Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed . UACC257 were serum starved stimulated with PBS or EDN at 100 nM for 60 min ( 5 min for CaMKII ) . Ten μg from RIPA lysates were analysed by Western blot . Kinase marker substrate phosphorylations corresponding loading controls are shown . For each kinase , two independent replicates were performed are shown . Each gel also had control lanes with increasing of kinase inhibitors to support phospho band

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Predicted kinases are indicated on top of each panel for which one of their marker substrate phosphorylations was assayed. UACC257 cells were serum starved and stimulated with PBS or EDN at 100 nM for 60 min (5 min for CaMKII). Ten μg protein from RIPA lysates were analysed by Western blot. Kinase marker substrate phosphorylations and corresponding loading controls are shown. For each kinase, two independent replicates were performed and are shown. Each gel also had control lanes with increasing concentration of kinase inhibitors to support phospho band identity


effect of kinase inhibitors on EDN induced migration of UACC257 was analysed using a scratch . Scratches were made in UACC257 monolayers . Serum was removed were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0 . 1 μM or 1 μM in triplicates . A Example pictures showing effect of EDN on migration . free areas are delimited by blue lines . Migration was quantified as change in free between 0 h 48 h . B four inhibitors that were effective at 1 µM are shown in detail , i . e . every point corresponds to one well . C Abundance of E - Cadherin ( normalized to GAPDH Actin ) was assessed by LC - SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM . Only single inhibitor which could reverse EDN induced E - Cadherin repression is shown . * p < 0 . 05 two - sided unpaired t - .

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The effect of kinase inhibitors on EDN induced cell migration of UACC257 cells was analysed using a scratch assay. Scratches were made in UACC257 monolayers. Serum was removed and cells were treated with PBS or 100 nM EDN in combination with DMSO or kinase inhibitors at 0.1 μM or 1 μM in triplicates. A Example pictures showing the effect of EDN on migration. Cell free areas are delimited by blue lines. Migration was quantified as the change in cell free area between 0 h and 48 h. B The four inhibitors that were effective at 1 µM are shown in detail, i.e. every point corresponds to one well. C Abundance of E-Cadherin (normalized to GAPDH and Actin) was assessed by LC-SRM after 48 h treatment with EDN or kinase inhibitors at 1 µM. Only the single inhibitor which could reverse EDN induced E-Cadherin repression is shown. * p < 0.05 two-sided unpaired t-test.


F Clonogenic growth of indicated HPAF - II left untreated or in presence of 150nM LGK974 for 10 days ; representative of three independent replicates .

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F Clonogenic growth assay of indicated HPAF-II cells left untreated or in the presence of 150nM LGK974 for 10 days; representative of three independent replicates.


G Immunoblot of βcatenin expression from lysates of HPAF - II from indicated genotype , treated with vehicle or 100nM LGK974 for 48h ; representative of three biological replicates .

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G Immunoblot of cytoplasmic βcatenin expression from lysates of HPAF-II cells from indicated genotype, treated with vehicle or 100nM LGK974 for 48h; representative of three biological replicates.


A Immunoblot of CCNL1 FBXW7 expression in HPAF - II wild - type FBXW7 - / - , representative blot of 3 independent replicates .

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A Immunoblot of CCNL1 and FBXW7 expression in HPAF-II wild-type and FBXW7-/- cells, representative blot of 3 independent replicates.


B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF - II wild - type FBXW7 - / - , representative blot of 3 independent replicates . C of cycloheximide chase in B , mean ± of three independent replicates .

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B Immunoblot of CCNL1 expression following a cycloheximide chase in HPAF-II wild-type and FBXW7-/- cells , representative blot of 3 independent replicates. C Quantification of cycloheximide chase in B, mean ± SEM of three independent replicates.


D Immunoblot of CCNL1 expression in HPAF - II wild - type dN - Cul1 expressing , representative blot of 3 independent replicates .

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D Immunoblot of CCNL1 expression in HPAF-II wild-type and dN-Cul1 expressing cells, representative blot of 3 independent replicates.


G Immunoblot of lysates following cycloheximide treatment of HEK293T expressing wild - type or degron - mutated CCNL1 , representative blot of 3 independent replicates . H of cycloheximide chase in G , mean ± of three independent replicates .

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G Immunoblot of lysates following cycloheximide treatment of HEK293T cells expressing wild-type or degron-mutated CCNL1, representative blot of 3 independent replicates. H Quantification of cycloheximide chase in G, mean ± SEM of three independent replicates.


A Immunoblot of immunoprecipitation of FLAG - FBXW7 overexpressed in HEK293T , detecting endogenous CCNL1 . Representative of three independent replicates .

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A Immunoblot of immunoprecipitation of FLAG-FBXW7 overexpressed in HEK293T cells, detecting endogenous CCNL1. Representative of three independent replicates.


B Immunoblot of immunoprecipitation of FLAG - CCNL1 overexpressed in HEK293T , detecting endogenous FBXW7 . Representative of three independent replicates .

B Immunoblot of immunoprecipitation of FLAG-CCNL1 overexpressed in HEK293T cells, detecting endogenous FBXW7. Representative of three independent replicates.


C Immunoblot of ubiquitination demonstrating requirement of 325 - TPALS - 329 degron for ubiquitination of CCNL1 . Representative of three independent replicates .

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C Immunoblot of cellular ubiquitination assay demonstrating the requirement of the 325-TPALS-329 degron for ubiquitination of CCNL1. Representative of three independent replicates.


D Immunoblot of ubiquitination demonstrating requirement of FBXW7 for ubiquitination of CCNL1 . Representative of three independent replicates .

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D Immunoblot of cellular ubiquitination assay demonstrating the requirement of FBXW7 for ubiquitination of CCNL1. Representative of three independent replicates.


E Immunoblot of ubiquitination showing re - expression of FBXW7 in knockout leads to increased CCNL1 ubiquitination . Representative of three independent replicates .

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E Immunoblot of cellular ubiquitination assay showing re-expression of FBXW7 in knockout cells leads to increased CCNL1 ubiquitination. Representative of three independent replicates.


B Immunoblot of HPAF - II wild - type FBXW7 - / - released from overnight nocodazole treatment at indicated time points ,

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B Immunoblot of HPAF-II wild-type and FBXW7-/- released from overnight nocodazole treatment at indicated time points,


D Representative of GFP - tubulin labeled wild - type , FBXW7 - / - , CCNL1OE HPAF - II monitoring progression through cytokinesis following arrest in prometaphase using monastrol ( 150μM for 18 hours ) . E of cytokinetic timing , n = 20 , 25 , 26 ( wild - type , FBXW7 - / , CCNL1OE ) pooled from three independent experiments F Immunoblot of wild - type , FBXW7 - / - CCNL1OE HPAF - II lines demonstrating varying of CCNL1 .

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D Representative images of GFP-tubulin labeled wild-type, FBXW7-/-, and CCNL1OE HPAF-II cells monitoring cellular progression through cytokinesis following arrest in prometaphase using monastrol (150μM for 18 hours). E Quantification of cytokinetic timing, n=20, 25, 26 (wild-type, FBXW7-/, CCNL1OE ) pooled from three independent experiments F Immunoblot of wild-type, FBXW7-/- and CCNL1OE HPAF-II cell lines demonstrating varying levels of CCNL1.


