C . The representative images showing the JC - 1 monomers ( green ) and aggregates ( red ) of mouse oocytes after treatment with DMSO ( control , Ctrl , n = 34 ) or U0126 ( n = 40 ) in in vitro culture medium for 16 h . Scale bar = 100 µm . D . The bar graph of ratio of intensities of JC - 1 aggregates to those of monomers in ( C ) . The intensities of JC - 1 were quantified by Image J software . The oocytes number used was labeled .

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C. The representative images showing the JC-1 monomers (green) and aggregates (red) of mouse oocytes after treatment with DMSO (control, Ctrl, n=34) or U0126 (n=40) in in vitro culture medium for 16 h. Scale bar=100 µm. D. The bar graph of ratio of intensities of JC-1 aggregates to those of monomers in (C). The intensities of JC-1 were quantified by Image J software. The oocytes number used was labeled. E


E . Representative images displaying JC - 1 monomers ( green ) , aggregates ( red ) and bright filed images of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies . Scale bar = 100 µm .

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E. Representative images displaying JC-1 monomers (green), aggregates (red) and bright filed images of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies. Scale bar=100 µm.


F . Confocal images of JC - 1 monomers ( green ) and aggregates ( red ) of GV and MII oocytes from wild - type ( WT ) and Erk1 / 2oo - / - oocytes ( n = 3 mice in each group ) . Erk1 / 2oo - / - oocytes exhibited big and multiple polar bodies , indicated by red arrows . Hoechst 33342 ( blue ) was co - stained to represent nucleus or chromosome . Scale bar = 10 µm .

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F. Confocal images of JC-1 monomers (green) and aggregates (red) of GV and MII oocytes from wild-type (WT) and Erk1/2oo-/- oocytes (n=3 mice in each group). Erk1/2oo-/- oocytes exhibited big and multiple polar bodies, indicated by red arrows. Hoechst 33342 (blue) was co-stained to represent nucleus or chromosome. Scale bar=10 µm.


( A Representative confocal images of HeLa and TRIM16KO cells treated with ( A ) MG132 ( 20 µM , 2h ) , scale bar : 7 . 5 µm . and the samples were processed for IF analysis with Ub and p62 antibody . ( B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with ( B ) MG132 ( 20 µM , 2h ) , where IF analysis was conducted with Ub and p62 antibodies . ( C The graph shows the the percentage of cells with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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(A Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 µM, 2h), scale bar: 7.5 µm. and the samples were processed for IF analysis with Ub and p62 antibody. (B Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. (C The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


D ) Representative confocal images of HeLa and TRIM16KO cells treated with ( D ) H2O2 ( 200 µM , 2h ) and the samples were processed for IF analysis with Ub and p62 antibody . E ) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with ( E ) H2O2 , ( 200 µM , 2h ) , where IF analysis was conducted with Ub and p62 antibodies . F ) The graph shows the the percentage of cells with Ub - p62 co - localized dots . Data from ≥ 10 fields ( 40X ) , n = 3 , Mean ± SD , * * p < 0 . 0003 , * p < 0 . 002 ( Student ' s unpaired t - test ) . Data information : Unless otherwise stated , scale bar : 10 µm .

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D) Representative confocal images of HeLa and TRIM16KO cells treated with (D) H2O2 (200 µM, 2h) and the samples were processed for IF analysis with Ub and p62 antibody. E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (E) H2O2, (200 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. F) The graph shows the the percentage of cells with Ub-p62 co-localized dots. Data from ≥10 fields (40X), n=3, Mean ±SD, **p < 0.0003, *p < 0.002 (Student's unpaired t-test). Data information: Unless otherwise stated, scale bar: 10 µm.


( G ) Western blot ( WB ) analysis of detergent - soluble and - insoluble fractions of HeLa and TRIM16KO cells treated with MG132 ( 50 µM , 1h ) and probed with indicated antibodies .

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(G) Western blot (WB) analysis of detergent-soluble and -insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 µM, 1h) and probed with indicated antibodies.


( H , I ) Densitometric analysis ( using ImageJ ) of WB normalized with α - Tubulin or Lamin - B1 . Data information : Unless otherwise stated , scale bar : 10 µm .

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(H, I) Densitometric analysis (using ImageJ) of WB normalized with α-Tubulin or Lamin-B1. Data information: Unless otherwise stated, scale bar: 10 µm.


