E Newly synthesized in eIF2αS / S eIF2αA / A MEF treated with indicated concentrations of Tm for 2 . 5 or 5 h .

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E Newly synthesized proteins in eIF2αS/S and eIF2αA/A MEF cells treated with indicated concentrations of Tm for 2.5 or 5 h.


F Newly synthesized in HeLa untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF .

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F Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of HF.


G Newly synthesized in HeLa treated with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

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G Newly synthesized proteins in HeLa cells treated with indicated concentrations of HF with or without proline supplementation (10 mM).


H Representative immunoblots of indicated in lysates from HeLa after treatment with indicated concentrations of HF with or without proline supplementation ( 10 mM ) .

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H Representative immunoblots of indicated proteins in lysates from HeLa cells after treatment with indicated concentrations of HF with or without proline supplementation (10 mM).


I Newly synthesized in HeLa untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol ( His ) or borrelidin ( Bor ) .

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I Newly synthesized proteins in HeLa cells untreated or treated with GCN2 siRNA 48 h before treatment with indicated concentrations of histidinol (His) or borrelidin (Bor).


G , H of IC50 values of HF on confluency at 72h endpoint of H441 ( G ) H23 ( H ) normalised to control conditions determined by nonlinear regression curve fit for inhibitory dose - response ( IC50 ) . Each point represents mean ( n = 3 biological replicates ) ± SD .

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G, H Analysis of IC50 values of HF on confluency at 72h endpoint of H441 (G) and H23 (H) cells normalised to control conditions and determined by nonlinear regression curve fit for inhibitory dose-response (IC50). Each point represents mean (n = 3 biological replicates) ± SD.


( A ) HMGB1 translocates from nucleus to cytosol during autophagy , but not apoptosis . Mouse embryonic fibroblasts ( MEFs ) human Panc2 . 03 were treated with 1 µM rapamycin ( Rap ) for 12 h , starvation ( HBSS ) for 3 h , or UV irradiation at 50 mJ / cm2 for 5 min before a 12 - h recovery then were immunostained with HMGB1 - specific antibody ( green ) Hoechst 33342 ( blue ) . mean ( Nuc ) ( Cyt ) HMGB1 per was determined by imaging cytometric as described in Materials methods . * , P < 0 . 05 * * , P < 0 . 005 versus untreated ( UT ) group ; n = 3 . Representative are depicted ( right ) .

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(A) HMGB1 translocates from the nucleus to the cytosol during autophagy, but not apoptosis. Mouse embryonic fibroblasts (MEFs) and the human Panc2.03 tumor cell line were treated with 1 µM rapamycin (Rap) for 12 h, starvation (HBSS) for 3 h, or UV irradiation at 50 mJ/cm2 for 5 min before a 12-h recovery and then were immunostained with HMGB1-specific antibody (green) and Hoechst 33342 (blue). The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was determined by imaging cytometric analysis as described in Materials and methods. *, P < 0.05 and **, P < 0.005 versus untreated (UT) group; n = 3. Representative images are depicted (right).


( B ) Inhibition of autophagy blocks HMGB1 translocation . were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5 - specific shRNA for 48 h were stimulated with starvation ( HBSS ) for 3 h immunostained with HMGB1 - or LC3 - specific antibody Hoechst 33342 . mean / HMGB1 LC3 per were determined by imaging cytometric . A representative Western blot of ATG5 level after shRNA HMGB1 is depicted ( right ) . In parallel , indicated were transfected with GFP - LC3 plasmid assayed for autophagy by quantifying of with GFP - LC3 . * , P < 0 . 05 , * * , P < 0 . 005 , * * * , P < 0 . 0005 versus HBSS group ; n = 3 . Ctrl , control .

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(B) Inhibition of autophagy blocks HMGB1 translocation. Cells were pretreated as indicated with 100 nM wortmannin or 10 µM Ly294002 for 1 h or ATG5-specific shRNA for 48 h and were stimulated with starvation (HBSS) for 3 h and immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. A representative Western blot of ATG5 level after shRNA and HMGB1 staining is depicted (right). In parallel, the indicated cells were transfected with GFP-LC3 plasmid and assayed for autophagy by quantifying the percentage of cells with GFP-LC3 punctae. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus HBSS group; n = 3. Ctrl, control.


( C ) Knockdown of ATG5 inhibits LC3 - II expression . Western blot of LC3 - I / II expression in Panc02 under conditions described in B . Actin was used as a loading control .

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(C) Knockdown of ATG5 inhibits LC3-II expression. Western blot analysis of LC3-I/II expression in Panc02 cells under the conditions described in B. Actin was used as a loading control.


( D ) Hmgb1 − / − does not influence ATG5 . Hmgb1 − / − Hmgb1 + / + MEFs were immunostained with HMGB1 - specific antibody ( green ) , ATG - 5 - specific antibody ( red ) , Hoechst 33342 ( blue ) . Representative are depicted in right panels .

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(D) Hmgb1−/− does not influence ATG5 staining. Hmgb1−/− and Hmgb1+/+ MEFs were immunostained with HMGB1-specific antibody (green), ATG-5-specific antibody (red), and Hoechst 33342 (dark blue). Representative images are depicted in the right panels.


( E ) mETC inhibitors promote ROS production , autophagy , HMGB1 translocation . Panc2 . 03 were stimulated with rotenone ( Rot ) , thenoyltrifluoroacetone ( TTFA ) , antimycin A ( AA ) at indicated doses for 12 h , ROS production was assessed by measuring of CM - H2DCFDA in a plate reader . In parallel experiments , were then immunostained with HMGB1 - or LC3 - specific antibody Hoechst 33342 . mean / HMGB1 LC3 per were determined by imaging cytometric . * , P < 0 . 05 , * * , P < 0 . 005 , * * * , P < 0 . 0005 versus untreated group ; n = 3 .

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(E) mETC inhibitors promote ROS production, autophagy, and HMGB1 translocation. Panc2.03 cells were stimulated with rotenone (Rot), thenoyltrifluoroacetone (TTFA), and antimycin A (AA) at indicated doses for 12 h, and ROS production was assessed by measuring the fluorescent intensity of CM-H2DCFDA in a fluorescent plate reader. In parallel experiments, cells were then immunostained with HMGB1- or LC3-specific antibody and Hoechst 33342. The mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell were determined by imaging cytometric analysis. *, P < 0.05, **, P < 0.005, and ***, P < 0.0005 versus untreated group; n = 3.


( F ) mETC inhibitors increase LC3 - II expression promote HMGB1 translocation . Western blot of LC3 - I / II / HMGB1 expression as indicated in E . Fibrillarin is a fraction control , tubulin is a fraction control .

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(F) mETC inhibitors increase LC3-II expression and promote HMGB1 translocation. Western blot analysis of LC3-I/II and nuclear/cytosolic HMGB1 expression as indicated in E. Fibrillarin is a nuclear fraction control, and tubulin is a cytoplasmic fraction control.


( G ) Antioxidant SOD RNAi limit starvation - induced autophagy HMGB1 translocation . Panc2 . 03 were pretreated with antioxidant ( NAC ) at indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h . Then were starved ( HBSS ) for 3 h analyzed by imaging cytometry to determine mean / HMGB1 LC3 per . * , P < 0 . 05 ; * * , P < 0 . 005 ; * * * , P < 0 . 0005 versus HBSS group ; n = 3 . A representative Western blot for SOD1 SOD2 level after siRNA is depicted here .

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(G) Antioxidant and SOD RNAi limit starvation-induced autophagy and HMGB1 translocation. Panc2.03 cells were pretreated with the antioxidant (NAC) at the indicated concentrations for 1 h or with SOD1 or SOD2 siRNA for 48 h. Then cells were starved (HBSS) for 3 h and analyzed by imaging cytometry to determine the mean nuclear/cytosolic HMGB1 intensity and LC3 punctae per cell. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus HBSS group; n = 3. A representative Western blot for SOD1 and SOD2 level after siRNA is depicted here.


( H ) Antioxidant SOD RNAi limit starvation - induced autophagy as measured by LC3 - II expression . Western blot of LC3 - I / II expression under conditions indicated in G . Actin was used as a loading control . Data are means ± .

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(H) Antioxidant and SOD RNAi limit starvation-induced autophagy as measured by LC3-II expression. Western blot analysis of LC3-I/II expression under the conditions indicated in G. Actin was used as a loading control. Data are means ± SEM.


( A ) HMGB1 knockout inhibits LC3 formation . HMGB1 − / − HMGB1 + / + MEFs were treated with autophagic stimuli as indicated , LC3 formation was detected by LC3 antibody or GFP - LC3 as described in Materials methods . * , P < 0 . 05 ; * * , P < 0 . 005 ; * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . UT , untreated . ( B ) Representative of LC3 in Hmgb1 − / − HMGB1 + / + MEFs with indicated treatments are depicted . of showing accumulation of LC3 was reported in A .

