( A - C ) of NRF2 ubiquitination in absence presence of TRIM16 by Co - IP assays using transiently transfected plasmid constructs as indicated . Two different variants of ubiquitin are used , one that can only be ubiquitinated at Lysine 48 residue ( HA - K48 - UB ) other that can be only ubiquitinated at Lysine 63 residue ( HA - K63 - UB ) . other lysine residues are mutated . L . E , Low exposure ; H . E , High Exposure .

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(A-C) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Co-IP assays using transiently transfected plasmid constructs as indicated. Two different variants of ubiquitin protein are used, one that can only be ubiquitinated at Lysine 48 residue (HA-K48-UB) and other that can be only ubiquitinated at Lysine 63 residue (HA-K63-UB). All other lysine residues are mutated. L.E, Low exposure; H.E, High Exposure.


of NRF2 ubiquitination in absence presence of TRIM16 by Ni - NTA pull - down assays using transiently transfected plasmid constructs as indicated . His - tagged ubiquitin which is mutated at lysines except 63 position is used in these assays .

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Analysis of NRF2 ubiquitination in absence and presence of TRIM16 by Ni-NTA pull-down assays using transiently transfected plasmid constructs as indicated. His-tagged ubiquitin which is mutated at all lysines except 63 position is used in these assays.


( F ) of NRF2 ubiquitination in absence presence of TRIM16 deletion variants by Co - IP assays using transiently transfected plasmid constructs as indicated .

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(F) Analysis of NRF2 ubiquitination in absence and presence of TRIM16 deletion variants by Co-IP assays using transiently transfected plasmid constructs as indicated.


( G ) Western blot of lysates from HeLa , TRIM16KO TRIM16KO complemented with TRIM16 deletion constructs where were treated with MG132 ( 20 µM , 2h ) blot is probed with indicated antibodies .

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(G) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( H ) Western blot of lysates from HeLa , TRIM16KO TRIM16KO complemented with TRIM16 deletion constructs where were treated with MG132 ( 20 µM , 2h ) blot is probed with indicated antibodies .

(H) Western blot analysis of lysates from HeLa, TRIM16KO cells and TRIM16KO cells complemented with TRIM16 deletion constructs where cells were treated with MG132 (20 µM, 2h) and the blot is probed with indicated antibodies.


( A ) Western blot of HeLa TRIM16KO lysates of treated with increasing concentrations of H2O2 as specified probed with antibodies as indicated .

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(A) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( B ) WB of HeLa TRIM16KO lysates of treated with 400 µM of H2O2 for different durations as indicated probed with antibodies as shown . red arrows indicate time point where were retreated with 400 µM of H2O2 . L . E , Low exposure ; H . E , High Exposure .

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(B) WB analysis of HeLa and TRIM16KO lysates of cells treated with 400 µM of H2O2 for different durations as indicated and probed with antibodies as shown. The red arrows indicate the time point where the cells were retreated with 400 µM of H2O2. L.E, Low exposure; H.E, High Exposure.


( C ) Densitometric of NRF2 band normalized with actin .

(C) Densitometric analysis of NRF2 band intensity normalized with actin.


( D ) WB of HeLa TRIM16KO lysates of treated with 2 . 5 µM ( 2 h ) of As2O3 .

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(D) WB analysis of HeLa and TRIM16KO lysates of cells treated with 2.5 µM (2 h) of As2O3.


( E - G ) isolated from HeLa TRIM16KO , untreated or treated with MG132 ( 20 µM , 2h ) were subjected to qRT - PCR with primers of genes as indicated . fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 , * * p < 0 . 005 ( Student ' s unpaired t - ) .

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(E-G) RNA isolated from HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h) 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, **p < 0.005 (Student's unpaired t-test).


isolated from control NRF2 knockdown HeLa transfected with control vector or Myc - TRIM16 subjected to qRT - PCR with primers of genes as indicated . Mean ± SD , n = 3 , * * p < 0 . 005 , * * * p < 0 . 0005 ( Student ' s unpaired t - ) .

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RNA isolated from control and NRF2 knockdown HeLa cells transfected with control vector or Myc-TRIM16 subjected to qRT-PCR with primers of genes as indicated. Mean ±SD, n=3, **p < 0.005, ***p < 0.0005 (Student's unpaired t-test).


( K ) Western blot of HeLa TRIM16KO lysates of treated with h increasing concentrations of H2O2 as specified probed with antibodies as indicated .