A Clonogenic growth assays for HPAF - II wild - type FBXW7 - / - polyclonal , FBXW7 - / - clones in presence of various OTS964 doses for 14 days . Representative of 4 independent replicates , quantified by crystal violet absorbance at A595 B Clonogenic growth assays for HPAF - II wild - type CCNL1OE polyclonal , CCNL1OE clones in presence of various doses of OTS964 for 14 days . Representative of 4 independent replicates , quantified by crystal violet absorbance at

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A Clonogenic growth assays for HPAF-II wild-type and FBXW7-/- polyclonal, and FBXW7-/- clones in presence of various OTS964 doses for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595 and B Clonogenic growth assays for HPAF-II wild-type and CCNL1OE polyclonal, and CCNL1OE clones in presence of various doses of OTS964 for 14 days. Representative images of 4 independent replicates, quantified by crystal violet absorbance at A595


C Immunoblot for indicated from lysates extracted from FBXW7 - / - CCNL1OE clones .

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C Immunoblot for the indicated proteins from lysates extracted from the individual FBXW7-/- and CCNL1OE clones.


B Immunoblot of lysates from cervical lines demonstrating C33A ( FBXW7R465H ) express high of CCNL1 , representative of three independent replicates .

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B Immunoblot of lysates from cervical cell lines demonstrating C33A cells (FBXW7R465H) express high levels of CCNL1, representative of three independent replicates.


C Clonogenic growth of C33A , Caski , SiHa in presence of various doses of OTS964 for 14 days . Representative of 3 independent replicates , quantified by crystal violet absorbance at

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C Clonogenic growth assay of C33A, Caski, and SiHa cells in presence of various doses of OTS964 for 14 days. Representative images of 3 independent replicates, quantified by crystal violet absorbance at A595


( A ) Detection of VgpA in supernatants from indicated strains by immunoblotting ( IB ) with anti - VgpA antisera .

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(A) Detection of VgpA in supernatants from indicated strains by immunoblotting (IB) with anti-VgpA antisera.


( B ) T3SS2 translocation of VgpA into Caco - 2 using CyaA - ; VopV is a known T3SS2 effector . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) .

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(B) T3SS2 translocation of VgpA into Caco-2 cells using CyaA-based assay; VopV is a known T3SS2 effector. Error bars represent mean ± standard deviation (n=3 biologically independent experiments).


( C ) Localization of translocated VgpA ( upper panel ) VgpAL10A ( lower panel ) during infection .

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(C) Localization of translocated VgpA (upper panel) and VgpAL10A (lower panel) during infection.


( A ) Silver - stained PAGE gel of from a extract that interact with either GST - VgpA or GST glutathione agarose . bound by GST - VgpA ( red arrow ) , but not GST , was identified as EBP2 by mass spectrometry ; GST - VgpA is labeled with a blue arrow .

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(A) Silver-stained PAGE gel of proteins from a nuclear extract that interact with either GST-VgpA or GST glutathione agarose. The protein bound by GST-VgpA (red arrow), but not GST, was identified as EBP2 by mass spectrometry; GST-VgpA is labeled with a blue arrow.


( B ) Western blot using anti - EBP2 as antibody confirmed that EBP2 binds VgpA .

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(B) Western blot using anti-EBP2 as antibody confirmed that EBP2 binds VgpA.


( C ) Interaction of VgpA or VgpA mutants ( VgpAΔ10 VgpAL10A ) with EBP2 .

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(C) Interaction of VgpA or VgpA mutants (VgpAΔ10 and VgpAL10A) with EBP2.


( D ) Localization of VgpA ( upper panel ) or in VgpAL10A ( lower panel ) in HeLa after transfection . EBP2 was detected with anti - EBP2 antibody ( red ) , VgpA VgpAL10A were detected with anti - VgpA sera ( green ) nuclei were stained with DAPI ( blue ) . arrows indicate co - localization of EBP2 VgpA ( upper panel ) . VgpAL10A localizes primarily to nucleoplasm ( arrow , lower panel ) .

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(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes primarily to the nucleoplasm (white arrow, lower panel).


( D ) Localization of VgpA ( upper panel ) or in VgpAL10A ( lower panel ) in HeLa after transfection . EBP2 was detected with anti - EBP2 antibody ( red ) , VgpA VgpAL10A were detected with anti - VgpA sera ( green ) nuclei were stained with DAPI ( blue ) . arrows indicate co - localization of EBP2 VgpA ( upper panel ) . VgpAL10A

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(D) Localization of VgpA (upper panel) or in VgpAL10A (lower panel) in HeLa cells after transfection. EBP2 was detected with anti-EBP2 antibody (red), VgpA and VgpAL10A were detected with anti-VgpA sera (green) and nuclei were stained with DAPI (blue). White arrows indicate co-localization of EBP2 and VgpA (upper panel). VgpAL10A localizes


( A ) of uninfected Caco - 2 or Caco - 2 infected with indicated V . parahaemolyticus strains . were stained with DAPI ( blue ) anti - BrdU antibody ( green ) .

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(A) Fluorescence micrographs of uninfected Caco-2 cells or Caco-2 cells infected with the indicated V. parahaemolyticus strains. Cells were stained with DAPI (blue) and anti-BrdU antibody (green).


( B ) of Caco - 2 transfected with siRNA targeting EBP2 ( siEBP2 ) or control siRNA ( siNC ) subsequently infected with vopZ ' ΔvscN1 . were stained with anti - BrdU antibody ( green ) DAPI ( blue ) .

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(B) Fluorescence micrographs of Caco-2 cells transfected with siRNA targeting EBP2 (siEBP2) or control siRNA (siNC) and subsequently infected with vopZ'ΔvscN1. Cells were stained with anti-BrdU antibody (green) and DAPI (blue).


( C ) of BrdU - positive ( green ) among total ( green + blue ) was analyzed for 100 in three experiments . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) . One - way of variance ( ANOVA ) was used for statistical . * P < 0 . 05 ( when compared to uninfected Caco - 2 ) .

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(C) The percentage of BrdU-positive cells (green) among the total cells (green + blue) was analyzed for 100 cells in three experiments. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). One-way analysis of variance (ANOVA) was used for statistical analysis. *P<0.05 (when compared to the uninfected Caco-2 cells).


( D ) of BrdU + was analyzed as above for treated with siNC or siEBP2 . Error bars represent mean ± standard deviation ( n = 3 biologically independent experiments ) . Two - way ANOVA was used for statistical . * P < 0 . 05 ( when compared to siNC - transfected that are infected with vopZ ' ΔvscN1 ) .

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(D) The percentage of BrdU+ cells was analyzed as above for cells treated with siNC or siEBP2. Error bars represent mean ± standard deviation (n=3 biologically independent experiments). Two-way ANOVA was used for statistical analysis. *P<0.05 (when compared to siNC-transfected cells that are infected with vopZ'ΔvscN1).


( E ) Immunoblot of EBP2 Actin in Caco - 2 transfected with siEBP2 or siNC .

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(E) Immunoblot of EBP2 and Actin in Caco-2 cells transfected with siEBP2 or siNC.


( A ) Localization of c - Myc in uninfected Caco - 2 ( first row ) , Caco - 2 infected with V . parahaemolyticus vopZ ' ΔvscN1 ( wild type VgpA , second row ) , Caco - 2 infected with vopZ ' ΔvscN1vgpA ' ( third row ) , Caco - 2 infected with vopZ ' ΔvscN1vgpA ' : pvgpA ( fourth row ) Caco - 2 infected with vopZ ' ΔvscN1vgpA ' : pvgpAL10A ( fifth row ) . Fixed were stained with DAPI ( blue ) , anti - EBP2 ( red ) , anti c - Myc ( green ) antibodies . arrows are examples indicating co - localization of EBP2 c - Myc .

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(A) Localization of c-Myc in uninfected Caco-2 cells (first row), Caco-2 cells infected with V. parahaemolyticus vopZ'ΔvscN1 (wild type VgpA, second row), Caco-2 cells infected with vopZ'ΔvscN1vgpA' (third row), Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpA (fourth row) and Caco-2 cells infected with vopZ'ΔvscN1vgpA':pvgpAL10A (fifth row). Fixed cells were stained with DAPI (blue), and anti-EBP2 (red), and anti c-Myc (green) antibodies. White arrows are examples indicating the co-localization of EBP2 and c-Myc.