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(A) HMGB1 knockout inhibits LC3 punctae formation. HMGB1−/− and HMGB1+/+ MEFs were treated with autophagic stimuli as indicated, and LC3 punctae formation was detected by LC3 antibody or GFP-LC3 as described in Materials and methods. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. UT, untreated. (B) Representative images of LC3 punctae in Hmgb1−/− and HMGB1+/+ MEFs with the indicated treatments are depicted. The percentage of cells showing accumulation of LC3 punctae was reported in A.


( C ) of LC3 processing by autophagy in presence or absence of lysosomal protease inhibitors pepstatin A ( pepA ) at 10 µg / ml E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 05 versus Hmgb1 + / + group ; n = 3 . Actin was used as a loading control . AU , arbitrary units .

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(C) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (pepA) at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.05 versus Hmgb1+/+ group; n = 3. Actin was used as a loading control. AU, arbitrary units.


( D ) Up - regulation of HMGB1 expression restores starvation - induced autophagy . Hmgb1 − / − MEFs were transfected with HMGB1 plasmid or empty vector then were treated with starvation for 3 h . LC3 formation was assayed by imaging cytometric . * * , P < 0 . 05 versus vector group ; n = 3 . Non , nontransfected .

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(D) Up-regulation of HMGB1 protein expression restores starvation-induced autophagy. Hmgb1−/− MEFs were transfected with HMGB1 plasmid or empty vector and then were treated with starvation for 3 h. LC3 punctae formation was assayed by imaging cytometric analysis. **, P < 0.05 versus vector group; n = 3. Non, nontransfected.


( E ) of p62 processing by autophagy in presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg / ml E64D at 10 µg / ml after starvation treatment for 3 h . * * , P < 0 . 005 * * * , P < 0 . 0005 versus Hmgb1 + / + group ; n = 3 . Representative are depicted ( left ) .

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(E) Analysis of p62 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A at 10 µg/ml and E64D at 10 µg/ml after starvation treatment for 3 h. **, P < 0.005 and ***, P < 0.0005 versus Hmgb1+/+ group; n = 3. Representative images are depicted (left).


( F ) Ultrastructural features in Hmgb1 − / − Hmgb1 + / + MEFs with or without starvation ( HBSS for 3 h ) treatment ( a - e point to autophagosomes autolysosomes ) . Data are means ± .

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(F) Ultrastructural features in Hmgb1−/− and Hmgb1+/+ MEFs with or without starvation (HBSS for 3 h) treatment (a-e point to autophagosomes and autolysosomes). Data are means ± SEM.


Inhibition of autophagy by HMGB1 . Hmgb1 − / − Hmgb1 + / + MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials methods then were treated with HBSS for 3 h , LC3 formation was detected by a confocal microscope . ( A ) Representative of LC3 ( arrows ) HMGB1 ( red arrows ) in cytoplasts of Hmgb1 − / − Hmgb1 + / + MEFs are depicted . ( B ) of showing accumulation of LC3 was reported ( * , P < 0 . 05 ; n = 3 ) . Data are means ± .

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Inhibition of autophagy by cytoplasmic HMGB1. Hmgb1−/− and Hmgb1+/+ MEFs were enucleated by centrifugation after cytochalasin B treatment as described in Materials and methods and then were treated with HBSS for 3 h, and LC3 punctae formation was detected by a confocal microscope. (A) Representative images of LC3 punctae (white arrows) and HMGB1 (red arrows) in cytoplasts of Hmgb1−/− and Hmgb1+/+ MEFs are depicted. (B) The percentage of cells showing accumulation of LC3 punctae was reported (*, P < 0.05; n = 3). Data are means ± SEM.


( A ) MEK inhibitors block starvation - induced p - ERK Bcl - 2 . HMGB1 − / − HMGB1 + / + MEFs were starved in presence or absence of 10 µM U0126 20 µM PD98059 for 6 h . expression were assessed as indicated by co - IP or Western blotting .

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(A) MEK inhibitors block starvation-induced p-ERK and Bcl-2. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 and 20 µM PD98059 for 6 h. Protein expression levels were assessed as indicated by co-IP or Western blotting.


( B ) Knockout of HMGB1 limits disassociation of Bcl - 2 - Beclin1 during treatment with autophagic stimuli . HMGB1 − / − HMGB1 + / + MEFs were starved in presence or absence of 10 µM U0126 for 3 h . expression were then assayed as indicated by co - IP or Western blotting .

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(B) Knockout of HMGB1 limits the disassociation of the Bcl-2-Beclin1 complex during treatment with autophagic stimuli. HMGB1−/− and HMGB1+/+ MEFs were starved in the presence or absence of 10 µM U0126 for 3 h. Protein expression levels were then assayed as indicated by co-IP or Western blotting.


( C ) HMGB1 interacts directly with Beclin1 during autophagy . HCT116 Panc02 were treated with HBSS for 3 h then assayed for expression as indicated by co - IP or Western blotting .

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(C) HMGB1 interacts directly with Beclin1 during autophagy. HCT116 and Panc02 cells were treated with HBSS for 3 h and then assayed for protein expression levels as indicated by co-IP or Western blotting.


( D ) HMGB1 direct interactions with Bcl - 2 are dependent on Beclin1 . Knockdown of Beclin1 Bcl - 2 by siRNA in HMGB1 wild - type MEFs was performed , expression were then assayed as indicated by co - IP or Western blotting . Ctrl , control .

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(D) HMGB1 direct interactions with Bcl-2 are dependent on Beclin1. Knockdown of Beclin1 and Bcl-2 by siRNA in HMGB1 wild-type MEFs was performed, and protein expression levels were then assayed as indicated by co-IP or Western blotting. Ctrl, control.


( E F ) Quantitative data demonstrating interaction between HMGB1 - Beclin1 Beclin1 - HMGB1 using densitometry software to relative band in co - IP experiments as shown in A - D . * * , P < 0 . 005 * * * , P < 0 . 0005 ; n = 3 . UT , untreated . Data are means ± .

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(E and F) Quantitative data demonstrating the interaction between HMGB1-Beclin1 and Beclin1-HMGB1 using densitometry software to assay the relative protein band intensity in co-IP experiments as shown in A-D. **, P < 0.005 and ***, P < 0.0005; n = 3. UT, untreated. Data are means ± SEM.


( A ) C106 mutation ( C106S ) of HMGB1 impairs its localization . Hmgb1 − / − MEFs were transfected with wild - type cysteine mutant HMGB1 - GFP plasmids as indicated then were treated with 1 µM rapamycin for 12 h or starved ( HBSS ) for 3 h . mean ( Nuc ) ( Cyt ) HMGB1 per was analyzed by imaging cytometric . * , P < 0 . 05 versus HMGB1 + / + group ; n = 3 . Representative of HMGB1 location are shown on left ( green , HMGB1 ; blue , nucleus ) . right panel is a schematic diagram of HMGB1 structure illustrating basic A box B box as well as acidic C - terminal domain , with cysteine mutation locations identified .

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(A) The C106 mutation (C106S) of HMGB1 impairs its nuclear localization. Hmgb1−/− MEFs were transfected with wild-type and cysteine mutant HMGB1-GFP plasmids as indicated and then were treated with 1 µM rapamycin for 12 h or starved (HBSS) for 3 h. The mean nuclear (Nuc) and cytosolic (Cyt) HMGB1 intensity per cell was analyzed by imaging cytometric analysis. *, P < 0.05 versus HMGB1+/+ group; n = 3. Representative images of HMGB1 location are shown on the left (green, HMGB1; blue, nucleus). The right panel is a schematic diagram of HMGB1 structure illustrating the basic A box and B box as well as the acidic C-terminal domain, with the cysteine mutation locations identified.


( B C ) HMGB1 enhances autophagy limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated then were starved ( HBSS ) for indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h then starved as indicated . LC3 formation was assayed by imaging cytometric ( B )

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(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+ MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. LC3 punctae formation was assayed by imaging cytometric analysis (B)


( B C ) HMGB1 enhances autophagy limits apoptosis . HMGB1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated then were starved ( HBSS ) for indicated time . In a parallel experiment , Hmgb1 + / + MEFs were pretreated with 5 mM ethyl pyruvate ( EP ) for 2 h then starved as indicated . Apoptosis was assayed by FACS ( C ) ( C ) as described in Materials methods . * , P < 0 . 05 * * , P < 0 . 005 ; n = 3 . UT , untreated . Non , nontransfection .

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(B and C) Cytoplasmic HMGB1 enhances autophagy and limits apoptosis. HMGB1−/−MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and then were starved (HBSS) for the indicated time. In a parallel experiment, Hmgb1+/+MEFs were pretreated with 5 mM ethyl pyruvate (EP) for 2 h and then starved as indicated. Apoptosis was assayed by FACS (C) (C) as described in Materials and methods. *, P < 0.05 and **, P < 0.005; n = 3. UT, untreated. Non, nontransfection.


( D ) C23 / C45 is required for binding of HMGB1 to Beclin1 . Hmgb1 − / − MEFs were transfected with wild - type or cysteine mutant HMGB1 - GFP plasmids as indicated stimulated with starvation ( HBSS ) for 3 h . These were then assayed for expression as indicated by IP or Western blotting . Blots are representative of three independent experiments with similar results .