( J ) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 ( 20 µM , 2h ) and processed for IF analysis with Ub and LC3B antibody .

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(J) Representative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.


( K , L ) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with ( K ) Bafilomycin A1 ( 300 nM , 3h ) alone or ( L ) with MG132 ( 10 µM , 4h ) as indicated . L . E , Low exposure ; H . E , high exposure .

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(K, L) WB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) Bafilomycin A1 (300 nM, 3h) alone or (L) with MG132 (10 µM, 4h) as indicated. L.E, Low exposure; H.E, high exposure.


( M ) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 ( 200 µM , 2h ) and processed for IF analysis with Proteostat dye , Ub , and p62 antibody . To show the cellular details , the brightness of TRIM16KO cells images was increased more than control

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(M) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye, Ub, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells


( A ) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies .

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(A) Western blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.


( B ) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies .

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(B) Western blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.


( C ) Left panel , western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies . Right panel , densitometric analysis ( mean ± SD ) of protein band intensity relative to actin , n = 3 , * * * p < 0 . 0005 ( Student ' s unpaired t - test ) .

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(C) Left panel, western blotting with HeLa and TRIM16KO cell lysates probed with indicated antibodies. Right panel, densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, ***p < 0.0005 (Student's unpaired t-test).


D - G ( D , F ) HEK293T cells were either transiently transfected with an empty vector or a Flag - tagged - TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies . L . E , Low exposure ; H . E , High Exposure . ( E , G ) Densitometric analysis ( mean ± SD ) of protein band intensity relative to actin , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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D-G (D, F) HEK293T cells were either transiently transfected with an empty vector or a Flag-tagged-TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. L.E, Low exposure; H.E, High Exposure. (E, G) Densitometric analysis (mean ±SD) of protein band intensity relative to actin, n=3, *p < 0.05 (Student's unpaired t-test).


( H ) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide ( 100 µM ) for the indicated period and probed with different antibodies as indicated . The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X - ray film . ( I ) Quantification of NRF2 , p62 , and KEAP1 band intensities relative to actin . n = 3 , * * p < 0 . 005 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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(H) WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 µM) for the indicated period and probed with different antibodies as indicated. The blots of control and TRIM16KO cells are exposed for the equal duration and developed together on the same X-ray film. (I) Quantification of NRF2, p62, and KEAP1 band intensities relative to actin. n=3, **p < 0.005, *p < 0.05 (Student's unpaired t-test).


( J ) Immunoprecipitation ( IP ) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 ( 10 µM , 4h ) .

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(J) Immunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 µM, 4h).


IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in ( L ) absence of MG132 ( 10 µM , 4h ) .

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IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence of MG132 (10 µM, 4h).


IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in ( M ) presence of MG132 ( 10 µM , 4h ) .

IP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (M) presence of MG132 (10 µM, 4h).


( N ) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates . Mr , Molecular Weight Marker .

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(N) IP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, Molecular Weight Marker.


( O ) Co - IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates .

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(O) Co-IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.


( P ) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates .

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(P) IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates.


( A , B ) Co - IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16 .

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(A, B) Co-IP analysis of the interaction between NRF2 and KEAP1 in absence and presence of TRIM16.


( C ) Co - IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16 .

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(C) Co-IP analysis of the interaction between NRF2 and p62 in absence and presence of TRIM16.


( D ) Co - IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16 .

(D) Co-IP analysis of the interaction between KEAP1 and p62 in absence and presence of TRIM16.


( 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 .

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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.


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 .

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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 ) Analysis of NRF2 ubiquitination in absence and 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 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 .

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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 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 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 analysis of HeLa and TRIM16KO lysates of cells treated with increasing concentrations of H2O2 as specified and 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 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 .

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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 analysis of NRF2 band intensity normalized with actin .

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


( D ) WB analysis of HeLa and TRIM16KO lysates of cells 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 ) 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 ) .

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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).


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 ) .

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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 analysis of HeLa and TRIM16KO lysates of cells treated with h increasing concentrations of H2O2 as specified and 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 analysis of Ub smear intensity normalized with actin . Mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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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 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 .