( B Detection of c - Myc or EBP2 in extracts from uninfected Caco - 2 or after infection with indicated strains . Following infection , extracts were obtained loaded on A / G beads cross - linked with anti - EBP2 ( B , IP : anti - EBP2 ) Input eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band ( normalized to uninfected ) from three independent experiments are presented under signals of blot of IP samples . n . d . , not determined .

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(B Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked with anti-EBP2 (B, IP: anti-EBP2) Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


C ) Detection of c - Myc or EBP2 in extracts from uninfected Caco - 2 or after infection with indicated strains . Following infection , extracts were obtained loaded on A / G beads cross - linked anti - C - Myc ( C , IP : anti - c - Myc ) antibodies , respectively . Input eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band ( normalized to uninfected ) from three independent experiments are presented under signals of blot of IP samples . n . d . , not determined .

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C) Detection of c-Myc or EBP2 in nuclear extracts from uninfected Caco-2 cells or after infection with indicated strains. Following infection, nuclear extracts were obtained and loaded on protein A/G beads cross-linked anti-C-Myc (C, IP: anti-c-Myc) antibodies, respectively. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to uninfected) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


( D ) Purified EBP2 , c - Myc GST - VgpA or GST - VgpAL10A were mixed then pulled down with A / G beads cross - linked with anti - EBP2 . Input eluates were blotted with anti - EBP2 , anti - c - Myc or anti - VgpA antibodies , respectively . Quantified value ( mean ± standard deviation ) as measured by band ( normalized to EBP2 + c - Myc sample without GST - VgpA GST - VgpAL10A ) from three independent experiments are presented under signals of blot of IP samples . n . d . , not determined .

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(D) Purified proteins EBP2, c-Myc and GST-VgpA or GST-VgpAL10A were mixed and then pulled down with protein A/G beads cross-linked with anti-EBP2. Input and eluates were blotted with anti-EBP2, anti-c-Myc or anti-VgpA antibodies, respectively. Quantified value (mean ± standard deviation) as measured by band intensity (normalized to EBP2+c-Myc sample without GST-VgpA and GST-VgpAL10A) from three independent experiments are presented under the signals of the blot of IP samples. n.d., not determined.


( E ) Caco - 2 were infected with indicated strains treated with cycloheximide ( CHX ) for indicated time . Whole lysates were analyzed by western blot using anti - c - Myc anti - Actin antibody .

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(E) Caco-2 cells were infected with the indicated strains and treated with cycloheximide (CHX) for the indicated time. Whole cells lysates were analyzed by western blot using anti-c-Myc and anti-Actin antibody.


( A ) Amounts of rRNA were from uninfected Caco - 2 or infected with indicated strains . * P < 0 . 05 ( when compared to uninfected Caco - 2 ) .

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(A) Amounts of rRNA were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).


( B ) Amounts of rRNA in uninfected or infected Caco - 2 transfected with control ( siNC ) or EBP2 - targeting ( siEBP2 ) siRNAs . * P < 0 . 05 ( when compared to siNC - transfected that are infected with vopZ ' ΔvscN1 ) .

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(B) Amounts of rRNA in uninfected or infected Caco-2 cells transfected with control (siNC) or EBP2-targeting (siEBP2) siRNAs. *P<0.05 (when compared to the siNC-transfected cells that are infected with vopZ'ΔvscN1).


( C ) Amounts of polymerase I ( POLR1A ) were from uninfected Caco - 2 or infected with indicated strains . * P < 0 . 05 ( when compared to uninfected Caco - 2 ) .

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(C) Amounts of RNA polymerase I (POLR1A) were from uninfected Caco-2 cells or cells infected with indicated strains. *P<0.05 (when compared to the uninfected Caco-2 cells).


( D ) . Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 38 h after infection . * P < 0 . 05 ( when compared to uninfected rabbits at 18 hours ) . ΔP < 0 . 05 ( when compared to uninfected rabbits at 38 hours post infection ) .

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(D). Amounts of rRNA in intestinal homogenates from infant rabbits infected with indicated strains 18 and 38 h after infection. *P<0.05 (when compared to the uninfected rabbits at 18 hours). ΔP<0.05 (when compared to the uninfected rabbits at 38 hours post infection).


( A ) VgpA ( upper panel : infected with WT ) VgpAL10A ( lower panel : infected with vgpAL10A ) localization in intestinal epithelial of infected . Arrows in upper panel indicate VgpA localized as in nucleus ; arrow in lower panel indicates VgpAL10A localized diffusively in nucleoplasm .

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(A) VgpA (upper panel: animals infected with WT) and VgpAL10A (lower panel: animals infected with vgpAL10A) localization in intestinal epithelial cells of infected animals. Arrows in upper panel indicate VgpA localized as punctae in the nucleus; the arrow in the lower panel indicates VgpAL10A localized diffusively in the nucleoplasm.


( B ) VgpA VgpAL10A interactions with EBP2 in intestinal epithelial isolated from infant rabbits infected with WT or vgpAL10A V . parahaemolyticus . Inputs represent detected in intestinal epithelial of uninfected rabbits or rabbits infected with indicated strains . Outputs represent eluates from A / G beads cross - linked with anti - EBP2 ( IP : anti - EBP2 ) or anti - VgpA ( IP : anti - VgpA ) antibodies analyzed with anti - VgpA anti - EBP2 antibodies .

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(B) VgpA and VgpAL10A interactions with EBP2 in intestinal epithelial cells isolated from infant rabbits infected with WT or vgpAL10A V. parahaemolyticus. Inputs represent proteins detected in intestinal epithelial cells of uninfected rabbits or rabbits infected with the indicated strains. Outputs represent eluates from protein A/G beads cross-linked with anti-EBP2 (IP: anti-EBP2) or anti-VgpA (IP: anti-VgpA) antibodies and analyzed with anti-VgpA and anti-EBP2 antibodies.


( C ) Frozen sections from uninfected rabbits or rabbits infected with indicated strains for 18 hours were stained with anti - Ki67 antibodies to detect actively dividing ( green ) , phalloidin to visualize F - actin ( red ) DAPI to detect nuclei ( blue ) .

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(C) Frozen sections from uninfected rabbits or rabbits infected with the indicated strains for 18 hours were stained with anti-Ki67 antibodies to detect actively dividing cells (green), phalloidin to visualize F-actin (red) and DAPI to detect nuclei (blue).


( D ) Eighteen hours after infant rabbits were infected with WT ( middle panel ) or vgpAL10A ( lower panel ) V . parahaemolyticus , fixed sections from small intestine were stained with antibodies to c - Myc ( green ) , EBP2 ( red ) , DAPI ( blue ) . Uninfected rabbits were processed similarly were included as a control ( upper panel ) . arrows indicate co - localization of c - Myc EBP2 ( middle panel ) .

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(D) Eighteen hours after infant rabbits were infected with WT (middle panel) or vgpAL10A (lower panel) V. parahaemolyticus, fixed sections from the small intestine were stained with antibodies to c-Myc (green), EBP2 (red), and DAPI (blue). Uninfected rabbits were processed similarly and were included as a control (upper panel). White arrows indicate co-localization of c-Myc and EBP2 (middle panel).


( A ) CFU in homogenates from distal small intestines of infant rabbits infected with indicated strains 18 , 28 38 h following inoculation . Black horizontal indicates mean CFU ( n = 7 - 10 / group ) . One - way ANOVA was used for statistical . * P < 0 . 05 for indicated comparison .

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(A) CFU in homogenates from the distal small intestines of infant rabbits infected with the indicated strains 18, 28 and 38 h following inoculation. Black horizontal line indicates mean CFU (n=7-10 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 for the indicated comparison.


( B ) H & E stained sections from distal small intestine from rabbits infected with indicated strains 18 hours after infection . Black arrows point to colonies closely attached to epithelium .

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(B) H&E stained sections from the distal small intestine from the rabbits infected with the indicated strains 18 hours after infection. Black arrows point to bacterial colonies closely attached to the epithelium.


( C ) Fluid accumulation in rabbits infected with indicated strains 38 hours post inoculation . Error bars represent mean ± standard deviation ( n = 7 / group ) . One - way ANOVA was used for statistical . * P < 0 . 05 ( when compared to rabbits infected with WT ) .

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(C) Fluid accumulation in rabbits infected with the indicated strains 38 hours post inoculation. Error bars represent mean ± standard deviation (n=7 animals/group). One-way ANOVA was used for statistical analysis. *P<0.05 (when compared to the rabbits infected with WT).


C , AltFUS ( arrow ) endogenous expression in Human tissues ( brain , kidney - 100 μg ) , in HEK293 HeLa cultured ( 100 μg ) using over - expression of altFUS CDS in HEK293 ( 50 μg ) as positive control ( representative image from n = 3 ) . asterisk indicates a species detected with anti - altFUS antibody specifically in brain .

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C, AltFUS (arrow) endogenous expression in Human tissues (brain, muscles and kidney - 100 μg), in HEK293 and HeLa cultured cells (100 μg) and using the over-expression of altFUS CDS in HEK293 cells (50 μg) as positive control (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


AltFUS ( arrow ) endogenous expression in motor cortex of three C9orf72 three sporadic ALS ( D ) ( representative image from n = 3 ) . asterisk indicates a species detected with anti - altFUS antibody specifically in brain .

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AltFUS (arrow) endogenous expression in the motor cortex of three C9orf72 and three sporadic ALS patients (D) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


AltFUS ( arrow ) endogenous expression in iPSC - derived motor neurons of three lines from controls from ALS ( E ) ( representative image from n = 3 ) . asterisk indicates a species detected with anti - altFUS antibody specifically in brain .

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AltFUS (arrow) endogenous expression in iPSC-derived motor neurons of three lines from controls and from ALS patients (E) (representative image from n=3). The asterisk indicates a protein species detected with the anti-altFUS antibody specifically in the brain.


I , Endogenous altFUS mitochondrial expression in HEK293 following fractionation ( representative image from n = 3 ) , with Tubulin as a marker of fraction VDAC as a marker of mitochondrial fraction . We used siFUS transfected as a negative control altFUS transfected as a positive control for altFUS expression ( WCL = Whole Lysate , Cyto = Cytosol fraction , Mito = mitochondrial fraction ) .

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I, Endogenous altFUS mitochondrial expression in HEK293 cells following fractionation (representative image from n=3), with Tubulin as a marker of the cytosolic fraction and VDAC as a marker of the mitochondrial fraction. We used siFUS transfected cells as a negative control and altFUS transfected cells as a positive control for altFUS expression (WCL = Whole Cell Lysate, Cyto = Cytosol fraction, Mito = mitochondrial fraction).


J , Representative of mitochondrial network ( Tomm20 in red ) in mock altFUS - Flag ( green ) transfected HeLa ( n = 3 ) . scale bar corresponds to 10μm . K , Proportion of tubules globules in mitochondrial network of mock HeLa HeLa transfected with altFUS - Flag was done over a minimum of 100 across a technical duplicate per independent experiments ( n = 3 , i . e . a minimum of 300 per biological conditions , p - value < 0 . 001 , Mann - Whitney U ) . boxes extend to 25th 75th percentiles , with median marked . whiskers correspond to 5th 95th percentiles .

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J, Representative images of the mitochondrial network (Tomm20 in red) in mock and altFUS-Flag (green) transfected HeLa cells (n=3). The white scale bar corresponds to 10μm. K, Proportion of tubules and globules in the mitochondrial network of mock HeLa cells and HeLa cells transfected with altFUS-Flag Quantification was done over a minimum of 100 cells across a technical duplicate per independent experiments (n=3, i.e. a minimum of 300 cells per biological conditions, p-value < 0.001, Mann-Whitney U test). The boxes extend to the 25th and 75th percentiles, with the median marked. The whiskers correspond to the 5th and 95th percentiles.


E , Scatter plot of identified by AP - MS indicating their enrichment ( Fold - change over control ) their SAINT probability score . above 0 . 8 threshold ( grey ) are indicated in black , others in grey . AltFUS is indicated in red ( bait ) preys known to regulate autophagy or stress response are indicated in green .

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E, Scatter plot of the proteins identified by AP-MS indicating their enrichment (Fold-change over control) and their SAINT probability score. Proteins above the 0.8 threshold (grey line) are indicated in black, others in grey. AltFUS is indicated in red (bait) and preys known to regulate the autophagy or the cellular stress response are indicated in green.


B , LC3 - II accumulation after bafilomycin treatment from mock , altFUS , FUS , FUS ( Ø ) , FUS - R495x , FUS ( Ø ) - R495x transfected FUS ( Ø ) - R495x altFUS co - transfected across 3 independent experiments ( n = 3 , mean ± SD ) . corresponds to treated / untreated of LC3 - II abundance Statistical significance is relative to mock condition unless otherwise indicated ( * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , n . s . = non - significant , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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B, LC3-II accumulation after bafilomycin treatment from mock, altFUS, FUS, FUS(Ø), FUS-R495x, FUS(Ø)-R495x transfected cells and FUS(Ø)-R495x and altFUS co-transfected cells across 3 independent experiments (n=3, mean ± SD). The quantification corresponds to the treated/untreated ratio of LC3-II abundance Statistical significance is relative to the mock condition unless otherwise indicated (**** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, n.s. = non-significant, two-way ANOVA with Tukey's multiple comparison correction).


D - E , of FUS granules , number ( D ) ( μm2 ) ( E ) in over - expressing bicistronic ( + ) or monocistronic ( - ) construct for FUS , FUS - G156E , FUS - R495x , FUS - K510E , FUS - Q519x , FUS - Q519I - fs527x , FUS - R521C , FUS - P525L . Statistical comparisons are made between bicistronic monocistronic versions of each construct ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , * * * = p value < 0 . 001 , * * = p value < 0 . 01 , * = p value < 0 . 05 , n . s . = non - significant , one - way ANOVA with Sidak ' s multiple comparison ) .

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D-E, Quantification of FUS cytoplasmic granules, number (D) and area (μm2) (E) in cells over-expressing the bicistronic (+) or monocistronic (-) construct for FUS, FUS-G156E, FUS-R495x, FUS-K510E, FUS-Q519x, FUS-Q519I-fs527x, FUS-R521C, and FUS-P525L. Statistical comparisons are made between bicistronic and monocistronic versions of each construct (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, *** = p value < 0.001, ** = p value < 0.01, * = p value < 0.05, n.s. = non-significant, one-way ANOVA test with Sidak's multiple comparison).


C - E , Locomotion represented by of climbing success in control RU - 486 - treated transgenic Drosophila expressing mCherry or altFUS ( C ) , bicistronic FUS or monocistronic FUS ( Ø ) ( D ) , bicistronic FUS - R495x or monocistronic FUS ( Ø ) - R495x ( E ) at day 1 , 10 20 post - induction . Statistical comparisons were made between each population ( n = 4 - biological replicates ) . Indicated significance is between monocistronic bicistronic transgenic flies of RU - 486 - treated population ( mean ± SD , n . s . = non - significant , * = p value < 0 . 05 , * * * = p value < 0 . 001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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C-E, Locomotion assay represented by the percentage of climbing success in control and RU-486-treated transgenic Drosophila expressing mCherry or altFUS (C), the bicistronic FUS or the monocistronic FUS(Ø) (D), and the bicistronic FUS-R495x or the monocistronic FUS(Ø)-R495x (E) at day 1, 10 and 20 post-induction. Statistical comparisons were made between each population (n=4 - biological replicates). Indicated significance is between the monocistronic and the bicistronic transgenic flies of the RU-486-treated population (mean ± SD, n.s. = non-significant, * = p value < 0.05, *** = p value < 0.001, two-way ANOVA with Tukey's multiple comparison correction).


