis test , data represent mean ± SD , n = 16 technical replicates per genotype in three independent experiments .

Quantification of the OCR analysis. Kruskal-Wallis test, data represent mean ± SD, n = 16 technical replicates per genotype in three independent experiments.


Blastocyst ( E4 . 5 ) and diapause embryos stained for Ronin , Sox2 and Sox17 . Egg cylinder stage ( E5 . 5 ) embryo stained for Ronin and Sox17 . Nuclei are counterstained with DAPI .

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Blastocyst (E4.5) and diapause embryos stained for Ronin, Sox2 and Sox17. Egg cylinder stage (E5.5) embryo stained for Ronin and Sox17. Nuclei are counterstained with DAPI.


Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4 . 5 , diapause and E5 . 5 embryo . Mitochondria analysed , E4 . 5 ( n mitochondria = 55 ) , diapause ( n mitochondria = 62 ) , E5 . 5 ( n mitochondria = 65 ) .

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Transmission electron microscopy analysis of mitochondrial morphology in the epiblast of E4.5, diapause and E5.5 embryo. Mitochondria analysed, E4.5 (n mitochondria = 55), diapause (n mitochondria = 62), E5.5 (n mitochondria = 65).


E4 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in E4 . 5 blastocysts , Mann - Whitney test , data represent mean ± SD ; Ronin fl / del n embryos = 26 , Ronin del / del n embryos = 27 . Diapause embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in diapause embryos , unpaired Student ' s t test , data represent mean ± SD , Ronin fl / del n embryos = 4 , Ronin del / del n embryos = 12 . E5 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in E5 . 5 embryos , unpaired Student ' s t test , data represent mean ± SD , Ronin fl / del n embryos = 21 , Ronin del / del n embryos = 15 . Reactivated E5 . 5 chimeric embryos stained for tdTomato and DAPI . Quantification of donor cells ' contribution in reactivated E5 . 5 embryos , Mann - Whitney test , data represent mean ± SD , Ronin fl / del n embryos = 17 , Ronin del / del n embryos = 12 .

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E4.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E4.5 blastocysts, Mann-Whitney test, data represent mean ± SD; Ronin fl/del n embryos = 26, Ronin del/del n embryos = 27.   Diapause embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in diapause embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 4, Ronin del/del n embryos = 12.   E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in E5.5 embryos, unpaired Student's t test, data represent mean ± SD, Ronin fl/del n embryos = 21, Ronin del/del n embryos = 15.   Reactivated E5.5 chimeric embryos stained for tdTomato and DAPI.   Quantification of donor cells' contribution in reactivated E5.5 embryos, Mann-Whitney test, data represent mean ± SD, Ronin fl/del n embryos = 17, Ronin del/del n embryos = 12.


Testing an assay for SARS - CoV - 2 using synthetic RNA targets . We show data from both fluorescent ( a ) and lateral flow detection ( b ) . Target concentrations ( in cp / μl ) are indicated . RPA NTC : water input into RPA ; Det NTC : water input into Cas13 detection reaction . In ( a ) , error bars indicate one standard deviation based on n = 3 technical replicates .

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Testing an assay for SARS-CoV-2 using synthetic RNA targets. We show data from both fluorescent (a) and lateral flow detection (b). Target concentrations (in cp/μl) are indicated. RPA NTC: water input into RPA; Det NTC: water input into Cas13 detection reaction. In (a), error bars indicate one standard deviation based on n=3 technical replicates.


A Representative 3D tile scan images of young adrenal gland , ovary , pituitary gland , testis , pancreas and thyroid gland stained with the antibodies indicated in the panel . Scale bars are 200 µm . The white and dashed lines represent the outlines of each organ .

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A Representative 3D tile scan images of young adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland stained with the antibodies indicated in the panel. Scale bars are 200 µm. The white and dashed lines represent the outlines of each organ.


B Representative 3D images at single - cell resolution level of adrenal gland , ovary , pituitary gland , testis , pancreas and thyroid gland . Insets ( * ) indicate higher magnification images of regions with single - cell resolution and surface rendering for all markers . Scale bars for tile scan 3D images are 200 µm , for single - cell resolution insets are 5 µm . The dashed lines represent the outlines of each organ .

B Representative 3D images at single-cell resolution level of adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland. Insets (*) indicate higher magnification images of regions with single-cell resolution and surface rendering for all markers. Scale bars for tile scan 3D images are 200 µm, for single-cell resolution insets are 5 µm. The dashed lines represent the outlines of each organ.


C Exemplar image illustrates the cell - cell interactome analysis with the distance estimates of CD68 + macrophages to blood vessels ( Endoglin ) in cortex , medulla and the transition zone ( TZ ) of an aged adrenal gland . The colour code indicates the distance which is very close in violet and very far in red . The scale bar is 50 µm . The white and dashed lines represent the outline of the transition zone .

C Exemplar image illustrates the cell-cell interactome analysis with the distance estimates of CD68+ macrophages to blood vessels (Endoglin) in cortex, medulla and the transition zone (TZ) of an aged adrenal gland. The colour code indicates the distance which is very close in violet and very far in red. The scale bar is 50 µm. The white and dashed lines represent the outline of the transition zone.


A Young and aged adrenal gland immunolabelled with HSPG2 , NG2 and Podocalyxin . Higher magnification insets show cortex ( Co ) and medulla ( Me ) . Combined box and whiskers , and scatter plot with quantification of vessel density in young and aged adrenal glands . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

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A Young and aged adrenal gland immunolabelled with HSPG2, NG2 and Podocalyxin. Higher magnification insets show cortex (Co) and medulla (Me). Combined box and whiskers, and scatter plot with quantification of vessel density in young and aged adrenal glands. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


B Young and aged ovary immunolabelled with Sca - 1 , PDGFRβ and Emcn . Higher magnification insets show cortex and medulla . Combo plot with quantification of vessel density in young and aged ovary . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

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B Young and aged ovary immunolabelled with Sca-1, PDGFRβ and Emcn. Higher magnification insets show cortex and medulla. Combo plot with quantification of vessel density in young and aged ovary. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm.