Figure 6 ( A ) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies . ( B , C ) RNA isolated from untreated or MG132 - treated ( 20 µM , 2h ) control or NRF2 siRNA transfected cells were subjected to qRT - PCR with primers of genes as indicated . The fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm . WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells , untreated or treated with MG132 ( 20 µM , 2h ) as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells , untreated or treated with H2O2 ( 200 µM , 2h ) as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel . Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( G ) H2O2 ( 200 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel . ( H ) Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) and subjected to WB analysis with Myc , GFP and indicated antibodies . ( I ) Representative confocal images of Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 which were treated with MG132 ( 20 µM , 2h ) and subjected to IF with Ub and p62 antibodies . ( J ) Graph represent % of cells with Ub - p62 dots , data from ≥ 10 microscopic fields ( 40X ) , n = 3 , mean ± SD , * p < 0 . 005 ( one - way ANOVA test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.(B, C) RNA isolated from untreated or MG132-treated (20 µM, 2h) control or NRF2 siRNA transfected cells were subjected to qRT-PCR with primers of genes as indicated. The fold induction in MG132 treated samples is calculated relative to untreated samples. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with MG132 (20 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (F) MG132 (20 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.(H) Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 or GFP-NRF2 were treated with MG132 (20 µM, 2h) and subjected to WB analysis with Myc, GFP and indicated antibodies.(I) Representative confocal images of Hela cells, TRIM16KO cells, and TRIM16KO cells complemented with Myc-NRF2 which were treated with MG132 (20 µM, 2h) and subjected to IF with Ub and p62 antibodies.(J) Graph represent % of cells with Ub-p62 dots, data from ≥ 10 microscopic fields (40X), n=3, mean ±SD, *p < 0.005 (one-way ANOVA test). Data information: Unless otherwise stated, scale bar: 10 µm.


Figure 7WB analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells expressing either ( A ) Flag - p62 probed with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric analysis of WB ' s normalized with α - Tubulin or Lamin - B1 . WB analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells expressing either ( C ) GFP - polyQ74 probed with or GFP or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric analysis of WB ' s normalized with α - Tubulin or Lamin - B1 . ( E ) Confocal images of GFP - TRIM16 expressing HeLa cells , untreated or treated with MG132 ( 10 µM , 4h ) and / or Bafilomycin A1 ( 300 nM , 3h ) and IF analysis is performed with antibodies as indicated . Right panel graphs : fluorescence intensity line tracing corresponding to a white line in zoom panel . Scale bar : 10 µm . ( F ) The graph shows the percentage of cells with TRIM16 - Ub or TRIM16 - Ub - p62 co - localized dots / aggregates . Data from ≥ 10 microscopic fields , n = 2 , mean ± SD . ( G ) Representative confocal images of GFP - TRIM16 expressing HeLa cells , untreated or treated with MG132 ( 10 µM , 4h ) and / or Bafilomycin A1 ( 300 nM , 3h ) and IF analysis is performed with antibodies as indicated . Right panel graphs : fluorescence intensity line tracing corresponding to a white line in zoom panel . Scale bar : 10 µm . ( H ) Co - IP analysis of the interaction between two differently tagged TRIM16 variants . ( I ) WB analysis of detergent - soluble and - insoluble DSS - cross - linked fractions of HeLa and TRIM16KO cells , untreated or treated with MG132 ( 10 µM , 4h ) and probed with indicated antibodies . ( J ) Co - IP analysis of the interaction between Flag - tagged TRIM16 and Myc - tagged TRIM16 full length and its domain deletion proteins .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (A) Flag-p62 probed with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.WB analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells expressing either (C) GFP-polyQ74 probed with or GFP or indicated antibodies. L.E, Low exposure; H.E, High Exposure. Densitometric analysis of WB's normalized with α-Tubulin or Lamin-B1.(E) Confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm. (F) The graph shows the percentage of cells with TRIM16-Ub or TRIM16-Ub-p62 co-localized dots/aggregates. Data from ≥ 10 microscopic fields, n=2, mean ±SD. (G) Representative confocal images of GFP-TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 µM, 4h) and/or Bafilomycin A1 (300 nM, 3h) and IF analysis is performed with antibodies as indicated. Right panel graphs: fluorescence intensity line tracing corresponding to a white line in zoom panel. Scale bar: 10 µm.(H) Co-IP analysis of the interaction between two differently tagged TRIM16 variants.(I) WB analysis of detergent-soluble and -insoluble DSS-cross-linked fractions of HeLa and TRIM16KO cells, untreated or treated with MG132 (10 µM, 4h) and probed with indicated antibodies.(J) Co-IP analysis of the interaction between Flag-tagged TRIM16 and Myc-tagged TRIM16 full length and its domain deletion proteins.


Figure 8 ( A ) MTT assays performed at different time points with HeLa and TRIM16KO cells , untreated or treated with H2O2 ( 400 µM , 2h ) . Data , mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) . ( B ) Images of HeLa and TRIM16KO cells , untreated or treated with As2O3 ( 2 . 5 µM , 4h ) . Scale bar : 400 µm . Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( C ) As2O3 ( 2 . 5 µM , 4h ) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( D ) H2O2 ( 400 µM , 2h ) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( E ) puromycin ( 5 µg / ml , 6h ) Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( F ) MG132 ( 20 µM , 8h ) . ( G ) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Arrowheads indicate the cleaved form of PARP - 1 and Casapse - 3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) MTT assays performed at different time points with HeLa and TRIM16KO cells, untreated or treated with H2O2 (400 µM, 2h). Data, mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).(B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (C) As2O3 (2.5 µM, 4h)Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (D) H2O2 (400 µM, 2h)Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h)Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (F) MG132 (20 µM, 8h).(G) Immunoblot blot analysis of HeLa and TRIM16KO lysates of cells treated with 5 µM of As2O3 for different durations as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure. Arrowheads indicate the cleaved form of PARP-1 and Casapse-3.


Figure 9 ( A , B ) Clonogenic assays were performed with HeLa and TRIM16KO cells , untreated or treated with As2O3 or MG132 . ( C ) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points . As2O3 is injected when tumor volume of all groups was > 100mm3 ( day 0 in the graph ) . Tumor volumes were measured on an interval as indicated . Mean ± SE , n = 6 ( each group ) , * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 ( ANOVA ) . ( D ) Graph shows the % tumor regression ( Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100 ) . Mean ± SE , n = 6 ( each group ) , * * p < 0 . 005 ( Student ' s unpaired t - test ) . ( E ) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3 . ( F ) Representative pictures of dissected tumors . ( G ) Immunohistochemistry analysis performed with KI - 67 cell proliferation marker and hematoxylin nuclear stain . The graph represents the % of cells with KI - 67 staining . > 400 cells were counted for this analysis from three different sections from different animals , mean ± SD , * p < 0 . 05 ( Student ' s unpaired t - test ) . Scale bar : 50 µm . ( H ) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated . L . E , Low exposure ; H . E , High Exposure .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 9(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132.(C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA). (D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test). (E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3. (F) Representative pictures of dissected tumors. (G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm.(H) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated. L.E, Low exposure; H.E, High Exposure.