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(D) C23/C45 is required for the binding of HMGB1 to Beclin1. Hmgb1−/− MEFs were transfected with wild-type or cysteine mutant HMGB1-GFP plasmids as indicated and stimulated with starvation (HBSS) for 3 h. These cells were then assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of three independent experiments with similar results.


( E ) Reducing reagents disrupt interaction between wild - type / C106 HMGB1 Beclin1 . As a control , before IP , samples were incubated with 50 mM DTT ( + DTT ) assayed for expression as indicated by IP or Western blotting . Blots are representative of two independent experiments with similar results .

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(E) Reducing reagents disrupt the interaction between wild-type/C106 HMGB1 and Beclin1. As a control, before IP, samples were incubated with 50 mM DTT (+DTT) and assayed for protein expression levels as indicated by IP or Western blotting. Blots are representative of two independent experiments with similar results.


( F ) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from nucleus to cytosol . were stimulated with HBSS , rapamycin ( Rap ) , rotenone ( Rot ) , or thenoyltrifluoroacetone ( TTFA ) for 3 h or 12 h , LC3 formation was assayed as indicated . * * , P < 0 . 005 versus Beclin1 shRNA group ; n = 3 . Ctrl , control . Data are means ± .

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(F) Knockdown of Beclin1 by siRNA inhibits autophagy under conditions of HMGB1 translocation from the nucleus to the cytosol. Cells were stimulated with HBSS, rapamycin (Rap), rotenone (Rot), or thenoyltrifluoroacetone (TTFA) for 3 h or 12 h, and LC3 punctae formation was assayed as indicated. **, P < 0.005 versus Beclin1 shRNA group; n = 3. Ctrl, control. Data are means ± SEM.


( A B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . as indicated were stimulated with 1 µg / ml HMGB1 ( rHMGB1 ) for 24 h , LC3 expression was detected by Western blotting ( A ) .

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(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, and LC3 expression was detected by Western blotting (A).


( A B ) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1 - induced autophagy . as indicated were stimulated with 1 µg / ml HMGB1 ( rHMGB1 ) for 24 h , LC3 formation ( arrows ) was assayed by immunofluorescence ( B ; n = 3 ; * , P < 0 . 05 ) .

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(A and B) Knockdown of Beclin1 in MEFs by shRNA inhibits exogenous HMGB1-induced autophagy. Cells as indicated were stimulated with 1 µg/ml HMGB1 protein (rHMGB1) for 24 h, LC3 punctae formation (arrows) was assayed by immunofluorescence (B; n = 3; *, P < 0.05).


( C D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with without pUNO1 - HMGB1 transfection . as indicated were stimulated with Earle ' s balanced salt solution for 3 h , LC3 expression was detected by Western blotting ( C ) .

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

(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 expression was detected by Western blotting (C).


( C D ) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS - induced autophagy with without pUNO1 - HMGB1 transfection . as indicated were stimulated with Earle ' s balanced salt solution for 3 h , LC3 formation ( arrows ) was assayed by immunofluorescence ( D ; n = 3 ; * , P < 0 . 05 ) . Data are means ± .

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(C and D) Knockdown of Beclin1 in MEFs by shRNA inhibits HBSS-induced autophagy with and without pUNO1-HMGB1 transfection. Cells as indicated were stimulated with Earle's balanced salt solution for 3 h, and LC3 punctae formation (arrows) was assayed by immunofluorescence (D; n = 3; *, P < 0.05). Data are means ± SEM.


Expression of HMGB1 or C106S mutant , but not C23S C45S mutants , restore autophagic flux in Hmgb1 − / − MEFs . ( A ) HMGB1 − / − MEFs were transfected with wild - type ( WT ) or cysteine mutant HMGB1 - GFP plasmids as indicated , starved ( HBSS ) for 3 h in presence or absence of 100 nM bafilomycin A1 , then immunostained with LAMP2 - specific antibody / Alexa Fluor 594 secondary antibody ( shown in red ) , LC3 - specific antibody / Alexa Fluor 647 secondary antibody ( shown in green ) , Hoechst 33342 ( shown in blue ) . were acquired digitally from a randomly selected pool of 10 - 15 fields under each condition . ( B ) Quantitative of of LAMP2 / LC3 colocalization was detected by Image - Pro Plus 5 . 1 software . Data are means ± .

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Expression of HMGB1 or the C106S mutant, but not C23S and C45S mutants, restore autophagic flux in Hmgb1−/− MEFs. (A) HMGB1−/− MEFs were transfected with wild-type (WT) or the cysteine mutant HMGB1-GFP plasmids as indicated, starved (HBSS) for 3 h in the presence or absence of 100 nM bafilomycin A1, and then immunostained with LAMP2-specific antibody/Alexa Fluor 594 secondary antibody (shown in red), LC3-specific antibody/Alexa Fluor 647 secondary antibody (shown in green), and Hoechst 33342 (shown in blue). Images were acquired digitally from a randomly selected pool of 10-15 fields under each condition. (B) Quantitative analysis of the percentage of LAMP2/LC3 colocalization was detected by Image-Pro Plus 5.1 software. Data are means ± SEM.


a , b , LDH release , IL - 1β secretion ( a ) immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 IL - 1α ( b )

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a, b, LDH release, IL-1β secretion (a) and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α(b)


immunoblots for caspase - 1 , caspase - 11 , IL - 1β , IL - 18 IL - 1α ( b ) from unprimed BMDMs infected for 16h with indicated ( grown to stationary phase ) .

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and immunoblots for caspase-1, caspase-11, IL-1β, IL-18 and IL-1α (b) from unprimed BMDMs infected for 16h with the indicated bacteria (grown to stationary phase).


c , Time course measuring LDH release from unprimed or IFN - γ - primed BMDMs infected with S .

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c, Time course measuring LDH release from unprimed or IFN-γ-primed BMDMs infected with S. typhimurium


d , e , LDH release IL - 1β secretion from primed BMDMs infected with SPI - 1 - expressing logarithmic phase S . typhimurium , treated with monosodium urate , alum nigericin or transfected with poly ( dA : dT ) LPS .

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d, e, LDH release and IL-1β secretion from primed BMDMs infected with SPI-1-expressing logarithmic phase S. typhimurium, treated with monosodium urate, alum and nigericin or transfected with poly(dA:dT) and LPS.


LDH release IL - 1β secretion from unprimed wild - type Gbp2 − / − BMDMs infected for 16h with indicated ( grown to stationary phase ) . Graphs show mean s . d . of quadruplicate wells data are representative of two ( b ) three ( a , c - f ) independent experiments . * Crossreactive band ; * * P < 0 . 01 ; NS , not significant ( two - t - ) .

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LDH release and IL-1β secretion from unprimed wild-type and Gbp2−/− BMDMs infected for 16h with the indicated bacteria (grown to stationary phase). Graphs show mean and s.d. of quadruplicate wells and data are representative of two (b) and three (a, c-f) independent experiments. *Crossreactive band; **P<0.01; NS, not significant (two-tailed t-test).


a , b , of live ( mCherry - positive ) dead ( mCherry - negative ) S . typhimurium per by immunofluorescence ( a ) or as of total by flow -

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a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry


a , b , of live ( mCherry - positive ) dead ( mCherry - negative ) S . typhimurium per by immunofluorescence ( a ) or as of total by flow - cytometry ( b ) in unprimed BMDMs at 16 h post - infection .

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a, b, Quantification of live (mCherry-positive) and dead (mCherry-negative) S. typhimurium per cell by immunofluorescence (a) or as percent of total by flow-cytometry (b) in unprimed BMDMs at 16 h post-infection.


c , Immunostaining for GBP2 of live dead Salmonella at 4 h post - infection . Arrowheads , shown in insets .

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c, Immunostaining for GBP2 and quantification of live and dead Salmonella at 4 h post-infection. Arrowheads, bacteria shown in insets.


d , of GBP - positive , LDH release IL - 1β secretion at indicated time points from BMDMs infected with Salmonella , live or killed by different means .

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d, Quantification of GBP-positive bacteria, LDH release and IL-1β secretion at indicated time points from BMDMs infected with Salmonella, live or killed by different means.


e , Immunohistochemistry for GBP2 Salmonella on spleen tissue from Salmonella ( mCherry - positive ) - infected mice ( representative of n = 3 per group ) . S . tm . , S . typhimurium .

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e, Immunohistochemistry for GBP2 and Salmonella on spleen tissue from Salmonella (mCherry-positive)-infected mice (representative of n = 3 per group). S. tm., S. typhimurium.


f , g , of GBP - positive Salmonella in anti - IFN - γ - treated or control ( f ) live dead among GBP2 - negative / - positive Salmonella ( g ) ( n = 3 per group ) . Scale bars , 10 µm ( c ) , 1 µm ( e ) . Graphs show mean 5 - 95 percentile ( box plots ) or s . d . of technical triplicates , data are representative of three independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - t - ) .