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(K) Western blot analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and probed with antibodies as indicated.


( L ) Densitometric of Ub smear normalized with actin . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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(L) Densitometric analysis of Ub smear intensity normalized with actin. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).


( M ) Representative confocal of control , Ubb Ube2n siRNA transfected treated with H2O2 ( 200 µM , 2h ) IF was performed with Ub p62 antibodies . Scale bar : 10 µm .

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(M) Representative confocal images of control, Ubb and Ube2n siRNA transfected cells treated with H2O2 (200 µM, 2h) and IF was performed with Ub and p62 antibodies. Scale bar: 10 µm.


( N ) graph shows of with . Data from ≥ 10 microscopic fields for each condition ( 40X ) , n = 2 , Mean ± SD .

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(N) The graph shows the percentage of cells with protein aggregates. Data from ≥ 10 microscopic fields for each condition (40X), n=2, Mean ±SD.


( O ) Western blot of Ubb Ube2n siRNA transfected lysates with antibodies as indicated .

(O) Western blot analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated.


( A ) Control or NRF2 siRNA transfected HeLa lysates were subjected to western blotting with indicated antibodies .

(A) Control or NRF2 siRNA transfected HeLa cells lysates were subjected to western blotting with indicated antibodies.


( B , C ) isolated from untreated or MG132 - treated ( 20 µM , 2h ) control or NRF2 siRNA transfected were subjected to qRT - PCR with primers of genes as indicated . fold induction in MG132 treated samples is calculated relative to untreated samples . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(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 of control siRNA or NRF2 siRNA transfected lysates of HeLa , untreated or treated with MG132 ( 20 µM , 2h ) as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure .

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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 of control siRNA or NRF2 siRNA transfected lysates of HeLa , untreated or treated with H2O2 ( 200 µM , 2h ) as indicated 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 of control siRNA NRF2 siRNA transfected treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub p62 antibodies . Right panels : tracing corresponding to a in zoom panel .

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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 of control siRNA NRF2 siRNA transfected treated with ( G ) H2O2 ( 200 µM , 2h ) , where IF was conducted with Ub p62 antibodies . Right panels : tracing corresponding to a 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 , TRIM16KO , TRIM16KO complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) subjected to WB with Myc , GFP indicated antibodies .

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(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 of Hela , TRIM16KO , TRIM16KO complemented with Myc - NRF2 which were treated with MG132 ( 20 µM , 2h ) subjected to IF with Ub p62 antibodies .

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(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 with Ub - p62 dots , data from ≥ 10 microscopic fields ( 40X ) , n = 3 , mean ± SD , * p < 0 . 005 ( one - way ANOVA ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(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.


WB of detergent - soluble - insoluble fractions of HeLa TRIM16KO expressing either ( A ) Flag - p62 probed with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric of WB ' s normalized with α - Tubulin or Lamin - B1 .

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WB 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 of detergent - soluble - insoluble fractions of HeLa TRIM16KO expressing either ( C ) GFP - polyQ74 probed with or GFP or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . Densitometric of WB ' s normalized with α - Tubulin or Lamin - B1 .

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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 of GFP - TRIM16 expressing HeLa , untreated or treated with MG132 ( 10 µM , 4h ) / or Bafilomycin A1 ( 300 nM , 3h ) IF is performed with antibodies as indicated . Right panel graphs : tracing corresponding to a in zoom panel . Scale bar : 10 µm . ( F ) graph shows of with TRIM16 - Ub or TRIM16 - Ub - p62 co - localized dots / . Data from ≥ 10 microscopic fields , n = 2 , mean ± SD .

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(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 of GFP - TRIM16 expressing HeLa , untreated or treated with MG132 ( 10 µM , 4h ) / or Bafilomycin A1 ( 300 nM , 3h ) IF is performed with antibodies as indicated . Right panel graphs : tracing corresponding to a in zoom panel . Scale bar : 10 µm .

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(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 of interaction between two differently tagged TRIM16 variants .

(H) Co-IP analysis of the interaction between two differently tagged TRIM16 variants.


( I ) WB of detergent - soluble - insoluble DSS - cross - linked fractions of HeLa TRIM16KO , untreated or treated with MG132 ( 10 µM , 4h ) probed with indicated antibodies .

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(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 of interaction between Flag - tagged TRIM16 Myc - tagged TRIM16 full its domain deletion .

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(J) Co-IP analysis of the interaction between Flag-tagged TRIM16 and Myc-tagged TRIM16 full length and its domain deletion proteins.


( A ) MTT assays performed at different time points with HeLa TRIM16KO , untreated or treated with H2O2 ( 400 µM , 2h ) . Data , mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - ) .

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(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 ) of HeLa TRIM16KO , untreated or treated with As2O3 ( 2 . 5 µM , 4h ) . Scale bar : 400 µm .

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(B) Images of HeLa and TRIM16KO cells, untreated or treated with As2O3 (2.5 µM, 4h). Scale bar: 400 µm.


Flow cytometry of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( C ) As2O3 ( 2 . 5 µM , 4h )

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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 of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( D ) H2O2 ( 400 µM , 2h )

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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 of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , 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 (E) puromycin (5 µg/ml, 6h)


Flow cytometry of HeLa TRIM16KO stained with Annexin - V / Propidium Iodide ( double ) , untreated or treated with ( F ) MG132 ( 20 µM , 8h ) .

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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 of HeLa TRIM16KO lysates of treated with 5 µM of As2O3 for different durations as indicated probed with antibodies as shown . L . E , Low exposure ; H . E , High Exposure . Arrowheads indicate form of PARP - 1 Casapse - 3 .

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(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.


( A , B ) Clonogenic assays were performed with HeLa TRIM16KO , untreated or treated with As2O3 or MG132 .

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(A, B) Clonogenic assays were performed with HeLa and TRIM16KO cells, untreated or treated with As2O3 or MG132.


( C ) volumes of HeLa TRIM16KO tumors at indicated time points . As2O3 is injected when volume of groups was > 100mm3 ( day 0 in graph ) . 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 % regression ( volume on day 1 of As2O3 treatment / volume on day of sacrifice X 100 ) . Mean ± SE , n = 6 ( each group ) , * * p < 0 . 005 ( Student ' s unpaired t - ) .

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(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 of HeLa TRIM16KO tumors formed in nude mice in absence presence of As2O3 . ( F ) Representative pictures of dissected tumors .

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(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 performed with KI - 67 proliferation marker hematoxylin stain . graph represents % of with KI - 67 . > 400 were counted for this from three different sections from different , mean ± SD , * p < 0 . 05 ( Student ' s unpaired t - ) . Scale bar : 50 µm .

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(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 of tissue lysates from two different mice with antibodies as indicated . L . E , Low exposure ; H . E , High Exposure .

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(H) WB analysis of tumor tissue lysates from two different mice with antibodies as indicated. L.E, Low exposure; H.E, High Exposure.


( A ) Western blotting for phospho - Thr172 - AMPK ( pAMPK ) , total AMPK , phospho - Ser79 - ACC ( pACC ) , total ACC 24h after treatment . represents pAMPK / AMPK pACC / ACC ratios relative to non - treated ( NT ) control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . 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

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(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 of 72h after treatment . Scale bars represent 0 . 25mm

(B) Phase contrast images of fibers 72h after treatment. Scale bars represent 0.25mm


( C ) Immunofluorescence for myoglobin myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . represents average myotube width Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . 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

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(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 of mRNA of MyoD ( D 24h after treatment relative to NT control . Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . 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

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(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 of mRNA o myogenin ( E ) 24h after treatment relative to NT control . Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent standard error of mean ( ) . 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

(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


( A - B ) Rates of glucose consumption ( A measured in media 24h after treatment relative to non - treated ( NT ) control from three independent experiments ( n = 3 ) . Error bars represent 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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


( A ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) total S6 . represents pS6K / S6K ( n = 3 ) pS6 / S6 ( n = 4 ) ratios relative to non - treated ( NT ) control Data information : Error bars represent . 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

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

(A) Western 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 of de novo synthesis using 35S - labelling . represents whole lane radiation signal density standardized to Coomassie relative to NT control ( n = 3 ) . Data information : Error bars represent . 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

(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


( A ) Immunofluorescence for myoglobin myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . represents average myotube width Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

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

(A) 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 media 24h after treatment relative to DMSO treated control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 media 24h after treatment relative to DMSO treated control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) total S6 . represents pS6K / S6K pS6 / S6 ratios relative to DMSO treated control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

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

(D) 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 ) total ACC . represents of iNOS normalized to tubulin relative to IFNγ / TNFα control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent For panels E through G , p - values were calculated using Student " s t - . * , 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

(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 of iNOS mRNA relative to IFNγ / TNFα control 24h after treatment Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent For panels E through G , p - values were calculated using Student " s t - . * , 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

(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 ) in culture media of relative to IFNγ / TNFα control 24h after treatment Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent For panels E through G , p - values were calculated using Student " s t - . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(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


( A ) Immunofluorescence for myoglobin myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . represents average myotube width Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . 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

(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 tubulin 24h after treatment . represents of iNOS normalized to tubulin relative to IFNγ / TNFα control Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . 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

(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 ) total S6 . represents pS6K / S6K pS6 / S6 ratios relative to NT control . Data information : quantifications are of three independent experiments ( n = 3 ) error bars represent . 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

(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


( A ) Representative of overall hind limb musculature . Scale bar represents 1cm

(A) Representative images of overall hind limb musculature. Scale bar represents 1cm


( B ) Western blotting for phospho - Ser79 - ACC ( pACC ) total ACC

(B) Western blotting for phospho-Ser79-ACC (pACC) and total ACC


( C ) Cross - sectional of gastrocnemius muscle calculated from 250 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 . 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

(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 of Atrogin - 1 / MAFbx ( D mRNA expression from tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent . 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

(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 o MuRF1 ( E ) mRNA expression from tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent . 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

(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


( A ) Cross - sectional of gastrocnemius muscle calculated from 250 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 . 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

(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 of Atrogin - 1 / MAFbx ( B mRNA expression from tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent . 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

(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 o MuRF1 ( C ) mRNA expression from tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent . 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

(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


Representative grey - scale of rat hippocampal neurons ( DIV 10 ) transfected with GFP indicated miRNA mimics . of mean number of intersections by Sholl . GFP - only transfected conditions were set to one in each experiment .

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

Representative 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 of GFP - transfected Cacna1c + / + ( WT ) or Cacna1c + / - rat hippocampal neurons ( DIV 10 ) . Scale bars = 50 μm . of mean number of intersections by Sholl . Data are represented as scattered dot plots with bar , mean ± S . D . ( n = 3 independent experiments ; Unpaired two - sample t - , * * p = 0 . 0096 ) . Fold changes represent changes in dendritic complexity relative to control condition .

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

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.


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

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

Representative 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.


A ) Representative of DIV 19 rat hippocampal neurons co - transfected with GFP ( green channel ) 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 surface Cav1 . 2 channels . Whole : scale bars = 20 μm ; dendrite insets : scale bars = 5 μm . B ) of occupied by surface Cav1 . 2 normalized to total ( GFP ) 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 - * p = 0 . 0318 ) .

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

A) Representative 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).


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

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

D) 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).


miR - 499 - 5p qPCR of total isolated from PBMCs of control ( = 26 , = 31 ) , BD ( = 26 , = 37 ) , or MDD ( = 18 , = 24 ) subjects . Wilcoxon rank sum after correction for antidepressant treatment ( linear model of form Fold Change ~ Group * Sex + + Antidepressant treatment ) . 2 - way ANOVA with correction for sex , antidepressant treatment ( linear model of form Fold Change ~ Group * Sex + + 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 data points .

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

miR-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 of total isolated from PBMCs of control ( = 8 , = 10 ) SZ ( = 8 , = 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 data points .

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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 of total isolated from PBMCs of control ( = 26 , = 31 ) , BD I ( = 9 , = 9 ) , or BDI II ( = 12 , = 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 data points .

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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 of total isolated from PBMCs of control subjects ( = 26 , = 31 ) , BD subjects in different mood states ( depressive ( = 7 , = 11 ) , euthymic ( = 7 , = 4 ) , hypomanic ( = 2 , = 6 ) , manic ( = 2 , = 1 ) , mixed ( = 1 , = 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 data points .

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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 of total isolated from PBMCs of healthy control subjects ( - CMT , n = 17 ) healthy subjects with a history of childhood maltreatment ( + CMT , n = 17 ) . ( * * * * p < 0 . 0001 , Mann - Whitney U ) . CMT : Childhood maltreatment . Data are presented as violin plots with median , quartiles data points .

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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.


Association of miR - 499 - 5p expression GMV in HC ( n = 23 ) : miR - 499 - 5p were negatively correlated with a GMV cluster comprising parts of left Wernicke language ( i . e . transverse temporal gyrus , left parietal operculum 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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Association 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).


( a ) Confocal time - lapse of an MCF10A - in - structure expressing GFP - LC3 H2B - mCherry . arrow marks recruitment of LC3 to entotic vacuole ( see Supplementary Movie S1 ) . Scale bar , 15 μm .

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(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 ) of MCF10A expressing GFP - LC3G120A mCherry - LC3 ; note no recruitment of GFP - LC3G120A to entotic vacuole . Scale bar , 15 μm . ( c ) LC3 is recruited from host - cytosol onto entotic vacuole . Data points are mean of GFP - LC3 on vacuoles versus cytoplasm for two independent - in - structures ( see Supplementary Movie S2 ) .

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(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 - - electron microscopy of an MCF10A GFP - LC3 - in - structure . were imaged by time - lapse microscopy , followed by fixation after LC3 recruitment ( see Supplementary Movie S3 ) . Top left , post - fixation of phase - contrast GFP - signals were taken demonstrating LC3 recruitment . Bottom left right , electron microscopy ( EM ) of same shows entotic vacuole is a single ( arrow in right image , which is a higher magnification of outlined in bottom left image )

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(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 ) of MCF10A - in - structures , with without autophagy inhibition , that show LC3 recruitment associated with of internalized . Data represent mean ± s . e . m . from at least three independent experiments ; n , total number of events ; * * * P0 . 0001 .

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(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 of MCF10A - in - structures expressing either 2 × FYVE - mCherry ( red ) GFP - LC3 ( green ; f ) or Lamp1 - GFP ( green ) mCherry - LC3 ( red ; g ) . arrows indicate recruitment to entotic vacuole ( see Supplementary Movies S4 S5 ) . Times are indicated as h : min : s ( a , f ) or min ( g ) . Scale bars , 5 μm .

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(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 of - in - structures in which only host express Cathepsin - B - mCherry . Left , a live inner with no Cathepsin B in vacuole ( arrow ) ; middle , a live internalized with Cathepsin B from host inside of vacuole ( arrowhead ) ; right , a dead internalized with Cathepsin B from host throughout corpse ( asterisk ) . Scale bar , 15 μm .

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(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 ) of internalized ( cross ) from time - lapse microscopy . ( j ) Initiation duration of 2 × FYVE domain ( red bar , n = 11 ) , LC3 ( green bar , n = 20 ) Lamp1 ( orange bar , n = 7 ) recruitment to entotic vacuoles , determined using double - expressing shown in f , g . Error bars represent s . d .

(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.


( a ) Left , model depicting multiple fates of internalized ; arrow thickness represents relative frequency of events . Right , of MCF10A - in - structures ( 1 , host ; 2 , internalized ) that show a ( top ) or release ( bottom ) event . Scale bar , 15 μm .

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(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 ) of MCF10A internalized fate over 20 h following autophagy inhibition . Data represent mean from at least three independent experiments ; n , total number of structures analysed ; * * * P0 . 007 , * * P0 . 004 , * P0 . 01 .

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(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 ) of LC3 recruitment ( c ) , fate ( d ) , of mixed - in - structures for which host or internalized 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 .

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(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 , of type of internalized after autophagy inhibition . Data represent mean of three independent experiments ; n , total events analysed ; * * P0 . 03 . Right , representative of non - apoptotic apoptotic of an internalized ; arrow points to fragmented apoptotic nuclei . Scale bars , 15 μm .

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(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 ) of mCherry / GFP of live MCF10A single or internalized expressing tandem GFP - mCherry - LC3 , with or without siRNA treatment as indicated . Data represent mean ± s . d . ; numbers of 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 .

(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 ) of autophagosome autolysosome in control MCF10A ( n = 10 ) internalized ( n = 10 ) cytoplasm imaged by electron microscopy ; * * P0 . 005 . ( h ) Representative electron of - in - structures ( left ) internalized cytoplasm ( middle right ) showing autophagosomes ( arrow ) autolysosomes ( arrowhead ) .

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(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).


( a ) fate of internalized ( control MCF10A MCF10A expressing E7 Bcl2 ) was measured with without autophagy inhibition . Data show of internalized release with autophagy inhibition , compared with each control without autophagy inhibition ; mean ± s . e . m . from at least three independent experiments ( total numbers analysed : ATG5 siRNA , 261 ; Bcl2 ATG5 siRNA , 97 ; 3 - MA , 170 ; Bcl2 3 - MA , 112 ) ; * P0 . 05 , * * P0 . 02 .