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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 ) The graph shows the percentage of cells with protein aggregates . 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 analysis of Ubb and Ube2n siRNA transfected cell lysates with antibodies as indicated .

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


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

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


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

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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 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 .

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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 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 .

WB analysis of control siRNA or NRF2 siRNA transfected lysates of HeLa cells, untreated or treated with H2O2 (200 µM, 2h) as indicated and probed with antibodies as shown. L.E, Low exposure; H.E, High Exposure.


Left panels : Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with ( F ) MG132 ( 20 µM , 2h ) , where IF was conducted with Ub and p62 antibodies . Right panels : fluorescence intensity line tracing corresponding to a white line in zoom panel .

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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 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 .

Left panels: Representative confocal images of control siRNA and NRF2 siRNA transfected cells treated with (G) H2O2 (200 µM, 2h), where IF was conducted with Ub and p62 antibodies. Right panels: fluorescence intensity line tracing corresponding to a white line in zoom panel.


( H ) Hela cells , TRIM16KO cells , and TRIM16KO cells complemented with Myc - NRF2 or GFP - NRF2 were treated with MG132 ( 20 µM , 2h ) and subjected to WB analysis with Myc , GFP and indicated antibodies .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 analysis of the interaction between two differently tagged TRIM16 variants .

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


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

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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 analysis of the interaction between Flag - tagged TRIM16 and Myc - tagged TRIM16 full length and its domain deletion proteins .

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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 and TRIM16KO cells , untreated or treated with H2O2 ( 400 µM , 2h ) . Data , mean ± SD , n = 3 , * p < 0 . 05 ( Student ' s unpaired t - test ) .

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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 ) Images of HeLa and TRIM16KO cells , 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 analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , 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 analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , 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 analysis of HeLa and TRIM16KO cells stained with Annexin - V / Propidium Iodide ( double staining ) , untreated or treated with ( E ) puromycin ( 5 µg / ml , 6h )

Flow cytometry analysis of HeLa and TRIM16KO cells stained with Annexin-V/Propidium Iodide (double staining), untreated or treated with (E) puromycin (5 µg/ml, 6h)


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

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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 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 .

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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 and TRIM16KO cells , 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 ) 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 ) .

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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 images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and 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 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 .

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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 analysis of tumor 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 ) , 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

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

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


( C ) Immunofluorescence staining for myoglobin and myosin heavy chain ( MyHC ) 48h after treatment . Scale bars represent 50µm . Quantification represents the average myotube width Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent standard error of the mean ( SEM ) . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from NT controls ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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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 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

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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 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

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(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 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

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(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 ) 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

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(A) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K (n = 3) and pS6/S6 (n = 4) ratios relative to the non-treated (NT) control Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from NT controls; ††, p < 0.01 from IFNγ/TNFα treated controls


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

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(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 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

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


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

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


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

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


( D ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the DMSO treated control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . For panels A through D , significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD

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(D) Western blotting of phospho-Thr389-p70S6K (pS6K), total p70S6K (S6K), phospho-Ser235/236-S6 (pS6) and total S6. Quantification represents the pS6K/S6K and pS6/S6 ratios relative to the DMSO treated control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. For panels A through D, significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD


( E ) Western blotting of iNOS , tubulin , phospho - Thr172 AMPKα ( pAMPK ) , total AMPKα ( AMPK ) , phospho - Ser79 - ACC ( pACC ) and total ACC . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(E) Western blotting of iNOS, tubulin, phospho-Thr172 AMPKα (pAMPK), total AMPKα (AMPK), phospho-Ser79-ACC (pACC) and total ACC. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( F ) RT - qPCR analysis of iNOS mRNA levels relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(F) RT-qPCR analysis of iNOS mRNA levels relative to the IFNγ/TNFα control 24h after treatment Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM For panels E through G, p-values were calculated using the Student"s t-test. *, p < 0.05; **, p < 0.01 from DMSO controls; ††, p < 0.01 from IFNγ/TNFα treated controls


( G ) Nitric oxide ( NO ) levels in the culture media of cells relative to the IFNγ / TNFα control 24h after treatment Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM For panels E through G , p - values were calculated using the Student " s t - test . * , p < 0 . 05 ; * * , p < 0 . 01 from DMSO controls ; † † , p < 0 . 01 from IFNγ / TNFα treated

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

(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 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