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f, g, Quantification of GBP-positive Salmonella in anti-IFN-γ-treated or control animals (f) and live and dead bacteria among GBP2-negative/-positive Salmonella (g) (n = 3 per group). Scale bars, 10 µm (c), 1 µm (e). Graphs show mean and 5-95 percentile (box plots) or s.d. of technical triplicates, and data are representative of three independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


a , b , Unprimed BMDMs infected with S . typhimurium for 4 h immunostained for LC3 GBP2 . Arrowheads , shown in insets . Scale bars , 10 & amp ; amp ; mgr ; m .

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a, b, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for LC3 and GBP2. Arrowheads, bacteria shown in insets. Scale bars, 10 &amp;mgr;m.


. c, Quantification of results from b.


b . d - g , LDH release immunoblots for caspase - 1 caspase - 11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3 - methyladenine ( 3 - MA ) .

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b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


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b. d-g, LDH release and immunoblots for caspase-1 and caspase-11 from BMDMs infected for 16 h or transfected with LPS in presence or absence of 3-methyladenine (3-MA).


h , i , LDH release IL - 1b secretion from BMDMs infected for 16 h or transfected with LPS . Graphs show mean s . d . of quadruplicate wells data are representative of two ( e , i ) three ( a - d , f - h ) independent experiments . * P 0 . 05 , * * P 0 . 01 ; NS , not significant ( two - t - ) .

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h, i, LDH release and IL-1b secretion from BMDMs infected for 16 h or transfected with LPS. Graphs show mean and s.d. of quadruplicate wells and data are representative of two (e, i) and three (a-d, f-h) independent experiments. *P  0.05, **P  0.01; NS, not significant (two-tailed t-test).


a , of galectin - 8 - positive Salmonella in unprimed BMDMs at 4 h post - infection .

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a, Quantification of galectin-8-positive Salmonella in unprimed BMDMs at 4 h post-infection. b


c , Unprimed BMDMs infected with S . typhimurium for 4 h immunostained for galectin - 8 , GBP2 LC3 . Arrowheads , shown in insets . Scale bars , 10 µm . d , of galectin - 8 / LC3 - double - positive Salmonella at indicated time points post - infection .

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c, Unprimed BMDMs infected with S. typhimurium for 4 h and immunostained for galectin-8, GBP2 and LC3. Arrowheads, bacteria shown in insets. Scale bars, 10 µm. d, Quantification of galectin-8/LC3-double-positive Salmonella at indicated time points post-infection.


e - h , of vacuolar by flow cytometry in BMDMs infected with mCherry - positive S . typhimurium ( e - g ) or S . flexneri ( h , wild - type or ΔT3SS ) for 4 h . Graphs show mean s . d . or 5 - 95 percentile ( Box plots ) of technical triplicates . Data are representative of 2 ( g , h ) , 3 ( a - d ) 4 ( e , f ) independent experiments . * P 0 . 05 , * * P 0 . 01 ( two - t - ) .

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e-h, Quantification of cytosolic and vacuolar bacteria by flow cytometry in BMDMs infected with mCherry-positive S. typhimurium (e-g) or S. flexneri (h, wild-type or ΔT3SS) for 4 h. Graphs show mean and s.d. or 5-95 percentile (Box plots) of technical triplicates. Data are representative of 2 (g, h), 3 (a-d) and 4 (e, f) independent experiments. *P  0.05, **P  0.01 (two-tailed t-test).


mRNA of TMX1 ( A ) NFAT1 ( B ) in melanocytes , keratinocytes melanoma lines . data are are normalized to expression of TBP are presented as mean ± ( n ≥ 3 ) .

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mRNA levels of TMX1 (A) and NFAT1 (B) in primary melanocytes, keratinocytes and melanoma cell lines. data are are normalized to the expression of TBP and are presented as mean ± SEM (n≥3).


Representative immunoblot ( from 3 ) depicting abundance of TMX1 NFAT1 in melanocytes melanoma lines ; actin was used as a loading control . Indicated band intensities were normalized to actin background signal was subtracted .

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Representative immunoblot (from 3) depicting protein abundance of TMX1 and NFAT1 in melanocytes and melanoma cell lines; actin was used as a loading control. Indicated band intensities were normalized to actin and background signal was subtracted.


Melan - A ( brown ) , TMX1 ( red - brown ) NFAT1 ( deep red ) ( ) in a stage 4 high risk nodular melanoma I ( out of four ) , enlarged regions show healthy tissue ( panels 2 , 5 8 ) tissue ( panels 3 , 6 9 ) . Arrows indicate Melan - A positive melanocytes .

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Melan-A (brown), TMX1 (red-brown) and NFAT1 (deep red) staining (IHC) in a primary stage 4 high risk nodular melanoma patient I (out of four), the enlarged regions show healthy tissue (panels 2, 5 and 8) and tumor tissue (panels 3, 6 and 9). Arrows indicate Melan-A positive melanocytes.


TMX1 ( red - brown ) NFAT1 ( deep red ) of healthy human skin increasing melanoma stages ; P1 - P13 refer to donor number .

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TMX1 (red-brown) and NFAT1 (deep red) staining of healthy human skin and increasing melanoma stages; P1-P13 refer to the donor patient number.


Insulin ( 50 µg / mL ) induced translocation of NFAT1 - GFP to nucleus of WM3734 . ( A ) ( F / F0 ) , ( B ) corresponding endpoint ( n = 5 ) .

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Insulin (50 µg/mL) induced the translocation of NFAT1-GFP to the nucleus of WM3734 cells. (A) Ratio images (F/F0), (B) corresponding endpoint quantification (n=5).


Reduced translocation of NFAT1 - GFP in TMX1 or TMX3 silenced ( siRNA ) melanoma . ( C ) show NFAT1 - GFP before after stimulation with thapsigargin ( Tg ; 1 µM ) in WM3734 . ( D ) Corresponding time - dependent import of NFAT1 as a change of F / F0 . ( E ) Normalized endpoint . Same was performed with Mel Juso with ( F ) , ( G ) time - dependent import ( H ) normalized endpoint . data are presented as mean ± ( n values : WM3734 , control = 142 , TMX1 kd = 116 , TMX3 kd = 148 ; Mel Juso , control = 75 , TMX1 kd = 47 , TMX3 kd = 67 ) .

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Reduced nuclear translocation of NFAT1-GFP in TMX1 or TMX3 silenced (siRNA) melanoma cells. (C) Images show NFAT1-GFP fluorescence intensity before and after stimulation with thapsigargin (Tg; 1 µM) in WM3734 cells. (D) Corresponding time-dependent nuclear import of NFAT1 as a change of F/F0. (E) Normalized endpoint quantification. Same analysis was performed with Mel Juso cells with (F) images, (G) time-dependent nuclear import and (H) normalized endpoint quantification. data are presented as mean ± SEM (n values: WM3734, control=142, TMX1 kd=116, TMX3 kd=148; Mel Juso, control=75, TMX1 kd=47, TMX3 kd=67).


Thapsigargin ( Tg ) induced Fura - 2 Ca2 + imaging in Ringer ' s buffer containing 0 . 25 mM Ca2 + . ( B ) of basal calcium ( C ) SOCE ( plateau - basal ) for WM3734 after stable silencing of TMX1 ( two clones ) . : In ( B - C ) , data are presented as mean ± ( n values : WM3734 , control = 939 , TMX1 kds 1 = 988 , TMX1 kds 2 = 508 ) .

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Thapsigargin (Tg) induced Fura-2 based cytosolic Ca2+ imaging in Ringer's buffer containing 0.25 mM Ca2+. (B) Quantification of basal cytosolic calcium levels and (C) SOCE quantification (plateau-basal) for WM3734 after stable silencing of TMX1 (two clones). : In (B-C), data are presented as mean ± SEM (n values: WM3734, control=939, TMX1 kds 1=988, TMX1 kds 2=508).


Ca2 + imaging ( Fura - 2 ) ( E ) SOCE ( plateau - basal ) for WM3734 after transient silencing of TMX1 or TMX3 . In ( E ) , data are presented as mean ± ( n values : WM3734 , control = 30 , TMX1 kd = 49 , TMX3 kd = 52 ) .

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Cytosolic Ca2+ imaging (Fura-2) and (E) SOCE quantification (plateau-basal) for WM3734 after transient silencing of TMX1 or TMX3. In (E), data are presented as mean ± SEM (n values: WM3734, control=30, TMX1 kd=49, TMX3 kd=52).


H2O2 ( HyPer ) was evaluated in two melanoma lines upon TMX1 kd TMX3 kd . ( F ) Exemplary ratiometric ( F505 nm / F420 nm ) are shown for WM3734 Mel Juso . of basal H2O2 in WM3734 ( G ) Mel Juso ( H ) . In ( G - H ) , data are presented as mean ± ( n values : WM3734 : control = 168 , TMX1 kd = 209 , TMX3 kd = 192 ; Mel Juso : control = 297 , TMX1 kd = 343 , TMX3 kd = 440 ) . In ( F ) scale bars : 10 µm ; color code : WM3734 : color code : blue = 0 , red = 3 ; Mel Juso : color code : blue = 0 , red = 1 . 5 .

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Cellular H2O2 (HyPer) was evaluated in two melanoma cell lines upon TMX1 kd and TMX3 kd. (F) Exemplary ratiometric images (F505 nm / F420 nm) are shown for WM3734 and Mel Juso. Quantification of basal cellular H2O2 in WM3734 (G) and Mel Juso (H) cells. In (G-H), data are presented as mean ± SEM (n values: WM3734: control=168, TMX1 kd=209, TMX3 kd=192; Mel Juso: control=297, TMX1 kd=343, TMX3 kd=440). In (F) scale bars: 10 µm; color code: WM3734: color code: blue=0, red=3; Mel Juso: color code: blue=0, red=1.5.


WM3734 were pre - incubated for 5 min with extracellular H2O2 ( 0 - 500 µM ) . Tg - stimulated NFAT1 - GFP translocation was measured as F / F0 .

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WM3734 cells were pre-incubated for 5 min with extracellular H2O2 (0 - 500 µM). Tg-stimulated NFAT1-GFP nuclear translocation was measured as F/F0.


IC50 of H2O2 inhibition of NFAT1 import was determined to be 13 . 2 µM ( red mark ) .

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The IC50 of H2O2 inhibition of NFAT1 nuclear import was determined to be 13.2 µM (red mark).


Tg - induced NFAT1 - GFP import in transient TMX1 silenced WM3734 after pre - incubation with 100 µM N - acetylcysteine ( NAC ) for 48 h .

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Tg-induced NFAT1-GFP nuclear import in transient TMX1 silenced WM3734 cells and after pre-incubation with 100 µM N-acetylcysteine (NAC) for 48 h.


Endpoint of data from ( C ) upon treatment with NAC , PEG - catalase ( 50 U / mL ) , or DTT ( 1 mM ) . data are presented as mean ± ( n values : control = 73 , control + NAC = 19 , TMX1 kd = 57 , TMX1 kd + NAC = 63 , TMX1 kd + catalase = 58 , TMX1 kd + DTT = 22 ) .

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Endpoint quantification of the data from (C) and upon treatment with NAC, PEG-catalase (50 U/mL), or DTT (1 mM). data are presented as mean ± SEM (n values: control=73, control+NAC= 19, TMX1 kd=57, TMX1 kd+NAC=63, TMX1 kd+catalase=58, TMX1 kd+DTT=22).


calcineurin activity ( CaNAR2 ) in WM3734 with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation .

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Cytosolic calcineurin activity (CaNAR2) in WM3734 cells with transient knockdown of TMX1 or TMX3 measured upon Tg stimulation.


of basal calcineurin activity maximal calcineurin activity ( plateau - basal ) . data are presented as mean ± ( n values : control = 49 , TMX1 kd = 48 , TMX3 kd = 63 ) .

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Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal). data are presented as mean ± SEM (n values: control=49, TMX1 kd=48, TMX3 kd=63).


Normalized Tg - induced calcineurin activity in Mel Juso after 5 min pre - incubation with extracellular H2O2 ( 0 - 200 µM ) . of maximum calcineurin activity ( plateau - basal ) .

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Normalized Tg-induced cytosolic calcineurin activity in Mel Juso cells after 5 min pre-incubation with extracellular H2O2 (0 - 200 µM). Quantification of the maximum cytosolic calcineurin activity (plateau-basal).


of basal calcineurin activity maximal calcineurin activity ( plateau - basal ) 8 h after treatment with antioxidant NAC ( 100 µM ) in WM3734 with transient knockdown of TMX1 . data are presented as mean ± ( n values : control = 15 , TMX1 kd = 24 , TMX1 kd + NAC = 19 ) .

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Quantification of the basal calcineurin activity and the maximal calcineurin activity (plateau-basal) 8 h after treatment with the antioxidant NAC (100 µM) in WM3734 with transient knockdown of TMX1. data are presented as mean ± SEM (n values: control=15, TMX1 kd=24, TMX1 kd+NAC=19).


Mitochondrial H2O2 ( mito - HyPer ) was measured in two lines upon transient TMX knockdown . Exemplary ratiometric ( F505 nm / F420 nm ) are shown ( A ) ; corresponding for ( B ) WM3734 ( C ) Mel Juso . , data are presented as mean ± ( n values : WM3734 : control = 546 , TMX1 kd = 510 , TMX3 kd = 621 ; Mel Juso : control = 416 , TMX1 kd = 418 , TMX3 kd = 442 ) .

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Mitochondrial H2O2 (mito-HyPer) was measured in two cell lines upon transient TMX knockdown. Exemplary ratiometric images (F505 nm / F420 nm) are shown (A); corresponding quantification for (B) WM3734 and (C) Mel Juso. , data are presented as mean ± SEM (n values: WM3734: control=546, TMX1 kd=510, TMX3 kd=621; Mel Juso: control=416, TMX1 kd=418, TMX3 kd=442).


Mitochondrial Ca2 + uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria - targeted calcium sensor ( 4mt - D3cpV ) ( D ) . were exposed to 0 . 25 mM Ca2 + containing Ringer ' s buffer Tg . ( E ) of basal mitochondrial Ca2 + ( F ) mitochondrial Ca2 + uptake ( basal - plateau ) . , data are presented as mean ± ( n values : control = 62 , TMX1 kds = 45 ) .

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Mitochondrial Ca2+ uptake in WM3734 with stable TMX1 knockdown was measured using a mitochondria-targeted calcium sensor (4mt-D3cpV) (D). Cells were exposed to 0.25 mM Ca2+ containing Ringer's buffer and Tg. (E) Quantification of basal mitochondrial Ca2+ levels and (F) mitochondrial Ca2+ uptake (basal-plateau). , data are presented as mean ± SEM (n values: control=62, TMX1 kds=45).


Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 with stable knockdown of TMX1 . mean mitochondrial volume was modeled on microscopy , of mean volume ( µm3 ) mean surface ( µm2 ) is depicted in ( G ) ( H ) respectively . data are presented as boxplots ( n values : control = 101 , TMX1 kds = 90 ; center : median ; box : 25 % 75 % percentile ; whiskers : 1 . 5 times interquartile range ; outliers are shown as dots ) .

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

Mitochondrial volume was determined using MitoTracker DeepRed in WM3734 cells with stable knockdown of TMX1. The mean mitochondrial volume was modeled based on microscopy, the quantification of mean volume (µm3) and mean surface (µm2) is depicted in (G) and (H) respectively. data are presented as boxplots (n values: control=101, TMX1 kds=90; center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range; outliers are shown as dots).


Peripheral mitochondria were quantified in WM3734 with stable knockdown of TMX1 . A peripheral mask was applied on ( CellMask Green ) mitochondria covered ( MitoTracker DeepRed ) was evaluated ( see Fig . 9 ) . Representative ( I ) ( J ) . In ( J ) , data are presented as mean ± ( n values : control = 163 , TMX1 kds = 116 ) .

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Peripheral mitochondria were quantified in WM3734 cells with stable knockdown of TMX1. A peripheral mask was applied based on the membrane staining (CellMask Green) and mitochondria covered area (MitoTracker DeepRed) was evaluated (see Fig. 9). Representative images (I) and quantification (J). In (J), data are presented as mean ± SEM (n values: control=163, TMX1 kds=116).


Representative electron of control 1205Lu with stable TMX1 knockdown ( K ) . Corresponding of between ( L ) mitochondria plasma , ( M ) mitochondria ER , ( N ) of MAM ( mitochondria - ER contact site ) . In ( L ) , data are presented as mean ± ( n values : control = 100 , TMX1 kds = 100 ) . In ( M - N ) , data are presented as mean ± ( n values : control = 60 , TMX1 kds = 60 ) .

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Representative electron micrographs of control and 1205Lu cells with stable TMX1 knockdown (K). Corresponding quantification of the distance between (L) mitochondria and plasma membrane, (M) mitochondria and ER, and (N) the length of the MAM (mitochondria-ER contact site). In (L), data are presented as mean ± SEM (n values: control=100, TMX1 kds= 100). In (M-N), data are presented as mean ± SEM (n values: control=60, TMX1 kds= 60).


H2O2 ( HyPer ) 48 h after transient silencing of TMX1 / or NOX4 in WM3734 or after inhibiting NOX4 with GKT137831 ( 140 nM ) ( O ) Representative ratiometric ( F505 nm / F420 nm ) ( P ) . In ( P ) , data are presented as mean ± ( n values : control = 837 , TMX1 kd = 888 , TMX1 kd + NOX4 kd = 793 , TMX1 kd + GKT = 844 ) .

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Cellular H2O2 concentration (HyPer) 48 h after transient silencing of TMX1 and / or NOX4 in WM3734 cells or after inhibiting NOX4 with GKT137831 (140 nM) (O) Representative ratiometric images (F505 nm / F420 nm) and (P) quantification. In (P), data are presented as mean ± SEM (n values: control=837, TMX1 kd=888, TMX1 kd + NOX4 kd=793, TMX1 kd + GKT=844).


Proliferation ( 48 h ) of melanoma lines measured after transient knockdown of TMX1 or TMX3 .