A , Graphical representation of FUS synonymous mutations ( yellow ) found in ALS . canonical FUS mRNA is represented in blue ( ENST00000254108 or NM _ 004960 ) . FUS coding is indicated in blue , altFUS coding is indicated in green . B , by confocal microscopy of TDP - 43 ( ) , FUS ( GFP tagged - green ) altFUS ( Flag tagged - red ) in HeLa over - expressing GFP - FUS ( Flag ) , GFP - FUS ( P31L - Flag ) - S44 = , GFP - FUS ( A38V - Flag ) - G51 = , GFP - FUS ( A46V - Flag ) - G59 = , GFP - FUS ( R64P - Flag ) - S77 = ( representative from n = 3 ) . arrows indicate some TDP - 43 . scale bar corresponds to 10 μm . C , of with TDP - 43 in HeLa ( see panel B ) . data are represented as fold - change compared to GFP - FUS ( Flag ) expressing . Statistical significance is indicated above bars ( n = 3 - biological replicates , mean ± SD , * * * * = p value < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparison correction ) .

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A, Graphical representation of FUS synonymous mutations (yellow) found in ALS patients. The canonical FUS mRNA is represented in dark blue (ENST00000254108 or NM_004960). The FUS protein coding sequence is indicated in light blue, and the altFUS protein coding sequence is indicated in green. B, Images by confocal microscopy of TDP-43 (white), FUS (GFP tagged - green) and altFUS (Flag tagged - red) in HeLa cells over-expressing GFP-FUS(Flag), GFP-FUS(P31L-Flag)-S44=, GFP-FUS(A38V-Flag)-G51=, GFP-FUS(A46V-Flag)-G59=, GFP-FUS(R64P-Flag)-S77= (representative images from n=3). White arrows indicate some TDP-43 aggregates. The white scale bar corresponds to 10 μm. C, Quantification of cells with TDP-43 aggregates in HeLa cells (see panel B). The data are represented as the fold-change compared to the GFP-FUS(Flag) expressing cells. Statistical significance is indicated above the bars (n=3 - biological replicates, mean ± SD, **** = p value < 0.0001, two-way ANOVA with Tukey's multiple comparison correction).


A . Representative pseudo colour of sypHy in WT BsnGT hippocampal neurons at , upon stimulation with 40 900 APs at 20Hz ( in presence of bafilomycin A1 ) upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation . B . Average traces of normalised change ( ΔF / F0 ) of sypHy in WT ( black ) BsnGT ( red ) neurons as described in A . Intensities were normalized to peak of NH4Cl response . C . Quantifications of mean RRP fraction in WT BsnGT . D . Frequency distribution histograms of response amplitudes of 1050 synaptic ( from 6 independent experiments ) to stimulation with 40 AP . distribution is shifted to lower values in BsnGT . E . Quantifications of mean TRP fraction in WT BsnGT . F . Frequency distribution histograms of synaptic response amplitudes in BsnGT WT neurons stimulated with 900 AP . Note shift in distribution between genotypes . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . In frequency distribution histograms ( D F ) black lines depict superimposed Gaussian fits for each group . sample n ( in parentheses ) corresponds to number of analysed imaging experiments ( B , C , E ) In D F , data of 1050 synapses per genotype were processed . Data is obtained from 6 ( A - F ) independent culture preparations . Significance was assessed with Student ' s t - ; * * * p < 0 . 001 . Scale bar is 2 µm in

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A. Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation. B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy in WT (black) and BsnGT (red) neurons as described in A. Intensities were normalized to the peak of NH4Cl response. C. Quantifications of mean RRP fraction in WT and BsnGT. D. Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta (from 6 independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT. E. Quantifications of mean TRP fraction in WT and BsnGT. F. Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) In D and F, the data of 1050 synapses per genotype were processed. Data is obtained from 6 (A-F) independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 2 µm in A


G , Representative of Syt1Ab uptake ( magenta ) driven by endogenous network activity in hippocampal neurons ( 18 DIV ) from WT BsnGT mice . VGLUT1 ( green ) marks excitatory presynapses ( upper panels ) VGAT inhibitory ones ( lower panels ) . H . of normalized IF of Syt1Ab uptake in excitatory ( red boxes ) inhibitory ( pink boxes ) synapses on experiments in G . I . of fraction of active ( Syt1Ab - labelled ) excitatory inhibitory synapses on experiments illustrated in G . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . dashed lines in H depict IF in WT that were used for normalization . sample n ( in parentheses ) corresponds to number of analysed ( G , Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - ; * * * p < 0 . 001 . Scale bar is 5 µm in

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G, Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and BsnGT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels). H. Quantification of normalized IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in G. I. Quantification of fraction of active (Syt1Ab-labelled) excitatory and inhibitory synapses on experiments illustrated in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in H depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images (G, Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in G


J . Representative of Syt1Ab uptake ( magenta ) upon depolarization with 50 mM KCl . Identical cultures were applied as in G . K . of normalized IF of Syt1Ab uptake on experiments from J . L . of fraction of active excitatory inhibitory synapses in J . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . dashed lines in K depict IF in WT that were used for normalization . sample n ( in parentheses ) corresponds to number of analysed J ) . Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - ; * * * p < 0 . 001 . Scale bar is 5 µm in J .

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J. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in G. K. Quantification of normalized IF of Syt1Ab uptake on experiments from J. L. Quantification of fraction of active excitatory and inhibitory synapses in J. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in K depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images J). Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in J.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted for WT BsnGT neurons treated with a CDK5 inhibitor roscovitine ( rosc , 100µM , gray pink trace ) or vehicle ( black red trace ) for 30 minutes before stimulation with 40 900 APs in presence of bafilomycin A . C , D . Plots show mean values of RRP ( C ) TRP ( D ) fraction for both genotypes before after treatment . E . Roscovitine treatment has a significantly higher effect on RRP TRP in BsnGT neurons compared to WT . Data information : n corresponding to number of imaging experiments done on 4 independent preparations is given in brackets for each . In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . Significance was assessed by two - way ANOVA with Tukey ´ s multiple comparison ( C , D ) by Student ' s t - ( E ) * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 2µm .

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

A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT. Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.


A . Representative immunoblot with antibody against CDK5 - dependent pSer551 of Syn1 ( pSer551Syn1 ) total Syn1 total stain used as loading control for of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT BsnGT mice . B , C . of pSer551Syn1 / Syn1 ( in B ) total Syn1 ( in C ) abundance on blots from A . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample n ( in brackets ) corresponds to number of independently processed in B C For statistics , student ' s t - in B C were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

A. Representative immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice. B, C. Quantification of pSer551Syn1 / Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from A. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C For statistics, student's t-test in B and C were used, *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative of 19 DIV cultured hippocampal neurons from WT BsnGT immunostained for pSer551Syn1 total Syn1 . F . of IF of pSer551Syn1 in synapses from WT , BsnGT , WT treated with roscovitine . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample n ( in brackets ) corresponds to number of independent ( E , quantified from two ( F ) independent cultures per condition . For statistics , one - way ANOVA with Sidak ´ s multiple comparisons in F were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm

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E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1. F. Quantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT, and WT cells treated with roscovitine. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images (E, quantified from two (F) independent cultures per condition. For statistics, one-way ANOVA with Sidak´s multiple comparisons test in F were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in


G . Representative of 21 DIV hippocampal WT BsnGT neurons at baseline activity , upon network activity silencing ( TTX , 1 µM , 72 h ) or in conditions of increased activity ( BIC , 30 µM , 48 h ) H . of experiment in G . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample n ( in brackets ) corresponds to number of independent , G ) quantified from four ( H ) independent cultures per condition . For statistics two - way ANOVA with Tukey ´ s multiple comparisons in H were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm in G .