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(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 VPS34 ATG5 siRNA on MCF10A - in - formation in suspension for 7 h ; > 300 per condition were scored for - in - formation ; ± s . e . m . from three independent experiments ; * * P0 . 004 .

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(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 of an MCF10A E7 + Bcl2 - in - structure formed in soft agar . arrow marks LC3 recruitment around internalized . Scale bar , 5 μm .

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(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 ) of MCF10A E7 + Bcl2 - in - structures 48 h after seeding into soft agar ( bars ) , colony formation after 2 ( 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 . insets show higher magnifications .

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(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 ) 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 . insets show higher magnifications . See also Supplementary Movie S6 .

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(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.


( a ) Time - lapse of MCF10A - in - structures with an internalized that undergoes apoptosis . arrows mark recruitment of LC3 around apoptotic fragments ( see Supplementary Movie S7 ) . Scale bar , 10 μm . ( b ) of LC3 recruitment to apoptotic internalized in presence of ATG5 or FIP200 siRNA ; n , number of analysed ; * * * P0 . 0001 , chi - squared .

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(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 expressing H2B - mCherry phagocytized by J774 macrophages expressing GFP - LC3 . Confocal show LC3 recruitment to an engulfed corpse ( arrow ; see Supplementary Movie S8 ) . Scale bar , 15 μm . ( d ) of LC3 recruitment to apoptotic phagosomes in control Atg5 - shRNA - expressing ; * * * P0 . 0001 , chi - squared .

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(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 - electron microscopy of an apoptotic phagosome with GFP - LC3 recruitment ( top left image , arrow ) . LC3 - loaded phagosome consists of a single ( black arrow in right image , which is a higher magnification of outlined in bottom left image ; EM , electron microscopy ; AP , apoptotic nucleus ) .

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(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 of apoptotic phagocytosis degradation in control ( n = 21 ) Atg5 shRNA ( n = 21 ) J774 GFP - LC3 ; * * * P0 . 0001 , chi - squared . Scale bar , 10 μm . See also Supplementary Fig . S3 .

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(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.


( a ) Representative of a J774 macrophage expressing GFP - LC3 incubated in media containing red dextran . arrow marks LC3 recruitment to a dextran - containing macropinosome . Scale bar , 2 μm . ( b ) of LC3 recruitment to macropinosomes in control Atg5 shRNA J774 macrophages ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared . See Supplementary Movie S9 .

(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 expressing GFP - LC3 incubated with red dextran . Scale bar , 2 μm . ( d ) of LC3 recruitment to macropinosomes in MCF10A treated with siRNA against FIP200 or ATG5 ; n , number of macropinosomes analysed ; * * * P0 . 0001 , chi - squared .

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(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 imaged by confocal microscopy for 1 h , followed by 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 .

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(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.


( a ) A C . elegans expressing mCherry : : RAB - 5 GFP : : LGG - 1 . arrows point to two apoptotic phagosomes . See Supplementary Movie S11 . Scale bar , 10 μm . ( b ) Cropped time - lapse showing RAB - 5 LGG - 1 recruitment to an apoptotic phagosome ( from a , left arrow ) .

(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 of apoptotic phagosomes in from control - RNAi - or bec - 1 - RNAi - fed worms . insets show higher magnifications of areas outlined in main panels . ( d ) of GFP : : LGG - 1 - mCherry : : RAB - 5 - positive phagosomes in control - or bec - 1 - RNAi determined by time - lapse imaging . Control , 21 , 52 phagosomes ; bec - 1 , 9 , 25 phagosomes ; * * * P0 . 0001 , chi - squared .

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(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 from control - bec - 1 - RNAi at different stages of development ( minutes after first division ) . yellow arrowheads mark apoptotic corpses . Scale bar , 10 μm . ( f ) of apoptotic corpses in control - bec - 1 - RNAi ; data show ± s . d . ; * * * P0 . 0001 .

(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 of CED - 1 : : GFP with control or bec - 1 RNAi . yellow arrowheads mark engulfed corpses surrounded by CED - 1 : : GFP . Scale bar , 5 μm .

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(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.


Mitochondria - enriched fractions were isolated from HdhQ7 or HdhQ111 . mtHSF1 was examined by WB . VDAC was analyzed as a loading control . ( n = 6 biological replicates ) .