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Proliferation (48 h) of melanoma cell lines measured after transient knockdown of TMX1 or TMX3.


Proliferation ( 48 h ) of melanoma lines with stable TMX1 knockdown ( two clones ) .

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Proliferation (48 h) of melanoma cell lines with stable TMX1 knockdown (two clones).


Proliferation ( 24 h ) of melanoma lines after transient knockdown of NFAT1 .

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Proliferation (24 h) of melanoma cell lines after transient knockdown of NFAT1.


Proliferation of WM3734 48 h after transient knockdown of TMX1 treatment with NAC ( 100 µM ) or mTEMPO ( 100 nM ) .

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Proliferation of WM3734 48 h after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (100 nM).


Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 or TMX3 .

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Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 or TMX3.


Transwell migration ( 48 h ) of WM3734 after transient knockdown of TMX1 treatment with NAC ( 100 µM ) or mTEMPO ( 1 µM ) .

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Transwell migration (48 h) of WM3734 after transient knockdown of TMX1 and treatment with NAC (100 µM) or mTEMPO (1 µM).


Transwell migration ( 48 h ) of melanoma lines after preventing NFAT - calcineurin interaction with dipyridamole ( 40 µM ) or inhibition of calcineurin with cyclosporine A ( CsA , 2 µM ) .

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Transwell migration (48 h) of melanoma cell lines after preventing NFAT-calcineurin interaction with dipyridamole (40 µM) or inhibition of calcineurin with cyclosporine A (CsA, 2 µM).


Invasion of WM3734 with stable TMX1 knockdown ( two clones ) through Matrigel for 96 h .

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Invasion of WM3734 with stable TMX1 knockdown (two clones) through Matrigel for 96 h.


Mouse xenograft model to assess growth of WM3734 with stable TMX1 knockdown ( TMX1 kds ; two clones ) in immunodeficient mice . ( I ) growth in first 19 days after injection ; ( J ) corresponding volume quantified after 19 days ( K ) after 45 days .

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Mouse xenograft model to assess tumor growth of WM3734 with stable TMX1 knockdown (TMX1 kds; two clones) in immunodeficient mice. (I) Tumor growth in the first 19 days after injection; (J) the corresponding tumor volume quantified after 19 days and (K) after 45 days.


Kaplan - Meier survival plots log rank for correlation between mRNA expression survival probability of melanoma , separated in groups with high or low expression of any of three genes of interest ( GOI ) , TMX1 , TMX3 NFAT1 .

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Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with high or low expression of any of the three genes of interest (GOI), TMX1, TMX3 and NFAT1.


mRNA expression of NFAT1 , TMX1 TMX3 in BRAF WT BRAF V600E melanoma . In ( B ) , expression are on FPKM values of TCGA samples ( center : median ; box : 25 % 75 % percentile ; whiskers : 1 . 5 times interquartile range ) .

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mRNA expression levels of NFAT1, TMX1 and TMX3 in BRAF WT and BRAF V600E melanoma patients. In (B), expression levels are based on the FPKM values of the TCGA samples (center line: median; box: 25 % and 75 % percentile; whiskers: 1.5 times interquartile range).


Kaplan - Meier survival plots log rank for correlation between mRNA expression survival probability of melanoma , separated in groups with ( C ) BRAF WT ( D ) BRAF V600E with high or low expression of NFAT1 .

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

Kaplan-Meier survival plots and log rank for the correlation between mRNA expression levels and survival probability of melanoma patients, separated in groups with (C) BRAF WT and (D) BRAF V600E and with high or low expression of NFAT1.


E . Double immunofluorescent of Fn12230 , fadA - deletion mutant US1 ( ∆ fadA ) spontaneous mutant lam in log stationary phases using mAb 7H7 at 1 : 800 dilution anti - human β - amyloid polyclonal antibody A11 at 1 : 500 dilution , followed by incubation with Alexa Fluor 680 - conjugated donkey anti - rabbit Alexa Fluor 555 - conjugated goat anti - mouse , both at 1 : 1 , 000 dilution . Co - of FadA A11 was observed in Fn in stationary phase , not in log phase , or in US1 or lam . imagines were taken under 60X objective . Scale bar equals 5 μm .

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E. Double immunofluorescent staining of Fn12230, fadA-deletion mutant US1 (∆fadA) and spontaneous mutant lam in log and stationary phases using mAb 7H7 at 1:800 dilution and anti-human β-amyloid polyclonal antibody A11 at 1:500 dilution, followed by incubation with Alexa Fluor 680-conjugated donkey anti-rabbit and Alexa Fluor 555-conjugated goat anti-mouse, both at 1:1,000 dilution. Co-staining of FadA and A11 was observed in Fn in stationary phase, not in log phase, or in US1 or lam. The imagines were taken under 60X objective. Scale bar equals 5 μm.


F . Scanning electron microscopy of Fn 12230 , US1 ( ∆ fadA ) lam in log stationary phase at 10 , 000x magnification . Note fibrous structure coating Fn in stationary phase , pointed by clear arrows , but not in log phase . Scale bar equals 1 μm .

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F. Scanning electron microscopy of Fn 12230, US1 (∆fadA) and lam in log and stationary phase at 10,000x magnification. Note the fibrous structure coating Fn in stationary phase, pointed by the clear arrows, but not in log phase. Scale bar equals 1 μm.


E . Thioflavin - T binding in presence of 0 . 1 % SDS . FadA ( 0 . 8 mg / ml ) were incubated with 10 μM Thioflavin - T at room for 10 min . was measured at excitation wavelength of 440 nm emission wavelength of 500 nm . experiment was performed in duplicate repeated three times . error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 ( compared to BSA , t - ) .

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E. Thioflavin-T binding assay in the presence of 0.1% SDS. FadA proteins (0.8 mg/ml) were incubated with 10 μM Thioflavin-T at room temperature for 10 min. The fluorescent intensity was measured at excitation wavelength of 440 nm and emission wavelength of 500 nm. The experiment was performed in duplicate and repeated three times. The error bars indicate SD. *p<0.05, **p< 0.01 (compared to BSA, t-test).


G . of amyloid - like FadA produced by Fn 23726 its ∆ fap2 mutant . An aliquot of 50 μg detergent - resistant pellets was loaded onto each lane , followed by SDD - Western blot as described above . mutant produced significantly less amyloid - like polymers than wild type .

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G. Analysis of amyloid-like FadA produced by Fn 23726 and its ∆fap2 mutant. An aliquot of 50 μg detergent-resistant pellets was loaded onto each lane, followed by SDD-AGE and Western blot analysis as described above. The mutant produced significantly less amyloid-like polymers than the wild type.


A . Detection of FadA in subgingival plaque samples by . Three with periodontitis are shown here who provided plaque samples from their healthy sites ( probing depth ≤ 3 mm , left panels ) as well as periodontal diseased sites ( probing depth ≥ 7 mm , middle panels ) . was performed as described using mAb 7H7 at 1 : 800 dilution . Anti - mouse IgG isotype control of diseased site is shown ( right panels ) . were taken using 100X objective . Scale bar equals 50 μm .

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A. Detection of FadA in subgingival plaque samples by IHC. Three patients with periodontitis are shown here who provided plaque samples from their healthy sites (probing depth ≤ 3 mm, left panels) as well as periodontal diseased sites (probing depth ≥ 7 mm, middle panels). IHC was performed as described using mAb 7H7 at 1:800 dilution. Anti-mouse IgG isotype control of the diseased site is shown (right panels). The images were taken using 100X objective. Scale bar equals 50 μm.


B . Double immunofluorescence of paired normal carcinoma tissues from two CRC . frozen tissue sections were incubated with mAb 7H7 at 1 : 50 dilution anti - amyloid antibody A11 at 1 : 25 dilution , or mouse rabbit IgG control , followed by incubation with Alexa Fluor 555 - conjugated goat anti - mouse Alexa Fluor 680 - conjugated donkey anti - rabbit , both at 1 : 1000 dilution . Co - of FadA ( red ) amyloid oligomers ( green ) was observed in carcinoma but not normal tissues . were taken under 60X objective . Scale bar equals 10 μm .

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B. Double immunofluorescence staining of paired normal and carcinoma tissues from two CRC patients. The frozen tissue sections were incubated with mAb 7H7 at 1:50 dilution and anti-amyloid antibody A11 at 1:25 dilution, or mouse and rabbit IgG control, followed by incubation with Alexa Fluor 555-conjugated goat anti-mouse and Alexa Fluor 680-conjugated donkey anti-rabbit, both at 1:1000 dilution. Co-staining of FadA (red) and amyloid oligomers (green) was observed in the carcinoma but not normal tissues. The images were taken under 60X objective. Scale bar equals 10 μm.


B . Inhibition of biofilm formation mediated by detergent - resistant Fn pellets by Congo Red . An aliquot of 10 μg detergent - resistant pellets prepared from Fn 12230 ( pellet ) was added in 96 - well plates in presence of 0 , 10 or 50 µg / ml Congo Red ( CR ) , followed by addition of Fn 12230 incubated as described above . results shown are average of four experiments each performed in duplicate . error bars indicate SD . A representative image following 72 hours of incubation is shown in top panel . * * p < 0 . 01 , * * * p < 0 . 001 ( t - ) .