Figure 1 ( A ) Western blotting for phospho - Thr172 - AMPK ( pAMPK ) , total AMPK , phospho - Ser79 - ACC ( pACC ) , and total ACC 24h after treatment . Quantification represents the pAMPK / AMPK and pACC / ACC ratios relative to the non - treated ( NT ) control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( B ) Phase contrast images of fibers 72h after treatment . Scale bars represent 0 . 25mm ( C ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( D - E ) RT - qPCR analysis of the mRNA levels of MyoD ( D 24h after treatment relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( D - E ) RT - qPCR analysis of the mRNA levels o myogenin ( E ) 24h after treatment relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

Figure 1 (A) Western blotting for phospho-Thr172-AMPK (pAMPK), total AMPK, phospho-Ser79-ACC (pACC), and total ACC 24h after treatment. Quantification represents the pAMPK/AMPK and pACC/ACC ratios relative to the non-treated (NT) control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (B) Phase contrast images of fibers 72h after treatment. Scale bars represent 0.25mm  (C) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (D-E) RT-qPCR analysis of the mRNA levels of MyoD (D 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls  (D-E) RT-qPCR analysis of the mRNA levels o myogenin (E) 24h after treatment relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent standard error of the mean (SEM). Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 2 ( A - B ) Rates of glucose consumption ( A measured in the media 24h after treatment relative to the non - treated ( NT ) control from three independent experiments ( n = 3 ) . Error bars represent the SEM Data information : Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls

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

Figure 2 (A-B) Rates of glucose consumption (A measured in the media 24h after treatment relative to the non-treated (NT) control from three independent experiments (n = 3). Error bars represent the SEM Data information: Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls


Figure 3 ( A ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K ( n = 3 ) and pS6 / S6 ( n = 4 ) ratios relative to the non - treated ( NT ) control Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( B ) Radiographic analysis of de novo protein synthesis using 35S - labelling . Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

Figure 3 (A) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K (n = 3) and pS6/S6 (n = 4) ratios relative to the non-treated (NT) control Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls (B) Radiographic analysis of de novo protein synthesis using 35S-labelling. Quantification represents whole lane radiation signal density standardized to Coomassie staining and relative to NT control levels (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 6 ( A ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( B - C ) Rates of glucose consumption ( B measured in the media 24h after treatment relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( B - C ) Rates o lactate production ( C ) measured in the media 24h after treatment relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( D ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD ( E ) Western blotting of iNOS , tubulin , phospho - Thr172 AMPKα ( pAMPK ) , total AMPKα ( AMPK ) , phospho - Ser79 - ACC ( pACC ) and total ACC . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( F ) RT - qPCR analysis of iNOS mRNA levels relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( G ) Nitric oxide ( NO ) levels in the culture media of cells relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 (A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (B-C) Rates of glucose consumption (B measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (B-C) Rates o lactate production (C) measured in the media 24h after treatment relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (D) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD  (E) Western blotting of iNOS, tubulin, phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC (pACC) and total ACC. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls (F) RT-qPCR analysis of iNOS mRNA levels relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls (G) Nitric oxide (NO) levels in the culture media of cells relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 7 ( A ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( B ) Western blotting for iNOS and tubulin protein levels 24h after treatment . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls ( C ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

Figure 7 (A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls  (B) Western blotting for iNOS and tubulin protein levels 24h after treatment. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls  (C) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the NT control. Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


Figure 8 ( A ) Representative images of overall hind limb musculature . Scale bar represents 1cm ( B ) Western blotting for phospho - Ser79 - ACC ( pACC ) and total ACC ( C ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls ( D - E ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( D mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls ( D - E ) RT - qPCR analysis o MuRF1 ( E ) mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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

Figure 8 (A) Representative images of overall hind limb musculature. Scale bar represents 1cm  (B) Western blotting for phospho-Ser79-ACC (pACC) and total ACC  (C) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls  (D-E) RT-qPCR analysis of Atrogin-1/MAFbx (D mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls  (D-E) RT-qPCR analysis o MuRF1 (E) mRNA expression from the tibialis anterior of three mice per cohort (n = 3). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from saline controls; †, p < 0.05; ††, p < 0.01 from C26 controls


Figure 9 ( A ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls ( B - C ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( B mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls ( B - C ) RT - qPCR analysis o MuRF1 ( C ) mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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

Figure 9 (A) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls  (B-C) RT-qPCR analysis of Atrogin-1/MAFbx (B mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls  (B-C) RT-qPCR analysis o MuRF1 (C) mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


Figure 1Representative grey - scale images of primary rat hippocampal neurons ( DIV 10 ) transfected with GFP and the indicated miRNA mimics . Quantification of the mean number of intersections by Sholl analysis . GFP - only transfected conditions were set to one in each experiment . Representative grey - scale images of GFP - transfected Cacna1c + / + ( WT ) or Cacna1c + / - primary rat hippocampal neurons ( DIV 10 ) . Scale bars = 50 μm . Quantification of the mean number of intersections by Sholl analysis . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Unpaired two - sample t - Test , * * p = 0 . 0096 ) . Fold changes represent changes in dendritic complexity relative to the control condition .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative grey-scale images of primary rat hippocampal neurons (DIV 10) transfected with GFP and the indicated miRNA mimics. Quantification of the mean number of intersections by Sholl analysis. GFP-only transfected conditions were set to one in each experiment.Representative grey-scale images of GFP-transfected Cacna1c+/+ (WT) or Cacna1c+/- primary rat hippocampal neurons (DIV 10). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Unpaired two-sample t-Test, **p=0.0096). Fold changes represent changes in dendritic complexity relative to the control condition.