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G. Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h) H. Quantification of experiment in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images , G) quantified from four (H) independent cultures per condition. For statistics two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in G.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted from WT BsnGT hippocampal neurons treated with calcineurin inhibitor FK506 . Data information : Scale bar is 2 µm .

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A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted from WT and BsnGT hippocampal neurons treated with calcineurin inhibitor FK506. Data information: Scale bar is 2 µm.


C , D . of RRP TRP fractions in WT BsnGT neurons from experiment in A , B . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets for each reflect number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison for C D significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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C, D. Quantification of the RRP and TRP fractions in WT and BsnGT neurons from experiment in A, B. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of analysed imaging experiments done on 3 independently prepared cultures per genotype Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test for C and D significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative Western blot on P2 fractions prepared from hippocampal tissue of WT BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 total SNAP25 . F . of blot in E shows lower pThr138SNAP25 to total SNAP25 IF in BsnGT compared to WT . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets for each reflect number of per genotype used for WB in F Statistical significance was assessed using Student ' s t - in F , significance is depicted as follows : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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E. Representative Western blot on P2 fractions prepared from hippocampal tissue of WT and BsnGT mice probed with antibodies against SNAP25 phosphorylated onThr138 and total SNAP25. F. Quantification of blot in E shows lower pThr138SNAP25 to total SNAP25 IF ratio in BsnGT compared to WT. Data information: In all plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets for each quantification and reflect the number of animals per genotype used for WB in F Statistical significance was assessed using Student's t-test in F, significance is depicted as follows: *p≤0.05, **p < 0.01, ***p< 0.001.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted for WT BsnGT neurons without treatment ( black red ) upon treatment with adenylyl cyclase activator forskolin ( gray pink ) or PKA inhibitor H89 ( grey orange ) . Data information : scale bar is 2 µm in

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A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons without treatment (black and red) and upon treatment with adenylyl cyclase activator forskolin (gray and pink) or PKA inhibitor H89 (light grey and orange). Data information: The scale bar is 2 µm in A


C , D . of RRP TRP fraction from experiment in A B . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample is given in brackets corresponds to number of analysed independent imaging experiments performed on three independently prepared culture batches in ( C D ) , statistical significance was assessed in C , D using two - way ANOVA with Tukey ´ s multiple comparison * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of the RRP and TRP fraction from experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments performed on three independently prepared culture batches in (C and D), The statistical significance was assessed in C, D using two-way ANOVA with Tukey´s multiple comparison test *p≤0.05, **p < 0.01, ***p< 0.001.


E . Representative immunoblot with antibody against pSer9Syn1 total Syn1 total stain of hippocampal P2 fraction from WT BsnGT mice prepared in presence of KN 93 to isolate PKA - dependent regulation of pSer9Syn1 . F , G . of pSer9Syn1 / Syn1 ( in F ) Syn1 ( in G ) on blots from E . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample is given in brackets corresponds to number of analysed independent imaging experiments samples prepared from in F G , statistical significance was assessed in F G using Student ' s t - as is depicted in graphs as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

E. Representative immunoblot with antibody against pSer9Syn1 and total Syn1 and total protein stain of hippocampal P2 fraction from WT and BsnGT mice prepared in the presence of KN 93 to isolate PKA-dependent regulation of pSer9Syn1. F, G. Quantification of pSer9Syn1/Syn1 ratio (in F) and Syn1 (in G) on blots from E. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the number of analysed independent imaging experiments samples prepared from individual animals in F and G, The statistical significance was assessed in F and G using Student's t-test as is depicted in graphs as *p≤0.05, **p < 0.01, ***p< 0.001.


H . Representative of hippocampal neurons from WT BsnGT mice labelled with antibodies against pSer9Syn1 total Syn1 . Neurons were pre - treated ( 4 µM , 1hr ) with KN93 to isolate PKA - dependent phosphorylation then treated in addition with forskolin or H89 before fixation . I . of in H . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers + indicates mean . sample is given in brackets corresponds to independent visual fields obtained from 2 independent culture preparations in I . statistical significance was assessed in I using two - way ANOVA with Tukey ´ s multiple comparison as * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . scale bar is 5 µm in H .

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H. Representative images of hippocampal neurons from WT and BsnGT mice labelled with antibodies against pSer9Syn1 and total Syn1. Neurons were pre-treated (4 µM, 1hr) with KN93 to isolate PKA-dependent phosphorylation and then treated in addition with forskolin or H89 before fixation. I. Quantification of staining in H. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size is given in brackets and corresponds to the independent visual fields obtained from 2 independent culture preparations in I. The statistical significance was assessed in I using two-way ANOVA with Tukey´s multiple comparison test as *p≤0.05, **p < 0.01, ***p< 0.001. The scale bar is 5 µm in H.


A , B . Representative pseudo colour ( A ) average traces ( B ) of sypHy plotted for untreated WT BsnGT neurons ( black red ) cultures treated with of 6 - Bnz - cAMPS ( 50 µM , 1 hour ; gray pink ) rolipram ( 1 µM , 30 min , grey orange ) . Data information : Scale bar is 2µm .

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A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for untreated WT and BsnGT neurons (black and red) and cultures treated with of 6-Bnz-cAMPS (50 µM, 1 hour; gray and pink) and rolipram (1 µM, 30 min, light grey and orange). Data information: Scale bar is 2µm.


C , D . Quantifications of RRP TRP fractions in experiment in A B . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets corresponds to number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two - way ANOVA with Tukey ´ s multiple comparison in C , D : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantifications of RRP and TRP fractions in experiment in A and B. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the number of imaging experiments performed on three independently prepared cultures Statistical significance was assessed using two-way ANOVA with Tukey´s multiple comparison test in C, D : *p≤0.05, **p < 0.01, ***p< 0.001.


H . of pSer145PDE4B to total PDE4B from experiment in I . Data information : In plots , interquartile range median are depicted as boxes , minimal maximal values as whiskers , + indicates mean . sample is given in brackets corresponds to used for sample preparation Statistical significance was assessed using Student ' s t - in H is given as : * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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H. Quantification of pSer145PDE4B to the total PDE4B ratio from experiment in I. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The sample size is given in brackets and corresponds to the individual animals used for sample preparation Statistical significance was assessed using Student's t-test in H and is given as: *p≤0.05, **p < 0.01, ***p< 0.001.


I . Representative immunoblot with antibody against CDK5 - dependent pSer145PDE4B total PDE4B on hippocampal tissue from WT BsnGT .

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I. Representative immunoblot with antibody against CDK5-dependent pSer145PDE4B and total PDE4B on hippocampal tissue from WT and BsnGT animals.


Representative of normal injured acinar . Scale bar , 2μm . Right ( insets ) - higher magnification of boxed regions from left show emergence of double - membraned autophagic vesicles engulfing mature granules , mitochondria , rough ER 24h after cerulein ( CER ) injury . Scale bar , 500nm . Arrowheads mark degradation events without double membranes . Graph of number of acinar granules per in vehicle vs . 24h CER treatment . Each data point = total granules in a in a single counted from one mouse . Red indicates mean . Graph of number of degradation events scored in vehicle vs . 24h CER treatment . Each data point = total events in a in a single counted from one mouse . Red indicates mean .