Mitochondria-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 were exposed to 5 mM 3 - NP for 2 h . mtHSF1 were determined by immunoblotting . VDAC was used as a loading control . ( n = 5 biological replicates ) .

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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 mitochondria - enriched fractions of HD transgenic YAC128 mice ( 9 months old ) or - matched . ( n = 5 biological replicates ) .

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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 fibroblasts ( GM04208 , GM04222 , GM21756 ) fibroblasts from healthy individuals . ( n = 5 for Con1 / GM04208 Con2 / GM04222 groups ; n = 3 for Con3 / GM21756 group ) .

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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 in mitochondria isolated from - derived iPSCs control iPSCs . ( n = 3 biological replicates ) .

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Representative immunoblot of HSF1 levels in mitochondria isolated from patient-derived iPSCs and control iPSCs. (n=3 biological replicates).


Representative of striatal organoids . Left , immunostaining for MSN marker DARPP32 ( green ) radial glial marker SOX2 ( red ) in striatal organoids at D60 . Middle , immunostaining for LGE progenitor markers Ctip2 ( green ) GSH2 ( red ) at D30 . Right panel , immunostaining for neuronal marker NeuN ( green ) proliferation marker Ki67 ( red ) at D30 . are representative of 12 independent differentiation experiments of H9 IMR90 - 4 . HO = Hoechst ( blue ) . scale bar represents 100 µm .

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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 scatterplot confirming increased HSF1 ( green ) translocation to mitochondria ( Tom20 , red ) . data were obtained from 3 independent biological experiments . Con : n = 15 organoids ; HD : n = 14 organoids . Two - unpaired t - ; Means ± SEMs . were taken using Structured Illumination Microscopy ( SIM ) .

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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).


subcellular localization of HSF1 in HD striatal organoids was observed by immunoelectron microscopy . arrows ( red ) mark HSF1 in mitochondria . negative control lacked HSF1 antibody . scale bar represents 200 nm .

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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 by lentiviral infection . of HSF1 were detected in mitochondrial fractions . ( n = 3 biological replicates ) .

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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).


HdhQ7 were transfected with Flag - mtHSF1 stained with anti - Tom20 ( green ) anti - Flag ( red ) antibodies . Mitochondrial was examined by confocal microscopy . Scatterplot with bar showed of with mitochondrial fragmentation . scale bar represents 40 µm . ( n = 3 biological replicates , at least 100 per group were counted ) .

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HdhQ7 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).


total of p - Drp1 S616 , p - Drp1S637 , Drp1 , OPA1 , MFN2 , Flag - mtHSF1 actin ( loading control ) were determined by WB . ( n = 3 biological replicates ) .

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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 ) DAPI ( blue ) . Mitochondrial was analyzed in Flag + or Flag - regions by confocal microscopy . scale bar presents 2 mm , enlarged image scale bar is 40 µm . ( n = 6 mice / group ) .

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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 ) .

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Striata injected with AAVs were lysed. p-Drp1 S616 was tested by immunoblotting. (n=5 mice/group).


Mitochondrial was analyzed by electron microscopy ( EM ) in mice injected with AAV - Con or AAV - mtHSF1 . Scatterplot showed of mitochondria . scale bar presents 4 µm , 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 .

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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 scatterplot of mitochondrial ( Tom20 , red ) in Flag + or Flag - . data were obtained from 3 independent biological experiments . Flag - : n = 154 mitochondria ; Flag + : n = 132 mitochondria .

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 scatterplot showing that number of p - Drp1 S616 in Flag + was greater than that in Flag - . data were obtained from 3 independent biological experiments . Flag - : n = 70 from 10 organoids ; Flag + : n = 55 from 10 organoids . were obtained using structured illumination microscopy ( SIM ) .

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).


mtDNA content was examined with primers of D - loop , mtCO1 , mtCO2 mtND2 by qPCR in control or Flag - mtHSF1 - expressing striatal . Scatterplot with bar showed relative of mtDNA content . ( n = 3 biological replicates ) .

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The 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 . ( n = 9 - 10 biological replicates )

Measurement of mitochondrial respiratory activity by Seahorse analysis. (n=9-10 biological replicates)


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 ) .

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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 . was determined by detection of LDH release into medium cleavage of PARP . Scatterplot with bar showed relative of LDH release . ( n = 3 biological replicates ) .