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B. Inhibition of biofilm formation mediated by detergent-resistant Fn pellets by Congo Red. An aliquot of 10 μg detergent-resistant pellets prepared from Fn 12230 (pellet) was added in 96-well plates in the presence of 0, 10 or 50 µg/ml Congo Red (CR), followed by addition of Fn 12230 and incubated as described above. The results shown are the average of four experiments each performed in duplicate. The error bars indicate SD. A representative image following 72 hours of incubation is shown in the top panel. ** p<0.01, ***p<0.001 (t-test).


D . Effects of Congo Red on Fn survival in acidic environment . Wild type Fn 12230 ( Fn ) or its fadA deletion mutant US1 ( ∆ fadA ) grown to stationary phase were washed incubated in PBS at pH4 in presence of absence of 50 μg / ml Congo Red ( CR ) for 1 or 2 hours . live at time 0 were designated as 100 % , those after 1 or 2 hrs of incubation were expressed relative to time 0 . results shown are average of 5 independent experiments , each performed in duplicate . error bars indicate SD . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( One - way ANOVA ) .

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D. Effects of Congo Red on Fn survival in acidic environment. Wild type Fn 12230 (Fn) or its fadA deletion mutant US1 (∆fadA) grown to stationary phase were washed and incubated in PBS at pH4 in the presence of absence of 50 μg/ml Congo Red (CR) for 1 or 2 hours. The live bacterial counts at time 0 were designated as 100%, and those after 1 or 2 hrs of incubation were expressed relative to time 0. The results shown are the average of 5 independent experiments, each performed in duplicate. The error bars indicate SD. *p<0.05, ** p<0.01, ***p<0.001 (One-way ANOVA).


F . Inhibition of Fn attachment by FadAc in presence or absence of anti - amyloid antibody OC . An aliquot of 50 μg purified recombinant FadAc was pre - incubated with DLD1 for 45 min either alone , or mixed with 20 μl OC or rabbit IgG control , before Fn was added attachment performed as above . attachment value by Fn alone was designated as 100 % , with other values expressed relative to it . FadA mutant L14A was included as a negative control . Data shown are mean values ± . experiment was performed in duplicate repeated three times . * * p < 0 . 01 ( One - way ANOVA ) .

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F. Inhibition of Fn attachment by FadAc in the presence or absence of anti-amyloid antibody OC. An aliquot of 50 μg purified recombinant FadAc was pre-incubated with DLD1 for 45 min either alone, or mixed with 20 μl OC or rabbit IgG control, before Fn was added and the attachment assay performed as above. The attachment value by Fn alone was designated as 100%, with all other values expressed relative to it. FadA mutant L14A was included as a negative control. Data shown are mean values ± SEM. The experiment was performed in duplicate and repeated three times. **p< 0.01 (One-way ANOVA).


D . Effect of detergent - resistant pellets prepared from Fn 12230 ( Fn ) US1 ( ∆ fadA ) on growth . HCT116 were inoculated into nude mice as described above . In one group of mice ( n = 3 ) , sarkosyl - resistant pellets prepared from Fn 12230 US1 ( ∆ fadA ) were injected into tumors on opposite sides . In another group ( n = 5 ) , sarkosyl - resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control ( rIgG ) before injecting into tumors on opposite sides . volumes were measured described above . vertical lines represent standard deviations . pairs are shown ( right panel ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 [ compared to US1 ( ∆ fadA ) , t - ] .

O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O B-GENEPROD O O B-EXP_ASSAY O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O

D. Effect of detergent-resistant pellets prepared from Fn 12230 (Fn) and US1 (∆fadA) on tumor growth. HCT116 cells were inoculated into the nude mice as described above. In one group of mice (n=3), sarkosyl-resistant pellets prepared from Fn 12230 and US1 (∆fadA) were injected into the tumors on opposite sides. In another group (n=5), sarkosyl-resistant pellets prepared from Fn 12230 were mixed with OC or rabbit IgG control (rIgG) before injecting into the tumors on opposite sides. The tumor volumes were measured described above. The vertical lines represent the standard deviations. The individual tumor pairs are shown (right panel). *p < 0.05, **p < 0.01, ***p< 0.001 [compared to US1 (∆fadA), t-test].


A . Approximately 1x109 CFU of Fn 12230 , US1 ( ∆ fadA ) , lam suspended in carboxymethylcellulose ( CMC ) were orally administered to C57BL / 6 mice three times a week for 10 . CMC alone was administered as a control . Maxillae from mice inoculated with CMC alone ( n = 5 ) , Fn 12230 ( n = 5 ) , US1 ( ∆ fadA ; n = 5 ) , lam ( n = 4 ) were harvested fixed in 4 % paraformaldehyde stored in 70 % ethanol , followed by microCT ( μCT ) scanning using a Scanco vivaCT 80 system at 55 kVp , 145 µA , 250 ms integration time . Shown in figure are reconstructed grayscale . ImageJ was used to measure difference in bone height from cementoenamel junction ( CEJ ) to alveolar crest between palatal roots of first second molars ( see arrows ) . B . average bone loss of each group shown in ( A ) , with lines above each bar representing standard deviations . * p < 0 . 05 . * * p < 0 . 01 ( t - ) .

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

A. Approximately 1x109 CFU of Fn 12230, US1 (∆fadA), and lam suspended in carboxymethylcellulose (CMC) were orally administered to C57BL/6 mice three times a week for 10 weeks. CMC alone was administered as a control. Maxillae from mice inoculated with CMC alone (n=5), Fn 12230 (n=5), US1 (∆fadA; n=5), and lam (n=4) were harvested and fixed in 4% paraformaldehyde and stored in 70% ethanol, followed by microCT (μCT) scanning using a Scanco vivaCT 80 system at 55 kVp, 145 µA, and 250 ms integration time. Shown in the figure are reconstructed grayscale images. ImageJ was used to measure difference in bone height from the cementoenamel junction (CEJ) to the alveolar crest between palatal roots of first and second molars (see arrows). B. The average bone loss of each group shown in (A), with the lines above each bar representing standard deviations. *p< 0.05. **p< 0.01 (t-test).


A . Relative mRNA expression in skeletal muscle from SBMA ( bars , n = 3 ) controls ( black bars , n = 3 ) ( mean + / − ) . * p < 0 . 05 , * * p < 0 . 01 by Student ' s t .

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A. Relative mRNA expression in skeletal muscle from SBMA patients (white bars, n = 3) and controls (black bars, n = 3) (mean +/− SEM). * p<0.05, ** p<0.01 by Student's t test.


B . Splicing of XBP1 mRNA was assessed by RT - PCR . Products from unspliced ( XBP1u ) spliced ( XBP1s ) transcripts were resolved on a nondenaturing polyacrylamide gel stained with SYBR green .

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B. Splicing of XBP1 mRNA was assessed by RT-PCR. Products from unspliced (XBP1u) and spliced (XBP1s) transcripts were resolved on a nondenaturing polyacrylamide gel and stained with SYBR green.


C . Relative mRNA expression in proximal hind limb muscle ( mean + / − ) . Mice evaluated were littermate WT ( n = 6 ) , AR113Q ( n = 6 ) , castrated WT ( C - WT , n = 6 ) castrated AR113Q ( C - AR113Q , n = 5 ) on a mixed C57BL / 6J - 129 genetic background . * p < 0 . 05 by ANOVA .

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C. Relative mRNA expression in proximal hind limb muscle (mean +/− SEM). Mice evaluated were littermate WT (n = 6), AR113Q (n = 6), castrated WT (C-WT, n = 6) and castrated AR113Q males (C-AR113Q, n = 5) on a mixed C57BL/6J-129 genetic background. *p<0.05 by ANOVA.


D . Western blot of BiP PDI expression in proximal hind limb muscle . Right panels show of signal relative to loading control ( mean + / − ) . * p < 0 . 05 by Student ' s t .

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D. Western blot of BiP and PDI expression in proximal hind limb muscle. Right panels show quantification of signal relative to loading control (mean +/− SEM). * p<0.05 by Student's t test.


E . Relative mRNA expression in proximal hind limb muscle of AR21Q ( n = 5 ) AR113Q ( n = 3 ) backcrossed to C57BL / 6J . * * p < 0 . 01 , * * * p < 0 . 001 by Student ' s t . F . Relative mRNA expression in spinal cord of AR21Q ( n = 5 ) AR113Q ( n = 3 ) ( mean + / − ) . n . s . = not significant by Student ' s t .

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E. Relative mRNA expression in proximal hind limb muscle of AR21Q (n = 5) and AR113Q (n = 3) males backcrossed to C57BL/6J. ** p<0.01, ***p<0.001 by Student's t test. F. Relative mRNA expression in spinal cord of AR21Q (n = 5) and AR113Q (n = 3) males (mean +/− SEM). n. s. = not significant by Student's t test.