Figure 2Representative images of DIV 10 hippocampal neurons co - transfected with control or miR - 499 - 5p mimics and a pMT2 - CACNB2 expression plasmid ( CACNB2 ) . Scale bars = 50 μm . Quantification of the mean number of intersections by Sholl analysis . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Two - way ANOVA : no main effect of the CACNB2 transfection p = 0 . 5027 or the miRNA mimics p = 0 . 0805 , main effect of the CACBN2 transfection by miRNA mimics interaction p = 0 . 0213 , Tukey ' s HSD : * p = 0 . 0303 ) . Fold changes represent the changes in dendritic complexity of transfected neurons compared to non - treated control neurons . ns = not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Representative images of DIV 10 hippocampal neurons co-transfected with control or miR-499-5p mimics and a pMT2-CACNB2 expression plasmid (CACNB2). Scale bars = 50 μm.   Quantification of the mean number of intersections by Sholl analysis. Data are represented as scattered dot plots with bar, mean±S.D. (n=3 independent experiments; Two-way ANOVA: no main effect of the CACNB2 transfection p=0.5027 or the miRNA mimics p=0.0805, main effect of the CACBN2 transfection by miRNA mimics interaction p=0.0213, Tukey's HSD: *p=0.0303). Fold changes represent the changes in dendritic complexity of transfected neurons compared to non-treated control neurons. ns = not significant.


Figure 3A ) Representative images of DIV 19 rat hippocampal neurons co - transfected with GFP ( green channel ) and Cav1 . 2 - HA ( red channel ) , together with either control or miR - 499 - 5p mimics . After 12 - 13 days of expression , labeling with Anti - HA antibodies was performed in live conditions to identify cell surface Cav1 . 2 channels . Whole cells : scale bars = 20 μm ; cell body and dendrite insets : scale bars = 5 μm . B ) Quantification of the area occupied by surface Cav1 . 2 fluorescence normalized to the total cell area ( GFP fluorescence ) from non - permeabilized neurons transfected as in A ) . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 4 independent experiments ; Paired two - sample t - Test * p = 0 . 0318 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A) Representative images of DIV 19 rat hippocampal neurons co-transfected with GFP (green channel) and Cav1.2-HA (red channel), together with either control or miR-499-5p mimics. After 12-13 days of expression, labeling with Anti-HA antibodies was performed in live conditions to identify cell surface Cav1.2 channels. Whole cells: scale bars = 20 μm; cell body and dendrite insets: scale bars= 5 μm. B) Quantification of the area occupied by surface Cav1.2 fluorescence normalized to the total cell area (GFP fluorescence) from non-permeabilized neurons transfected as in A). Data are represented as scattered dot plots with bar, mean±S.D. (n=4 independent experiments; Paired two-sample t-Test *p=0.0318).


Figure 4D ) Representative Western blot images of CACNB2 ( upper panel ) and GAPDH ( lower panel ) protein expression levels in the hippocampus of WT rats and Cacna1c + / - rats injected with the AAV - Control or AAV - miR - 499 hairpin viruses . GAPDH was used as a loading control E ) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers and data points ( + : mean , line : median ; whiskers : minimum and maximum values ) . ( * p = 0 . 0103 , Unpaired two - sample t - Test ; n = 7 rats in the AAV - Control group , n = 6 rats in the AAV - miR - 499 group ) . F ) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c + / - rats injected with either AAV - control or AAV - miR - 499 . Data are represented as box plot with whiskers and data points ( + : mean , line : median ; whiskers : minimum and maximum values ) . ( * * p = 0 . 0044 , Unpaired two - sample t - Test ; n = 8 rats in the AAV - Control group , n = 7 rats in the AAV - miR - 499 group ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D) Representative Western blot images of CACNB2 (upper panel) and GAPDH (lower panel) protein expression levels in the hippocampus of WT rats and Cacna1c +/- rats injected with the AAV-Control or AAV-miR-499 hairpin viruses. GAPDH was used as a loading control E) Quantification of CACNB2 Western blots using hippocampal protein lysate from wild type rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (*p=0.0103, Unpaired two-sample t-Test; n=7 rats in the AAV-Control group, n=6 rats in the AAV-miR-499 group). F) Quantification of CACNB2 Western blots using hippocampal protein lysate from Cacna1c +/- rats injected with either AAV-control or AAV-miR-499. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). (**p=0.0044, Unpaired two-sample t-Test; n=8 rats in the AAV-Control group, n=7 rats in the AAV-miR-499 group).


Figure 5miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 26 , male = 31 ) , BD ( female = 26 , male = 37 ) , or MDD ( female = 18 , male = 24 ) subjects . Wilcoxon rank sum test after correction for age and antidepressant treatment ( linear model of the form Fold Change ~ Group * Sex + Age + Antidepressant treatment ) . 2 - way ANOVA with correction for sex , age and antidepressant treatment ( linear model of the form Fold Change ~ Group * Sex + Age + Antidepressant treatment ) . Control vs . BD : * * * * p = 8 . 89e - 07 ; control vs . MDD : n . s . p = 0 . 12674 . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 8 , male = 10 ) and SZ ( female = 8 , male = 15 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 9785 ; main effect Group : p = 0 . 0924 ; main effect Interaction : p = 0 . 9802 . Tukey ' s HSD : p = 0 . 0924 , ns . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control ( female = 26 , male = 31 ) , BD I ( female = 9 , male = 9 ) , or BDI II ( female = 12 , male = 16 ) subjects . 2 - way ANOVA , Main effect Sex : p = 0 . 174 ; main effect Group : * * * * p = 4 . 07e - 09 ; Interaction Group x Sex : p = 0 . 311 . Tukey ' s HSD : control vs . BDI : * * * * p = 0 . 0000037 . control vs . BDII : p = 0 . 0000002 . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of control subjects ( female = 26 , male = 31 ) , and BD subjects in different mood states ( depressive ( female = 7 , male = 11 ) , euthymic ( female = 7 , male = 4 ) , hypomanic ( female = 2 , male = 6 ) , manic ( female = 2 , male = 1 ) , mixed ( female = 1 , male = 1 ) . 2 - way ANOVA , main effect Sex : p = 0 . 260 ; main effect Group : p = 1 . 26e - 07 ; Interaction Group x Sex : p = 0 . 059 ; Tukey ' s HSD : control vs . depressive * * * p = 0 . 0002621 , control vs . euthymic * * * p = 0 . 0008533 , control vs . hypomanic * * * * p = 0 . 0000470 . Data are presented as violin plots with median , quartiles and data points . miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects ( - CMT , n = 17 ) and healthy subjects with a history of childhood maltreatment ( + CMT , n = 17 ) . ( * * * * p < 0 . 0001 , Mann - Whitney U test ) . CMT : Childhood maltreatment . Data are presented as violin plots with median , quartiles and data points .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD (female= 26, male= 37), or MDD (female= 18, male=24) subjects. Wilcoxon rank sum test after correction for age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). 2-way ANOVA with correction for sex, age and antidepressant treatment (linear model of the form Fold Change ~ Group*Sex + Age + Antidepressant treatment). Control vs. BD: ****p=8.89e-07; control vs. MDD: n.s. p=0.12674. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 8, male= 10) and SZ (female= 8, male= 15) subjects. 2-way ANOVA, Main effect Sex: p=0.9785; main effect Group: p= 0.0924; main effect Interaction: p= 0.9802. Tukey's HSD: p=0.0924, ns. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control (female= 26, male= 31), BD I (female= 9, male= 9), or BDI II (female= 12, male=16) subjects. 2-way ANOVA, Main effect Sex: p=0.174; main effect Group: ****p=4.07e-09; Interaction Group x Sex: p=0.311. Tukey's HSD: control vs. BDI: ****p=0.0000037. control vs. BDII:p=0.0000002. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of control subjects (female= 26, male= 31), and BD subjects in different mood states (depressive (female= 7, male=11), euthymic (female= 7, male=4), hypomanic (female=2, male=6), manic (female=2, male=1), mixed (female=1, male=1). 2-way ANOVA, main effect Sex: p=0.260; main effect Group: p=1.26e-07; Interaction Group x Sex: p=0.059; Tukey's HSD: control vs. depressive ***p=0.0002621, control vs. euthymic ***p=0.0008533, control vs. hypomanic ****p=0.0000470. Data are presented as violin plots with median, quartiles and data points.miR-499-5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects (-CMT, n=17) and healthy subjects with a history of childhood maltreatment (+CMT, n=17). (****p<0.0001, Mann-Whitney U test). CMT: Childhood maltreatment. Data are presented as violin plots with median, quartiles and data points.


Figure 6Association of miR - 499 - 5p expression and GMV in HC ( n = 23 ) : miR - 499 - 5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area ( i . e . transverse temporal gyrus , the left parietal operculum and the left superior temporal gyrus ) ( in blue ) at p < . 05 cluster - level family wise error - corrected ( FWE ) for multiple comparisons after an initial threshold of p < . 001 uncorrected ( k = 1090 , x / y / z = - 42 / - 30 / 15 , t = 4 . 7 , p = . 045 ) .

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Figure 6Association of miR-499-5p expression and GMV in HC (n=23): miR-499-5p levels were negatively correlated with a GMV cluster comprising parts of the left Wernicke language area (i.e. transverse temporal gyrus, the left parietal operculum and the left superior temporal gyrus) (in blue) at p<.05 cluster-level family wise error-corrected (FWE) for multiple comparisons after an initial threshold of p<.001 uncorrected (k=1090, x/y/z=-42/-30/15, t= 4.7, p=.045).


figf1 ( a ) Confocal time - lapse images of an MCF10A cell - in - cell structure expressing GFP - LC3 and H2B - mCherry . The arrow marks recruitment of LC3 to the entotic vacuole ( see Supplementary Movie S1 ) . Scale bar , 15 μm . ( b ) Images of MCF10A cells expressing GFP - LC3G120A and mCherry - LC3 ; note no recruitment of GFP - LC3G120A to the entotic vacuole . Scale bar , 15 μm . ( c ) LC3 is recruited from the host - cell cytosol onto the entotic vacuole membrane . Data points are the mean fluorescence intensity of GFP - LC3 on vacuoles versus cytoplasm for two independent cell - in - cell structures ( see Supplementary Movie S2 ) . ( d ) Correlative video - light - electron microscopy of an MCF10A GFP - LC3 cell - in - cell structure . Cells were imaged by time - lapse microscopy , followed by fixation after LC3 recruitment ( see Supplementary Movie S3 ) . Top left , post - fixation images of phase - contrast and GFP - fluorescence signals were taken demonstrating LC3 recruitment . Bottom left and right , electron microscopy ( EM ) of the same cell shows the entotic vacuole is a single membrane ( arrow in right image , which is a higher magnification of the area outlined in the bottom left image ) ( e ) Percentage of MCF10A cell - in - cell structures , with and without autophagy inhibition , that show LC3 recruitment associated with death of the internalized cell . Data represent mean ± s . e . m . from at least three independent experiments ; n , total number of death events ; * * * P0 . 0001 . ( f , g ) Time - lapse images of MCF10A cell - in - cell structures expressing either 2 × FYVE - mCherry ( red ) and GFP - LC3 ( green ; f ) or Lamp1 - GFP ( green ) and mCherry - LC3 ( red ; g ) . The arrows indicate recruitment to the entotic vacuole ( see Supplementary Movies S4 and S5 ) . Times are indicated as h : min : s ( a , f ) or min ( g ) . Scale bars , 5 μm . ( h ) Representative images of cell - in - cell structures in which only host cells express Cathepsin - B - mCherry . Left , a live inner cell with no Cathepsin B in the vacuole ( arrow ) ; middle , a live internalized cell with Cathepsin B from the host inside of the vacuole ( arrowhead ) ; right , a dead internalized cell with Cathepsin B from the host throughout the corpse ( asterisk ) . Scale bar , 15 μm . ( i ) Five representative timings of GFP - LC3 recruitment to entotic vacuoles ( green bar ) and death of internalized cells ( cross ) from time - lapse microscopy . ( j ) Initiation and duration of 2 × FYVE domain ( red bar , n = 11 ) , LC3 ( green bar , n = 20 ) and Lamp1 ( orange bar , n = 7 ) recruitment to entotic vacuoles , determined using double - expressing cells shown in f , g . Error bars represent s . d .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) Confocal time-lapse images of an MCF10A cell-in-cell structure expressing GFP-LC3 and H2B-mCherry. The arrow marks recruitment of LC3 to the entotic vacuole (see Supplementary Movie S1). Scale bar, 15 μm.(b) Images of MCF10A cells expressing GFP-LC3G120A and mCherry-LC3; note no recruitment of GFP-LC3G120A to the entotic vacuole. Scale bar, 15 μm. (c) LC3 is recruited from the host-cell cytosol onto the entotic vacuole membrane. Data points are the mean fluorescence intensity of GFP-LC3 on vacuoles versus cytoplasm for two independent cell-in-cell structures (see Supplementary Movie S2).(d) Correlative video-light-electron microscopy of an MCF10A GFP-LC3 cell-in-cell structure. Cells were imaged by time-lapse microscopy, followed by fixation after LC3 recruitment (see Supplementary Movie S3). Top left, post-fixation images of phase-contrast and GFP-fluorescence signals were taken demonstrating LC3 recruitment. Bottom left and right, electron microscopy (EM) of the same cell shows the entotic vacuole is a single membrane (arrow in right image, which is a higher magnification of the area outlined in the bottom left image)(e) Percentage of MCF10A cell-in-cell structures, with and without autophagy inhibition, that show LC3 recruitment associated with death of the internalized cell. Data represent mean±s.e.m. from at least three independent experiments; n, total number of death events; ***P0.0001.(f,g) Time-lapse images of MCF10A cell-in-cell structures expressing either 2×FYVE-mCherry (red) and GFP-LC3 (green; f) or Lamp1-GFP (green) and mCherry-LC3 (red; g). The arrows indicate recruitment to the entotic vacuole (see Supplementary Movies S4 and S5). Times are indicated as h:min:s (a,f) or min (g). Scale bars, 5 μm.(h) Representative images of cell-in-cell structures in which only host cells express Cathepsin-B-mCherry. Left, a live inner cell with no Cathepsin B in the vacuole (arrow); middle, a live internalized cell with Cathepsin B from the host inside of the vacuole (arrowhead); right, a dead internalized cell with Cathepsin B from the host throughout the corpse (asterisk). Scale bar, 15 μm.(i) Five representative timings of GFP-LC3 recruitment to entotic vacuoles (green bar) and death of internalized cells (cross) from time-lapse microscopy. (j) Initiation and duration of 2×FYVE domain (red bar, n=11), LC3 (green bar, n=20) and Lamp1 (orange bar, n=7) recruitment to entotic vacuoles, determined using double-expressing cells shown in f,g. Error bars represent s.d.