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

(A) Immunofluorescence staining for myoglobin and myosin heavy chain (MyHC) 48h after treatment. Scale bars represent 50µm. Quantification represents the average myotube width Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( B ) Western blotting for iNOS and tubulin protein levels 24h after treatment . Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ / TNFα control Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

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

(B) Western blotting for iNOS and tubulin protein levels 24h after treatment. Quantification represents the levels of iNOS protein normalized to tubulin and relative to the IFNγ/TNFα control Data information: All quantifications are of three independent experiments (n = 3) and error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from non-treated (NT) controls; †, p < 0.05; ††, p < 0.01 from IFNγ/TNFα treated controls


( C ) Western blotting of phospho - Thr389 - p70S6K ( pS6K ) , total p70S6K ( S6K ) , phospho - Ser235 / 236 - S6 ( pS6 ) and total S6 . Quantification represents the pS6K / S6K and pS6 / S6 ratios relative to the NT control . Data information : All quantifications are of three independent experiments ( n = 3 ) and error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from non - treated ( NT ) controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from IFNγ / TNFα treated controls

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-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 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 images 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 ) and total ACC

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

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


( C ) Cross - sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort ( n = 4 ) . Cross - sections were stained by H & E . Scale bars represent 100µm Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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

O O O O O O O O O O B-MEASURED_VAR I-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 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 analysis o MuRF1 ( E ) mRNA expression from the tibialis anterior of three mice per cohort ( n = 3 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from saline controls ; † , p < 0 . 05 ; † † , p < 0 . 01 from C26 controls

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

(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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cross-sectional analysis of the gastrocnemius muscle calculated from 250 fibers per mouse from four mice per cohort (n = 4). Cross-sections were stained by H&E. Scale bars represent 100µm Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


( B - C ) RT - qPCR analysis of Atrogin - 1 / MAFbx ( B mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

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

(B-C) RT-qPCR analysis of Atrogin-1/MAFbx (B mRNA expression from the tibialis anterior of four mice per cohort (n = 4). Data information: Error bars represent the SEM. Significance between means was first determined using ANOVA. Significance p-values were calculated using Fisher"s LSD. *, p < 0.05; **, p < 0.01 from PF controls


( B - C ) RT - qPCR analysis o MuRF1 ( C ) mRNA expression from the tibialis anterior of four mice per cohort ( n = 4 ) . Data information : Error bars represent the SEM . Significance between means was first determined using ANOVA . Significance p - values were calculated using Fisher " s LSD . * , p < 0 . 05 ; * * , p < 0 . 01 from PF controls

O O O O O O O O O O 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

(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 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 .

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

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

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

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

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

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

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

O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 .

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

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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 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 .

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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 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 .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 subjects (female= 26, male= 31), and BD subjects in different mood states (depressive (female= 7, male=11), euthymic (female= 7, male=4), hypomanic (female=2, male=6), manic (female=2, male=1), mixed (female=1, male=1). 2-way ANOVA, main effect Sex: p=0.260; main effect Group: p=1.26e-07; Interaction Group x Sex: p=0.059; Tukey's HSD: control vs. depressive ***p=0.0002621, control vs. euthymic ***p=0.0008533, control vs. hypomanic ****p=0.0000470. Data are presented as violin plots with median, quartiles and data points.


miR - 499 - 5p qPCR analysis of total RNA isolated from PBMCs of healthy control subjects ( - CMT , n = 17 ) and healthy subjects with a history of childhood maltreatment ( + CMT , n = 17 ) . ( * * * * p < 0 . 0001 , Mann - Whitney U test ) . CMT : Childhood maltreatment . Data are presented as violin plots with median , quartiles and data points .

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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 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 ) .

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

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 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 .

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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 ) 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 ) .

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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 - 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 )

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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 ) 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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(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 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 .

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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 ) 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 .

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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 ) 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 .

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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 , 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 .

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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 ) 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 .

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(f) Quantification of mCherry/GFP ratio of live MCF10A single cells or internalized cells expressing tandem GFP-mCherry-LC3, with or without siRNA treatment as indicated. Data represent mean±s.d.; numbers of cells analysed for each data point (from left to right) 30, 30, 30, 25, 30, 25, 25, 25, 30, 27; *P0.03, **P0.003, ***P0.0004.


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

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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 ) 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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.