A . Muscle fiber ( 100 / mouse ) was quantified from proximal hind limb muscle of AR113Q ( black ) or AR113Q , CHOP − / − mice ( ) at 12 wks . Left panel shows fiber distribution , middle panel shows cumulative of as a function of fiber , right panel shows relative fiber cross sectional ( mean + / − ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney . Right panel , p < 0 . 001 by Student ' s t .

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A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limb muscle of AR113Q (black) or AR113Q, CHOP −/− mice (white) at 12 wks. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.


B . Representative image of muscle following NADH stain . Bar = 20 µM .

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B. Representative image of muscle fibers following NADH stain. Bar = 20 µM.


C . Distribution of proximal hind limbmuscle fiber from wt ( black ) CHOP − / − ( ) mice at 12 wks . Difference not significant by Mann - Whitney .

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C. Distribution of proximal hind limbmuscle fiber size from wt (black) and CHOP −/− (white) mice at 12 wks. Difference not significant by Mann-Whitney test.


A . Relative expression of MurRF1 MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice ( n = 5 - 6 / genotype ) . * p < 0 . 05 by ANOVA , n . s . = not significant .

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A. Relative expression of MurRF1 and MAFbx mRNAs in proximal hind limbmuscle of 12 wk mice (n = 5-6/genotype). *p<0.05 by ANOVA, n. s. = not significant.


B . LC3 p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot . Right panels show of signal relative to GAPDH . * * p < 0 . 01 by Student ' s t .

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B. LC3 and p62 expression in proximal hind limbmuscle of 12 wk mice was assessed by western blot. Right panels show quantification of signal relative to GAPDH. **p<0.01 by Student's t test.


C . Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle . * p < 0 . 05 by Student ' s t .

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C. Relative expression of mRNAs encoding autophagy regulators in proximal hind limbmuscle. *p<0.05 by Student's t test.


Androgen receptor expression in skeletal muscle of 12 wk mice . Hsp90 serves as a loading control . Right panel shows of relative signal ( n = 3 / genotype ) .

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Androgen receptor protein expression in skeletal muscle of 12 wk mice. Hsp90 serves as a loading control. Right panel shows quantification of relative signal intensity (n = 3/genotype).


D . Proximal hind limb stained for androgen receptor ( in red ) exhibit intranuclear inclusions . Nuclei are stained by DAPI ( in blue ) .

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D. Proximal hind limb muscles stained for the androgen receptor (in red) exhibit intranuclear inclusions. Nuclei are stained by DAPI (in blue).


E . P - JNK P - eIF2 alpha expression ( top , middle ) XBP1 mRNA splicing ( bottom ) in proximal hind limbmuscle of 12 wk mice . Right panels show of signal relative to loading control . * * p < 0 . 01 by Student ' s t . n . s . = not significant .

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E. P-JNK and P-eIF2 alpha expression (top, middle) and XBP1 mRNA splicing (bottom) in proximal hind limbmuscle of 12 wk mice. Right panels show quantification of signal relative to loading control. **p<0.01 by Student's t test. n. s. = not significant.


Denervated gastrocnemius or intact controls were harvested at indicated times following unilateral sciatic nerve transection in 6 wk mice . A . Western blot shows enhanced eIF2 alpha phosphorylation ( top ) RT - PCR demonstrates increased XBP1 mRNA splicing ( bottom ) in denervated muscle . Right panels show relative of signal . * p < 0 . 05 by Student ' s t .

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Denervated gastrocnemius muscles or contralateral intact controls were harvested at the indicated times following unilateral sciatic nerve transection in 6 wk male mice. A. Western blot shows enhanced eIF2 alpha phosphorylation (top) and RT-PCR demonstrates increased XBP1 mRNA splicing (bottom) in denervated muscle. Right panels show relative quantification of signal intensity. *p<0.05 by Student's t test.


. B . Relative expression of BiP , ATF4 CHOP mRNA ( n = 3 ) . * p < 0 . 05 by Student ' s t .

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. B. Relative expression of BiP, ATF4 and CHOP mRNA (n = 3). *p<0.05 by Student's t test.


C . Following surgical denervation of wild type or CHOP − / − mice , LC3 p62 expression was assessed by western blot . Right panels show of signal relative to GAPDH . * * * p < 0 . 001 by ANOVA , n . s . = not significant .

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C. Following surgical denervation of wild type or CHOP−/− mice, LC3 and p62 expression was assessed by western blot. Right panels show quantification of signal relative to GAPDH. ***p<0.001 by ANOVA, n. s. = not significant.


D . Muscle fiber ( 100 / mouse ) was quantified from wild type ( black , n = 5 ) , CHOP − / − ( , n = 3 ) Beclin - 1 + / − ( grey , n = 3 ) mice 7 days post sciatic nerve transection . Shown is relative fiber cross sectional ( mean + / − ) . * * * p < 0 . 001 by ANOVA .

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D. Muscle fiber size (100 fibers/mouse) was quantified from wild type (black, n = 5), CHOP −/− (white, n = 3) and Beclin-1 +/− (grey, n = 3) mice 7 days post sciatic nerve transection. Shown is relative fiber cross sectional area (mean +/− SEM). ***p<0.001 by ANOVA.


E . Muscle fiber ( 100 / mouse ) was quantified from CHOP − / − ( black , n = 6 ) or CHOP − / − , Beclin - 1 + / − mice ( , n = 6 ) 7 days post sciatic nerve transection . Left panel shows fiber distribution , middle panel shows cumulative of as a function of fiber , right panel shows relative fiber cross sectional ( mean + / − ) . Left , middle panels , p < 0 . 0001 by Mann - Whitney . Right panel , p < 0 . 001 by Student ' s t .

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E. Muscle fiber size (100 fibers/mouse) was quantified from CHOP −/− (black, n = 6) or CHOP −/−, Beclin-1 +/− mice (white, n = 6) 7 days post sciatic nerve transection. Left panel shows fiber size distribution, middle panel shows cumulative percent of fibers as a function of fiber area, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left, middle panels, p<0.0001 by Mann-Whitney test. Right panel, p<0.001 by Student's t test.


A . Muscle fiber ( 100 / mouse ) was quantified from proximal hind limbmuscle of AR113Q ( red , n = 6 ) or AR113Q , Beclin - 1 + / − ( blue , n = 6 ) mice at 16 wks . Left panel shows fiber distribution , right panel shows relative fiber cross sectional ( mean + / − ) . Left panel , p < 0 . 0001 by Mann - Whitney . Right panel , p < 0 . 0001 by Student ' s t .

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A. Muscle fiber size (100 fibers/mouse) was quantified from proximal hind limbmuscle of AR113Q (red, n = 6) or AR113Q, Beclin-1 +/− (blue, n = 6) mice at 16 wks. Left panel shows fiber size distribution, and right panel shows relative fiber cross sectional area (mean +/− SEM). Left panel, p<0.0001 by Mann-Whitney test. Right panel, p<0.0001 by Student's t test.


B . AR expression in skeletal muscle of 16 wk mice was assessed by western blot . GAPDH controls for loading . Right panel shows of signal relative to GAPDH ( mean + / − ) . Differences not significant ( n . s . ) .

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B. AR expression in skeletal muscle of 16 wk mice was assessed by western blot. GAPDH controls for loading. Right panel shows quantification of signal relative to GAPDH (mean +/− SEM). Differences not significant (n. s.).


A . Left panel , Kaplan - Meyer survival curve of AR113Q ( red , n = 12 ) AR113Q , Beclin - 1 + / − ( blue , n = 15 ) . * p < 0 . 05 by log - rank . Right panel , mean survival + / − . * p < 0 . 05 by Student ' s t - .

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A. Left panel, Kaplan-Meyer survival curve of AR113Q males (red line, n = 12) and AR113Q, Beclin-1 +/− males (blue line, n = 15). *p<0.05 by log-rank analysis. Right panel, mean survival +/− SEM. *p<0.05 by Student's t-test.


B , C . ( panel B ) grip strength ( panel C ) at different ages for wild type ( wt , green , n = 7 ) , Beclin - 1 + / − ( yellow n = 9 ) , AR113Q ( red , n = 12 ) , AR113Q , Beclin - 1 + / − ( blue , n = 15 ) mice .

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B, C. Body weight (panel B) and grip strength (panel C) at different ages for wild type (wt, green line, n = 7), Beclin-1 +/− (yellow line n = 9), AR113Q (red line, n = 12), and AR113Q, Beclin-1 +/− (blue line, n = 15) male mice.


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D . of disease onset as measured by a decrease of 5 % or more in forelimb grip strength ( not significant by log rank ) .

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D. Age of disease onset as measured by a decrease of 5% or more in forelimb grip strength (not significant by log rank analysis).


Tfeb expression in vasculature ( mice n = 10 ) . ( A ) Representative of retinal vessels ( p5 ) ( A ) ( scale bar : 50 µm ) of Tfeb - EGFP mice stained with anti - iB4 ( A ) anti -

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Tfeb expression in the vasculature (mice n=10). (A) Representative images of retinal vessels (p5) (A) (scale bar: 50 µm) of Tfeb-EGFP mice stained with anti-iB4 (A) and anti-GFP