figf2 ( a ) Left , model depicting the multiple cell fates of internalized cells ; the arrow thickness represents the relative frequency of events . Right , images of MCF10A cell - in - cell structures ( 1 , host cell ; 2 , internalized cell ) that show a cell death ( top images ) or release ( bottom images ) event . Scale bar , 15 μm . ( b ) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition . Data represent the mean from at least three independent experiments ; n , total number of structures analysed ; * * * P0 . 007 , * * P0 . 004 , * P0 . 01 . ( c , d ) Quantification of LC3 recruitment ( c ) , and cell fate ( d ) , of mixed cell - in - cell structures for which host or internalized cells were treated with ATG5 siRNA . Data represent mean ± s . e . m . from three separate experiments ; n , total number of structures analysed ; * P0 . 01 , * * P0 . 004 . ( e ) Left , quantification of type of internalized cell death after autophagy inhibition . Data represent the mean of three independent experiments ; n , total death events analysed ; * * P0 . 03 . Right , representative images of non - apoptotic and apoptotic death of an internalized cell ; the arrow points to fragmented apoptotic nuclei . Scale bars , 15 μm . ( f ) Quantification of mCherry / GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP - mCherry - LC3 , with or without siRNA treatment as indicated . Data represent mean ± s . d . ; numbers of cells analysed for each data point ( from left to right ) 30 , 30 , 30 , 25 , 30 , 25 , 25 , 25 , 30 , 27 ; * P0 . 03 , * * P0 . 003 , * * * P0 . 0004 . ( g ) Quantification of autophagosome and autolysosome area in control MCF10A ( n = 10 ) and internalized cell ( n = 10 ) cytoplasm imaged by electron microscopy ; * * P0 . 005 . ( h ) Representative electron micrographs of cell - in - cell structures ( left ) and internalized cell cytoplasm ( middle and right ) showing autophagosomes ( arrow ) and autolysosomes ( arrowhead ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) Left, model depicting the multiple cell fates of internalized cells; the arrow thickness represents the relative frequency of events. Right, images of MCF10A cell-in-cell structures (1, host cell; 2, internalized cell) that show a cell death (top images) or release (bottom images) event. Scale bar, 15 μm.(b) Quantification of MCF10A internalized cell fate over 20 h following autophagy inhibition. Data represent the mean from at least three independent experiments; n, total number of structures analysed; ***P0.007, **P0.004, *P0.01.(c,d) Quantification of LC3 recruitment (c), and cell fate (d), of mixed cell-in-cell structures for which host or internalized cells were treated with ATG5 siRNA. Data represent mean±s.e.m.from three separate experiments; n, total number of structures analysed; *P0.01, **P0.004.(e) Left, quantification of type of internalized cell death after autophagy inhibition. Data represent the mean of three independent experiments; n, total death events analysed; **P0.03. Right, representative images of non-apoptotic and apoptotic death of an internalized cell; the arrow points to fragmented apoptotic nuclei. Scale bars, 15 μm.(f) Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP-mCherry-LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27; *P0.03, **P0.003, ***P0.0004.(g) Quantification of autophagosome and autolysosome area in control MCF10A (n=10) and internalized cell (n=10) cytoplasm imaged by electron microscopy; **P0.005. (h) Representative electron micrographs of cell-in-cell structures (left) and internalized cell cytoplasm (middle and right) showing autophagosomes (arrow) and autolysosomes (arrowhead).


figf3 ( a ) The fate of internalized cells ( control MCF10A and MCF10A expressing E7 and Bcl2 ) was measured with and without autophagy inhibition . Data show the percentage of internalized cell release with autophagy inhibition , compared with each control without autophagy inhibition ; mean ± s . e . m . from at least three independent experiments ( total cell numbers analysed : ATG5 siRNA , 261 cells ; Bcl2 ATG5 siRNA , 97 cells ; 3 - MA , 170 cells ; Bcl2 3 - MA , 112 cells ) ; * P0 . 05 , * * P0 . 02 . ( b ) Effects of Y27632 , 3 - MA and VPS34 and ATG5 siRNA on MCF10A cell - in - cell formation in suspension for 7 h ; > 300 cells per condition were scored for cell - in - cell formation ; ± s . e . m . from three independent experiments ; * * P0 . 004 . ( c ) Representative images of an MCF10A E7 + Bcl2 cell - in - cell structure formed in soft agar . The arrow marks LC3 recruitment around internalized cells . Scale bar , 5 μm . ( d ) Quantification of MCF10A E7 + Bcl2 cell - in - cell structures 48 h after seeding into soft agar ( white bars ) , and colony formation after 2 weeks ( grey bars ) . Data represent mean ± s . e . m . from three independent experiments ; * P0 . 03 , * * P0 . 004 , * * * P0 . 001 . ( e ) Representative wells for data in d . The insets show higher magnifications . ( f ) Quantification of MCF10A E7 + Bcl2 colony formation with Atg5 knockdown with two separate siRNAs . Data represent mean ± s . e . m . from three independent experiments ; * P0 . 001 . ( g ) Representative wells for data in f . The insets show higher magnifications . See also Supplementary Movie S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) The fate of internalized cells (control MCF10A and MCF10A expressing E7 and Bcl2) was measured with and without autophagy inhibition. Data show the percentage of internalized cell release with autophagy inhibition, compared with each control without autophagy inhibition; mean±s.e.m. from at least three independent experiments (total cell numbers analysed: ATG5 siRNA, 261 cells; Bcl2 ATG5 siRNA, 97 cells; 3-MA, 170 cells; Bcl2 3-MA, 112 cells); *P0.05, **P0.02.(b) Effects of Y27632, 3-MA and VPS34 and ATG5 siRNA on MCF10A cell-in-cell formation in suspension for 7 h; >300 cells per condition were scored for cell-in-cell formation; ±s.e.m.from three independent experiments; **P0.004.(c) Representative images of an MCF10A E7+Bcl2 cell-in-cell structure formed in soft agar. The arrow marks LC3 recruitment around internalized cells. Scale bar, 5 μm.(d) Quantification of MCF10A E7+Bcl2 cell-in-cell structures 48 h after seeding into soft agar (white bars), and colony formation after 2 weeks (grey bars). Data represent mean±s.e.m.from three independent experiments; *P0.03, **P0.004, ***P0.001. (e) Representative wells for data in d. The insets show higher magnifications.(f) Quantification of MCF10A E7+Bcl2 colony formation with Atg5 knockdown with two separate siRNAs. Data represent mean±s.e.m.from three independent experiments; *P0.001. (g) Representative wells for data in f. The insets show higher magnifications. See also Supplementary Movie S6.