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Representative TEM images of normal and injured acinar cells. Scale bar, 2μm. Right (insets)- higher magnification images of boxed regions from left show emergence of double-membraned autophagic vesicles engulfing mature granules, mitochondria, and rough ER 24h after cerulein (CER) injury. Scale bar, 500nm. Arrowheads mark degradation events without double membranes. Graph of the number of acinar cell granules per cell in vehicle vs. 24h CER treatment. Each data point=total granules in a cell in a single cell counted from one mouse. Red line indicates mean. Graph of the number of degradation events scored in vehicle vs. 24h CER treatment. Each data point=total events in a cell in a single cell counted from one mouse. Red line indicates mean.


mRNA expression by qRT - PCR of Rab5 , Rab7a , Rab7b , Rab9 . Significance determined by one - way ANOVA , Tukey post hoc . Each data point represents mean of triplicate technical replicates from a single mouse , n = 3 - 4 mice in independent experiments . Error bars denote standard deviation .

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mRNA expression by qRT-PCR of Rab5, Rab7a, Rab7b, and Rab9. Significance determined by one-way ANOVA, Tukey post hoc. Each data point represents the mean of triplicate technical replicates from a single mouse, n=3-4 mice in independent experiments. Error bars denote standard deviation.


Top - Representative RAB7 expression in base of stomach units ( ZCs ) in vehicle or 24h HDT by immunohistochemistry . Arrowheads point to RAB7 vesicle . Scale bar 20µm . Bottom - Representative ATF3 expression in base of stomach units ( ZCs ) by immunohistochemistry . ATF3 appears in nuclei following 24h high - dose tamoxifen ( HDT ) . Scale bar 20µm . Stomach unit base outlined by dashed black .

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Top - Representative RAB7 expression in base of stomach units (ZCs) in vehicle or 24h HDT by immunohistochemistry. Arrowheads point to RAB7 vesicle aggregates. Scale bar 20µm. Bottom - Representative ATF3 expression in base of stomach units (ZCs) by immunohistochemistry. ATF3 appears in nuclei following 24h high-dose tamoxifen (HDT). Scale bar 20µm. Stomach unit base outlined by dashed black line.


Western blot of ATF3 expression over HDT CER timecourses with β - Actin loading control .

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Western blot of ATF3 expression over HDT and CER timecourses with β-Actin loading control.


Top - Representative RAB7 in pancreatic acinar . Arrowheads indicate RAB7 vesicles . Scale bar 20µm . Bottom - Representative ATF3 expression in pancreatic acinar . Nuclei express ATF3 after 24h cerulein ( CER ) injury . Scale bar 20µm .

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Top - Representative RAB7 staining in pancreatic acinar cells. Arrowheads indicate RAB7 vesicles. Scale bar 20µm. Bottom - Representative ATF3 expression in pancreatic acinar cells. Nuclei express ATF3 after 24h cerulein (CER) injury. Scale bar 20µm.


ChIP - PCR on mouse stomach tissue at vehicle 6 hour HDT . probed with ATF3 , Histone H3 antibodies , or a Rabbit IgG control . Also included is 2 % input water to control for contamination . Rab7b amplicon is from a conserved , putative ATF3 - binding site in first intron . Amplicon from Atf3 is a positive control from a previously characterized ATF3 - binding site ; negative control is from a site in Rab9a lacking putative ATF3 binding motifs .

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ChIP-PCR on mouse stomach tissue at vehicle and 6 hour HDT. Chromatin probed with ATF3, Histone H3 antibodies, or a Rabbit IgG control. Also included is the 2% chromatin input and water to control for DNA contamination. Rab7b amplicon is from a conserved, putative ATF3-binding site in the first intron. Amplicon from Atf3 is a positive control from a previously characterized ATF3-binding site; negative control is from a site in Rab9a lacking putative ATF3 binding motifs.


mRNA expression of ATF3 by qRT - PCR in control , ATF3 knockdown ( KD ) , ATF3 overexpression ( OE ) AGS lines generated using a PiggybacTM transposon system . Significance by one - way ANOVA , Dunnett post - hoc . Datapoint = mean of technical triplicates in a single mouse . n = 3 mice . Error bars denote standard deviation . mRNA expression of RAB7B by qRT - PCR in AGS lines from 3A . Statistics plotting as for panel A .

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mRNA expression of ATF3 by qRT-PCR in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cell lines generated using a PiggybacTM transposon system. Significance by one-way ANOVA, Dunnett post-hoc. Datapoint = mean of technical triplicates in a single mouse. n=3 mice. Error bars denote standard deviation. mRNA expression of RAB7B by qRT-PCR in AGS cell lines from 3A. Statistics and plotting as for panel A.


Top - Immunofluorescence for RAB7 ( red ) DAPI ( blue , nuclei ) in control , ATF3 knockdown ( KD ) , ATF3 overexpression ( OE ) AGS . Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue . Bottom - Immunofluorescence for ATF3 ( purple ) Cathepsin D ( CTSD , green , lysosomes ) in AGS . Scale bar , 20µm .

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Top - Immunofluorescence for RAB7 (red) and DAPI (blue, nuclei) in control, ATF3 knockdown (KD), and ATF3 overexpression (OE) AGS cells. Arrowhead indicates aggregated RAB7 vesicular formation similar to those observed in tissue. Bottom - Immunofluorescence for ATF3 (purple) and Cathepsin D (CTSD, green, lysosomes) in AGS cells. Scale bar, 20µm.


Representative immunofluorescence of gastric chief after vehicle or 24h HDT treatment stained for Pepsinogen C ( PGC , red , granules ) MAP1LC3B ( green , autophagy marker ) . Top row is wild - type , bottom row is Atf3 − / − . Scale bar , 50μm . Stomach unit base outlined by dashed .

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Representative immunofluorescence images of gastric chief cells after vehicle or 24h HDT treatment stained for Pepsinogen C (PGC, red, granules) and MAP1LC3B (green, autophagy marker). Top row is wild-type, bottom row is Atf3−/−. Scale bar, 50μm. Stomach unit base outlined by dashed white line.


Transmission electron micrograph of zymogenic chief ( ZCs ) after 24h vehicle or HDT treatment in WT ( top ) Atf3 − / − mice ( bottom ) . Scale bar , 1μm . Arrowheads indicate components being degraded . of degradation events per counted from EM of ZCs after 24h HDT treatment in WT Atf3 − / − mice as shown in panel 3D . Each data point = total granules in a in a single section with from 2 separate mice plotted together ( data point colors indicate from a single mouse ) . Red indicates mean across mice .

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Transmission electron micrograph images of zymogenic chief cells (ZCs) after 24h vehicle or HDT treatment in WT (top) and Atf3−/− mice (bottom). Scale bar, 1μm. Arrowheads indicate cell components being degraded. Quantification of degradation events per cell counted from EM images of ZCs after 24h HDT treatment in WT and Atf3−/−mice as shown in panel 3D. Each data point=total granules in a cell in a single TEM section with cells from 2 separate mice plotted together (data point colors indicate cells from a single mouse). Red line indicates mean across all the mice.


Representative immunofluorescence of gastric units after vehicle or 72h high - dose tamoxifen ( HDT ) treatment stained for GIF ( red , granules ) , GSII ( green , neck SPEM marker ) , BRDU ( , proliferation ) DAPI ( blue , nuclei ) . Top row is wild - type , bottom row is Atf3 − / − . Yellow arrowheads point to areas of base dropout where ZCs have been lost . Scale bar , 50μm . Stomach units outlined by dashed .

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Representative immunofluorescence images of gastric units after vehicle or 72h high-dose tamoxifen (HDT) treatment stained for GIF (red, granules), GSII (green, neck cells and SPEM marker), BRDU (white, proliferation) and DAPI (blue, nuclei). Top row is wild-type, bottom row is Atf3−/−. Yellow arrowheads point to areas of base dropout where ZCs have been lost. Scale bar, 50μm. Stomach units outlined by dashed white line.