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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 of mtDNA content of AAV - Con - injected mice or AAV - mtHSF1 - injected mice . Scatterplot with bar showed relative of mtDNA content ( n = 3 mice / group ) .

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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).


- Caspase3 in AAV - Con - or AAV - mtHSF1 - injected mice was detected by WB . ( n = 5 mice / group ) .

Cleaved-Caspase3 in AAV-Con- or AAV-mtHSF1-injected mice was detected by WB analysis. (n=5 mice/group).


Representative scatterplot showing number of mtDNA ( purple ) colocalized with mitochondria ( Tom20 , green ) in striatal organoids injected with AAV - Con or AAV - mtHSF1 . data were obtained from 3 independent biological experiments . Con : n = 65 from 20 organoids ; Flag - mtHSF1 : n = 70 from 20 organoids . Two - unpaired t - was used . were captured by Structured Illumination Microscopy ( SIM ) .

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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).


typical fragmentation of AAV - Con - ( GFP ) or AAV - mtHSF1 - injected striatal organoids was measured . Scatterplot showed quantitative result of fragmentation . arrows ( yellow ) mark fragmented nuclei . Two - unpaired t - was used . data were obtained from 3 independent biological experiments . Con : n = 14 organoids ; Flag - mtHSF1 : n = 15 organoids .

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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.


Total lysates were subjected to immunoprecipitation with an anti - HSF1 antibody followed by WB . ( n = 3 biological replicates ) .

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Total cell lysates were subjected to immunoprecipitation with an anti-HSF1 antibody followed by WB analysis. (n=3 biological replicates).


HdhQ7 or HdhQ111 extracts were incubated with DSS ( 1 mM ) for 30 min at RT . SSBP1 oligomers were detected by immunoblotting with an anti - SSBP1 antibody . ( n = 3 biological replicates ) .

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HdhQ7 or HdhQ111 cell extracts were incubated with DSS (1 mM) for 30 min at RT. SSBP1 oligomers were detected by immunoblotting with an anti-SSBP1 antibody. (n=3 biological replicates).


SSBP1 oligomers in control fibroblasts ( GM04208 GM21756 ) were analyzed . ( n = 3 biological replicates ) .

SSBP1 oligomers in control and patient fibroblasts (GM04208 and GM21756) were analyzed. (n=3 biological replicates).


SSBP1 oligomers were measured in WT mice or YAC128 mice . ( n = 3 mice / group ) .

SSBP1 oligomers were measured in WT mice or YAC128 mice. (n=3 mice/group).


were transfected with an empty vector or Flag - mtHSF1 for 48 h . total lysates were harvested crosslinked with DSS . SSBP1 oligomers were tested by immunoblotting . ( n = 4 biological replicates ) .

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Cells were transfected with an empty vector or Flag-mtHSF1 for 48 h. The total cell lysates were harvested and crosslinked with DSS. SSBP1 oligomers were tested by immunoblotting. (n=4 biological replicates).


Flag - mtHSF1 was expressed in striata of WT mice for three . SSBP1 oligomers were determined by immunoblotting . ( n = 5 mice / group ) .

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Flag-mtHSF1 was expressed in the striata of WT mice for three weeks. SSBP1 oligomers were determined by immunoblotting. (n=5 mice/group).


Flag - mtHSF1 or sh - SSBP1 was delivered into via lentiviral infection . expression of SSBP1 mtHSF1 was detected by WB . mtDNA content was examined by qPCR . ( n = 3 biological replicates ) .

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Flag-mtHSF1 or sh-SSBP1 was delivered into cells via lentiviral infection. The expression of SSBP1 and mtHSF1 was detected by WB analysis. The mtDNA content was examined by qPCR analysis. (n=3 biological replicates).


neurons expressing Flag - mtHSF1 were stained with anti - DARPP32 ( green ) anti - Flag ( red ) antibodies . arrows ( ) mark Flag + or Flag - neurons . neurite of Flag + or Flag - neurons was measured by Image J showed by scatterplot . scale bar represents 50 µm . ( n = 155 for Flag - group n = 154 for Flag + group ) .

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Primary neurons expressing Flag-mtHSF1 were stained with anti-DARPP32 (green) and anti-Flag (red) antibodies. The arrows (white) mark Flag+ or Flag- primary neurons. The neurite length of Flag+ or Flag- neurons was measured by Image J and showed by scatterplot. The scale bar represents 50 µm. (n = 155 for Flag- group and n = 154 for Flag+ group).