figf4 ( a ) Time - lapse images of MCF10A cell - in - cell structures with an internalized cell that undergoes apoptosis . The arrows mark the recruitment of LC3 around apoptotic fragments ( see Supplementary Movie S7 ) . Scale bar , 10 μm . ( b ) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA ; n , number of cells analysed ; * * * P0 . 0001 , chi - squared test . ( c ) Apoptotic U937 cells expressing H2B - mCherry phagocytized by J774 macrophages expressing GFP - LC3 . Confocal images show LC3 recruitment to an engulfed corpse ( arrow ; see Supplementary Movie S8 ) . Scale bar , 15 μm . ( d ) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5 - shRNA - expressing cells ; * * * P0 . 0001 , chi - squared test . ( e ) Correlative light - electron microscopy of an apoptotic phagosome with GFP - LC3 recruitment ( top left image , white arrow ) . The LC3 - loaded phagosome consists of a single membrane ( black arrow in right image , which is a higher magnification of the area outlined in the bottom left image ; EM , electron microscopy ; AP , apoptotic cell nucleus ) . ( f ) Representative images of apoptotic cell phagocytosis and degradation in control ( n = 21 ) and Atg5 shRNA ( n = 21 ) J774 GFP - LC3 cells ; * * * P0 . 0001 , chi - squared test . Scale bar , 10 μm . See also Supplementary Fig . S3 .

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figf4(a) Time-lapse images of MCF10A cell-in-cell structures with an internalized cell that undergoes apoptosis. The arrows mark the recruitment of LC3 around apoptotic fragments (see Supplementary Movie S7). Scale bar, 10 μm. (b) Quantification of LC3 recruitment to apoptotic internalized cells in the presence of ATG5 or FIP200 siRNA; n, number of cells analysed; ***P0.0001, chi-squared test.(c) Apoptotic U937 cells expressing H2B-mCherry phagocytized by J774 macrophages expressing GFP-LC3. Confocal images show LC3 recruitment to an engulfed corpse (arrow; see Supplementary Movie S8). Scale bar, 15 μm. (d) Quantification of LC3 recruitment to apoptotic phagosomes in control and Atg5-shRNA-expressing cells; ***P0.0001, chi-squared test.(e) Correlative light-electron microscopy of an apoptotic phagosome with GFP-LC3 recruitment (top left image, white arrow). The LC3-loaded phagosome consists of a single membrane (black arrow in right image, which is a higher magnification of the area outlined in the bottom left image; EM, electron microscopy; AP, apoptotic cell nucleus).(f) Representative images of apoptotic cell phagocytosis and degradation in control (n=21) and Atg5 shRNA (n=21) J774 GFP-LC3 cells; ***P0.0001, chi-squared test. Scale bar, 10 μm. See also Supplementary Fig. S3.


figf5 ( a ) Representative images of a J774 macrophage expressing GFP - LC3 incubated in media containing red dextran . The arrow marks LC3 recruitment to a dextran - containing macropinosome . Scale bar , 2 μm . ( b ) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared test . See Supplementary Movie S9 . ( c ) Representative image of LC3 recruitment to macropinosomes ( arrows ) in MCF10A cells expressing GFP - LC3 incubated with red dextran . Scale bar , 2 μm . ( d ) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5 ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared test . ( e ) J774 GFP - LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h , followed by the addition of LysoTracker red to mark acidified compartments . A macropinosome recruited LC3 ( arrow ) whereas engulfed latex beads did not ( arrowheads ) . Scale bar , 5 μm . See Supplementary Movie S10 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Representative images of a J774 macrophage expressing GFP-LC3 incubated in media containing red dextran. The arrow marks LC3 recruitment to a dextran-containing macropinosome. Scale bar, 2 μm. (b) Quantification of LC3 recruitment to macropinosomes in control and Atg5 shRNA J774 macrophages; n, number of macropinosomes analysed; ***P0.0001, chi-squared test. See Supplementary Movie S9.(c) Representative image of LC3 recruitment to macropinosomes (arrows) in MCF10A cells expressing GFP-LC3 incubated with red dextran. Scale bar, 2 μm. (d) Quantification of LC3 recruitment to macropinosomes in MCF10A cells treated with siRNA against FIP200 or ATG5; n, number of macropinosomes analysed; ***P0.0001, chi-squared test.(e) J774 GFP-LC3 macrophages were incubated with 3 μm uncoated latex beads and imaged by confocal microscopy for 1 h, followed by the addition of LysoTracker red to mark acidified compartments. A macropinosome recruited LC3 (arrow) whereas engulfed latex beads did not (arrowheads). Scale bar, 5 μm. See Supplementary Movie S10.


figf6 ( a ) A C . elegans embryo expressing mCherry : : RAB - 5 and GFP : : LGG - 1 . The arrows point to two apoptotic phagosomes . See Supplementary Movie S11 . Scale bar , 10 μm . ( b ) Cropped time - lapse images showing RAB - 5 and LGG - 1 recruitment to an apoptotic phagosome ( from a , left arrow ) . ( c ) Representative images of apoptotic phagosomes in embryos from control - RNAi - or bec - 1 - RNAi - fed worms . The insets show higher magnifications of the areas outlined in the main panels . ( d ) Quantification of GFP : : LGG - 1 - and mCherry : : RAB - 5 - positive phagosomes in control - or bec - 1 - RNAi embryos determined by time - lapse imaging . Control , 21 embryos , 52 phagosomes ; bec - 1 , 9 embryos , 25 phagosomes ; * * * P0 . 0001 , chi - squared test . ( e ) Representative DIC images from control - and bec - 1 - RNAi embryos at different stages of development ( minutes after first division ) . The yellow arrowheads mark apoptotic corpses . Scale bar , 10 μm . ( f ) Quantification of apoptotic corpses in control - and bec - 1 - RNAi embryos ; data show ± s . d . ; * * * P0 . 0001 . ( g ) Representative images of CED - 1 : : GFP embryos with control or bec - 1 RNAi . The yellow arrowheads mark engulfed corpses surrounded by CED - 1 : : GFP . Scale bar , 5 μm .