Representative immunohistochemistry of - Caspase 3 in pancreatic acinar marking apoptosis in vehicle or 24h cerulein ( CER ) treatment . Top row is WT , bottom row is Atf3 − / − . Scale bar , 50μm . Apoptosis quantified following 24h CER injury by number of - Caspase 3 + from a 40x field . Each data point is mean of positive in 4 - 5 40x fields in an mouse , n = 3 mice per treatment group . Significance by unpaired two - t - . Error bars denote standard deviation .

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Representative immunohistochemistry of Cleaved-Caspase 3 in pancreatic acinar cells marking apoptosis in vehicle or 24h cerulein (CER) treatment. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Apoptosis quantified following 24h CER injury by the number of Cleaved-Caspase 3+ cells from a 40x field. Each data point is mean of positive cells in 4-5 40x fields in an individual mouse, n=3 mice per treatment group. Significance by unpaired two-tailed t-test. Error bars denote standard deviation.


Representative immunohistochemistry of BrdU in pancreatic acinar marking proliferation in vehicle or 7 days of repeated cerulein injury . Top row is WT , bottom row is Atf3 − / − . Scale bar , 50μm . of proliferation at peak ADM as counted by number of BrdU + from a 20x field following 7D cerulein treatment . Plotting statistical as per panel F .

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Representative immunohistochemistry of BrdU in pancreatic acinar cells marking proliferation in vehicle or 7 days of repeated cerulein injury. Top row is WT, bottom row is Atf3−/−. Scale bar, 50μm. Quantification of proliferation at peak ADM as counted by the number of BrdU+ cells from a 20x field following 7D cerulein treatment. Plotting and statistical analysis as per panel F.


Serial sections of human tissue . Representative immunohistochemistry of GSII ( top ; spasmolytic polypeptide - expressing metaplasia [ SPEM ] marker neck ) counterstained with hematoxylin ( blue ) eosin ( pink ) . Representative of ATF3 ( bottom ) counterstained with eosin ( pink ) . Tissue was taken from regions adjacent to gastric adenocarcinoma . Scale bars left - 50μm right - 20μm . Arrowheads indicate strongly expressing ATF3 corresponding region in same labeled with GSII in a serial tissue section . GSII at base of gastric in of stomach indicate transition to SPEM .

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Serial sections of human tissue. Representative immunohistochemistry of GSII (top; spasmolytic polypeptide-expressing metaplasia [SPEM] marker and neck cells) counterstained with hematoxylin (blue) and eosin (pink). Representative staining of ATF3 (bottom) counterstained with eosin (pink). Tissue was taken from regions adjacent to gastric adenocarcinoma. Scale bars left - 50μm right - 20μm. Arrowheads indicate strongly expressing ATF3 cells and the corresponding region in the same gland labeled with GSII in a serial tissue section. GSII at the base of gastric glands in the body of the stomach indicate transition to SPEM.


( A ) abundance is normalized signal ( LFQ ) for a divided by its molecular . Specificity ( enrichment ) is of LFQ in MIA40FLAG fraction to control samples . LFQ for that were not detected in control samples was arbitrarily set to 1 for calculation purposes . LFQ , Label - Free - ; M . W . , molecular .

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(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight.


( B ) Flp - In T - REx 293 with induced MIA40FLAG expression were solubilized , affinity purification of MIA40FLAG was performed . Fractions were analyzed by SDS - PAGE Western blot . Load : 2 . 5 % . Eluate : 100 % . Unbound : 2 . 5 % .

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(B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %.


( C ) Flp - In T - REx 293 with induced MIA40FLAG expression were solubilized , affinity purification of MIA40FLAG was performed . Eluate fractions were solubilized under reducing ( DTT ) or non - reducing ( IAA ) conditions analyzed by SDS - PAGE Western blot . DTT , dithiothreitol ; IAA , iodoacetamide . Eluate : 100 % .

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(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.


( D ) extracts from Flp - In T - REx 293 with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification . Load eluate fractions were analyzed by reducing SDS - PAGE Western blot . Load : 2 . 5 % . Eluate : 100 % .

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(D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.


( A ) Schematic representation of thiol trapping . Mitochondria were solubilized in sample buffer with either dithiothreitol ( DTT ) , iodoacetamide ( IAA ) , or 4 - acetamido - 4 - maleimidylstilbene - 2 , 2 - disulfonic acid ( AMS ) . samples were analyzed by SDS - PAGE Western blot .

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(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.


( B ) Indirect thiol trapping . Mitochondria were pretreated with IAA as indicated to block free cysteine residues , disulfide bonds were subsequently reduced by DTT . Mitochondria were solubilized in sample buffer with AMS .

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(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.


( C ) Localization of mitochondrial analyzed by limited degradation by proteinase K in intact mitochondria ( 250 mM sucrose ) , mitoplasts ( 5 mM sucrose ) , mitochondrial lysates ( 1 % Triton X - 100 ) . samples were analyzed by SDS - PAGE Western blot . Mitos , mitochondria ; Sup , post - mitochondria supernatant ; OM , outer ; IM , inner ; IMS , intermembrane space .

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(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.


( D ) N - SIM super - resolution of one Z - stack ( 0 . 15 μm ) orthogonal section ( XYZ ) of HeLa or HeLa that stably expressed COA7 - HA transfected with different markers TOMM20 - DsRed ( OM ) , COX8A - DsRed ( IM ) , matrix targeted photoactivatable GFP ( Matrix target ) labeled with anti - HA , anti - COA7 Smac / Diablo ( IMS ) antibodies . picture represents majority population of from three independent experiments . Scale bar = 2 μm , scale bar in magnified insert = 0 . 5 μm . panel shows Pearson ' s coefficient in a co - localized volume of different subcompartment combinations with anti - HA or anti - COA7 . data are expressed as a mean ± SD ( n = 5 ) . * * * p < 0 . 001 [ IMSxEnd vs . IMxEnd p = 0 . 0001 ; IMSxEnd vs . MxEnd p = 0 . 0002 ] , * * * * p < 0 . 0001 [ OMxHA vs . IMSxHA p < 0 . 0001 ; IMSxHA vs . IMxHA p < 0 . 0001 ] ( one - way ANOVA ) . M , matrix ; End , endogenous COA7 ; HA , COA7 - HA .

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(D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.


( A ) Mitochondria were isolated from that were transfected with a plasmid that encoded COA7HIS or an empty vector . Mitochondria were solubilized analyzed by reducing SDS - PAGE Western blot . Mitos , mitochondria .

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(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria.


( B ) Radiolabeled [ 35S ] TIMM8A [ 35S ] COX19 precursors were imported into mitochondria that were isolated from that were transfected with a plasmid that encoded COA7HIS or an empty vector . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 30 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

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(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( C ) Mitochondria were isolated from that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing ( DTT ) non - reducing ( IAA ) conditions analyzed for of MIA40 by Western blot .

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(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.


( D ) from HEK293 transfected with empty plasmid or RESA1HIS were modified with PEG - PCMal . Control were pretreated with thiol oxidizing agent diamide ( DAM ) or reductant DTT . samples were analyzed by SDS - PAGE Western blot .

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(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.


( E ) Flp - In T - REx 293 induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector . affinity purification of MIA40FLAG was performed , eluate fractions were analyzed by SDS - PAGE Western blot .

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(E) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.


Radiolabeled [ 35S ] COA7 ( A ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 induced to express MIA40FLAG . samples were analyzed by reducing SDS - PAGE autoradiography . results of three biological replicates were analyzed , quantified , normalized to control mitochondria at 45 min . data are expressed as a mean ± ( n = 3 ) . IAA , iodoacetamide .

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Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.