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figf6(a) A C. elegans embryo expressing mCherry::RAB-5 and GFP::LGG-1. The arrows point to two apoptotic phagosomes. See Supplementary Movie S11. Scale bar, 10 μm. (b) Cropped time-lapse images showing RAB-5 and LGG-1 recruitment to an apoptotic phagosome (from a, left arrow).(c) Representative images of apoptotic phagosomes in embryos from control-RNAi- or bec-1-RNAi-fed worms. The insets show higher magnifications of the areas outlined in the main panels. (d) Quantification of GFP::LGG-1- and mCherry::RAB-5-positive phagosomes in control- or bec-1-RNAi embryos determined by time-lapse imaging. Control, 21 embryos, 52 phagosomes; bec-1, 9 embryos, 25 phagosomes; ***P0.0001, chi-squared test.(e) Representative DIC images from control- and bec-1-RNAi embryos at different stages of development (minutes after first division). The yellow arrowheads mark apoptotic corpses. Scale bar, 10 μm. (f) Quantification of apoptotic corpses in control- and bec-1-RNAi embryos; data show ± s.d.; ***P0.0001.(g) Representative images of CED-1::GFP embryos with control or bec-1 RNAi. The yellow arrowheads mark engulfed corpses surrounded by CED-1::GFP. Scale bar, 5 μm.


Figure 1Mitochondria - enriched fractions were isolated from HdhQ7 or HdhQ111 cells . mtHSF1 was examined by WB analysis . VDAC was analyzed as a loading control . ( n = 6 biological replicates ) . HdhQ7 WT striatal cells were exposed to 5 mM 3 - NP for 2 h . mtHSF1 levels were determined by immunoblotting . VDAC was used as a loading control . ( n = 5 biological replicates ) . HSF1 was measured in the mitochondria - enriched fractions of the HD transgenic YAC128 mice ( 9 months old ) or age - matched littermates . ( n = 5 biological replicates ) . mtHSF1 was analyzed in HD patient fibroblasts ( GM04208 , GM04222 , and GM21756 ) and fibroblasts from healthy individuals . ( n = 5 for Con1 / GM04208 and Con2 / GM04222 groups ; n = 3 for Con3 / GM21756 group ) . Representative immunoblot of HSF1 levels in mitochondria isolated from patient - derived iPSCs and control iPSCs . ( n = 3 biological replicates ) . Representative images of striatal organoids . Left , immunostaining for the MSN marker DARPP32 ( green ) and the radial glial marker SOX2 ( red ) in striatal organoids at D60 . Middle , immunostaining for the LGE progenitor markers Ctip2 ( green ) and GSH2 ( red ) at D30 . Right panel , immunostaining for the neuronal marker NeuN ( green ) and the proliferation marker Ki67 ( red ) at D30 . The images are representative of 12 independent differentiation experiments of H9 and IMR90 - 4 cells . HO = Hoechst ( blue ) . The scale bar represents 100 µm . Representative images and scatterplot confirming the increased HSF1 ( green ) translocation to mitochondria ( Tom20 , red ) . The data were obtained from 3 independent biological experiments . Con : n = 15 organoids ; HD : n = 14 organoids . Two - tailed unpaired t - test ; Means ± SEMs . Images were taken using Structured Illumination Microscopy ( SIM ) . The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy . The arrows ( red ) mark HSF1 in mitochondria . The negative control lacked the HSF1 antibody . The scale bar represents 200 nm . HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection . The protein levels of HSF1 were detected in mitochondrial fractions . ( n = 3 biological replicates ) .

O B-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Mitochondria-enriched fractions were isolated from HdhQ7 or HdhQ111 cells. mtHSF1 was examined by WB analysis. VDAC was analyzed as a loading control. (n=6 biological replicates).HdhQ7 WT striatal cells were exposed to 5 mM 3-NP for 2 h. mtHSF1 levels were determined by immunoblotting. VDAC was used as a loading control. (n=5 biological replicates).HSF1 was measured in the mitochondria-enriched fractions of the HD transgenic YAC128 mice (9 months old) or age-matched littermates. (n=5 biological replicates).mtHSF1 was analyzed in HD patient fibroblasts (GM04208, GM04222, and GM21756) and fibroblasts from healthy individuals. (n = 5 for Con1/ GM04208 and Con2/ GM04222 groups; n = 3 for Con3/ GM21756 group).Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).Representative images of striatal organoids. Left, immunostaining for the MSN marker DARPP32 (green) and the radial glial marker SOX2 (red) in striatal organoids at D60. Middle, immunostaining for the LGE progenitor markers Ctip2 (green) and GSH2 (red) at D30. Right panel, immunostaining for the neuronal marker NeuN (green) and the proliferation marker Ki67 (red) at D30. The images are representative of 12 independent differentiation experiments of H9 and IMR90-4 cells. HO= Hoechst (blue). The scale bar represents 100 µm.Representative images and scatterplot confirming the increased HSF1 (green) translocation to mitochondria (Tom20, red). The data were obtained from 3 independent biological experiments. Con: n=15 organoids; HD: n=14 organoids. Two-tailed unpaired t-test; Means ± SEMs. Images were taken using Structured Illumination Microscopy (SIM).The subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy. The arrows (red) mark HSF1 in mitochondria. The negative control lacked the HSF1 antibody. The scale bar represents 200 nm.HTT was knocked down in HdhQ7 or HdhQ111 cells by lentiviral infection. The protein levels of HSF1 were detected in mitochondrial fractions. (n=3 biological replicates).


Figure 2HdhQ7 cells were transfected with Flag - mtHSF1 and stained with anti - Tom20 ( green ) and anti - Flag ( red ) antibodies . Mitochondrial morphology was examined by confocal microscopy . Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation . The scale bar represents 40 µm . ( n = 3 biological replicates , at least 100 cells per group were counted ) . The total protein levels of p - Drp1 S616 , p - Drp1S637 , Drp1 , OPA1 , MFN2 , Flag - mtHSF1 and actin ( loading control ) were determined by WB analysis . ( n = 3 biological replicates ) . Brain sections were stained with an anti - Tom20 antibody ( red ) , an anti - Flag antibody ( green ) and DAPI ( blue ) . Mitochondrial morphology was analyzed in Flag + or Flag - regions by confocal microscopy . The scale bar presents 2 mm , and the enlarged image scale bar is 40 µm . ( n = 6 mice / group ) . Striata injected with AAVs were lysed . p - Drp1 S616 was tested by immunoblotting . ( n = 5 mice / group ) . Mitochondrial morphology was analyzed by electron microscopy ( EM ) in mice injected with AAV - Con or AAV - mtHSF1 . Scatterplot showed the length of mitochondria . The scale bar presents 4 µm , and the enlarged image scale bar is 400 nm . ( n = 3 mice / group ) . AAV - Con group , n = 268 mitochondria were measured ; AAV - Flag - mtHSF1 group , n = 179 mitochondria were measured . Representative images and scatterplot of mitochondrial length ( Tom20 , red ) in Flag + or Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 154 mitochondria ; Flag + : n = 132 mitochondria . Representative images and scatterplot showing that the number of p - Drp1 S616 puncta in Flag + cells was greater than that in Flag - cells . The data were obtained from 3 independent biological experiments . Flag - : n = 70 cells from 10 organoids ; Flag + : n = 55 cells from 10 organoids . The images were obtained using structured illumination microscopy ( SIM ) .

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Figure 2HdhQ7 cells were transfected with Flag-mtHSF1 and stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was examined by confocal microscopy. Scatterplot with bar showed the percentage of cells with mitochondrial fragmentation. The scale bar represents 40 µm. (n=3 biological replicates, at least 100 cells per group were counted).The total protein levels of p-Drp1 S616, p-Drp1S637, Drp1, OPA1, MFN2, Flag-mtHSF1 and actin (loading control) were determined by WB analysis. (n=3 biological replicates).Brain sections were stained with an anti-Tom20 antibody (red), an anti-Flag antibody (green) and DAPI (blue). Mitochondrial morphology was analyzed in Flag+ or Flag- regions by confocal microscopy. The scale bar presents 2 mm, and the enlarged image scale bar is 40 µm. (n=6 mice/group).Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).Mitochondrial morphology was analyzed by electron microscopy (EM) in mice injected with AAV-Con or AAV-mtHSF1. Scatterplot showed the length of mitochondria. The scale bar presents 4 µm, and the enlarged image scale bar is 400 nm. (n=3 mice/group). AAV-Con group, n=268 mitochondria were measured; AAV-Flag-mtHSF1 group, n=179 mitochondria were measured.Representative images and scatterplot of mitochondrial length (Tom20, red) in Flag+ or Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=154 mitochondria; Flag+: n=132 mitochondria.Representative images and scatterplot showing that the number of p-Drp1 S616 puncta in Flag+ cells was greater than that in Flag- cells. The data were obtained from 3 independent biological experiments. Flag-: n=70 cells from 10 organoids; Flag+: n=55 cells from 10 organoids. The images were obtained using structured illumination microscopy (SIM).


Figure 3The mtDNA content was examined with primers of D - loop , mtCO1 , mtCO2 and mtND2 by qPCR in control or Flag - mtHSF1 - expressing striatal cells . Scatterplot with bar showed the relative percentage of mtDNA content . ( n = 3 biological replicates ) . Measurement of mitochondrial respiratory activity by Seahorse analysis . ( n = 9 - 10 biological replicates ) Cells were treated with EtBr for six days to delete mtDNA . mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr . ( n = 3 biological replicates ) . D - E . Cell death was determined by detection of LDH release into the medium and cleavage of PARP . Scatterplot with bar showed the relative percentage of LDH release . ( n = 3 biological replicates ) . qPCR analysis of the mtDNA content of AAV - Con - injected mice or AAV - mtHSF1 - injected mice . Scatterplot with bar showed the relative percentage of mtDNA content ( n = 3 mice / group ) . Cleaved - Caspase3 in AAV - Con - or AAV - mtHSF1 - injected mice was detected by WB analysis . ( n = 5 mice / group ) . Representative images and scatterplot showing the number of mtDNA ( purple ) colocalized with mitochondria ( Tom20 , green ) in striatal organoids injected with AAV - Con or AAV - mtHSF1 . The data were obtained from 3 independent biological experiments . Con : n = 65 cells from 20 organoids ; Flag - mtHSF1 : n = 70 cells from 20 organoids . Two - tailed unpaired t - test was used . Images were captured by Structured Illumination Microscopy ( SIM ) . The typical nuclear fragmentation morphology of AAV - Con - ( GFP ) or AAV - mtHSF1 - injected striatal organoids was measured . Scatterplot showed the quantitative result of nuclear fragmentation . The arrows ( yellow ) mark fragmented nuclei . Two - tailed unpaired t - test was used . The data were obtained from 3 independent biological experiments . Con : n = 14 organoids ; Flag - mtHSF1 : n = 15 organoids .

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Figure 3The mtDNA content was examined with primers of D-loop, mtCO1, mtCO2 and mtND2 by qPCR in control or Flag-mtHSF1-expressing striatal cells. Scatterplot with bar showed the relative percentage of mtDNA content. (n=3 biological replicates).Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)Cells were treated with EtBr for six days to delete mtDNA. mtDNA repopulation efficiency was measured by qPCR after withdrawal of EtBr. (n=3 biological replicates).D-E. Cell death was determined by detection of LDH release into the medium and cleavage of PARP. Scatterplot with bar showed the relative percentage of LDH release. (n=3 biological replicates).qPCR analysis of the mtDNA content of AAV-Con-injected mice or AAV-mtHSF1-injected mice. Scatterplot with bar showed the relative percentage of mtDNA content (n=3 mice/group).Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).Representative images and scatterplot showing the number of mtDNA (purple) colocalized with mitochondria (Tom20, green) in striatal organoids injected with AAV-Con or AAV-mtHSF1. The data were obtained from 3 independent biological experiments. Con: n=65 cells from 20 organoids; Flag-mtHSF1: n=70 cells from 20 organoids. Two-tailed unpaired t-test was used. Images were captured by Structured Illumination Microscopy (SIM).The typical nuclear fragmentation morphology of AAV-Con- (GFP) or AAV-mtHSF1-injected striatal organoids was measured. Scatterplot showed the quantitative result of nuclear fragmentation. The arrows (yellow) mark fragmented nuclei. Two-tailed unpaired t-test was used. The data were obtained from 3 independent biological experiments. Con: n=14 organoids; Flag-mtHSF1: n=15 organoids.