Fig 2: TRIM16 mediates selective clearance of aggregated autophagy cargoes A–D(A, C) WB analysis of detergent‐soluble and ‐insoluble fractions of HeLa and TRIM16KO cells expressing either (A) Flag‐p62 or (C) GFP‐polyQ74 probed with Flag or GFP or indicated antibodies. L.E, low exposure; H.E, high exposure. (B, D) Densitometric analysis of WB's (shown in panels A and C) normalized with α‐tubulin or lamin‐B1.EConfocal images of GFP‐TRIM16‐expressing HeLa cells, untreated or treated with MG132 (10 μM, 4 h) and/or bafilomycin A1 (300 nM, 3 h) and IF analysis is performed with antibodies as indicated. Right panel graphs: Fluorescence intensity line is tracing corresponding to a white line in zoom panel. Scale bar: 10 μm.FThe 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.GRepresentative confocal images of GFP‐TRIM16 expressing HeLa cells, untreated or treated with MG132 (10 μM, 4 h) and/or bafilomycin A1 (300 nM, 3 h) and IF analysis is performed with antibodies as indicated. Right panel graphs: Fluorescence intensity line is tracing corresponding to a white line in zoom panel. Scale bar: 10 μm.HCo‐IP analysis of the interaction between two differently tagged TRIM16 variants.IWB analysis of detergent‐soluble and detergent‐insoluble DSS cross‐linked fractions of HeLa and TRIM16KO cells, untreated or treated with MG132 (10 μM, 4 h) and probed with indicated antibodies.JCo‐IP analysis of the interaction between Flag‐tagged TRIM16 and Myc‐tagged TRIM16 full length and its domain deletion proteins. Source data are available online for this figure.

Fig 3: TRIM16 is required for assembly of protein aggregates A–F(A, D) Representative confocal images of HeLa and TRIM16KO cells treated with (A) MG132 (20 μM, 2 h, scale bar: 7.5 μm) or (D) H2O2 (200 μM, 2 h) and the samples were processed for IF analysis with Ub and p62 antibody. (B, E) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with (B) MG132 (20 μM, 2 h), (E) H2O2, (200 μM, 2 h), where IF analysis was conducted with Ub and p62 antibodies. (C, F) The graph shows the percentage of cells with Ub‐p62 co‐localized dots. Data from ≥ 10 fields (40×), n = 3, mean ± SD, **P < 0.0003, *P < 0.002 (Student's unpaired t‐test).GWestern blot (WB) analysis of detergent‐soluble and detergent‐insoluble fractions of HeLa and TRIM16KO cells treated with MG132 (50 μM, 1 h) and probed with indicated antibodies.H, IDensitometric analysis (using ImageJ) of WB normalized with α‐tubulin or lamin‐B1.JRepresentative confocal images of HeLa and TRIM16KO cells treated with MG132 (20 μM, 2 h) and processed for IF analysis with Ub and LC3B antibody.K, LWB analysis of LC3B levels in lysates of HeLa and TRIM16KO cells treated with (K) bafilomycin A1 (300 nM, 3 h) alone or (L) with MG132 (10 μM, 4 h) as indicated. L.E, low exposure; H.E, high exposure.MRepresentative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 μM, 2 h) and processed for IF analysis with ProteoStat dye, and p62 antibody. To show the cellular details, the brightness of TRIM16KO cells images was increased more than control cells.NPictorial representation of data presented in Figs 1 and EV1.Data information: Unless otherwise stated, scale bar: 10 μm.Source data are available online for this figure.

Fig 4: TRIM16 interacts with and regulates the expression of p62, KEAP1, and NRF2 Left panel, HEK293T cells were either transiently transfected with an empty vector or a Flag‐tagged‐TRIM16 expressing vector and immunoblotted with Flag or indicated antibodies. Right panel, densitometric analysis (mean ± SD) of protein band intensity relative to actin, n = 3, *P < 0.05 (Student's unpaired t‐test).Western blot analysis of cytoplasmic and nuclear fraction isolated from MG132‐treated control and TRIM16KO cells.WB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 μg/ml) 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.WB analysis of control or Flag‐TRIM16 vector transfected HeLa cells treated with cycloheximide (100 μg/ml) for the indicated period and probed with different antibodies as indicated. The blots of control and overexpressed cells are exposed for the equal duration and developed together on the same X‐ray film.Quantification of KEAP1, p62, and NRF2 band intensities (from panel D) relative to actin.Co‐IP analysis of the interaction between TRIM16 and NRF2 in HEK293T lysates of cells expressing GFP or GFP‐NRF2 and Flag‐TRIM16.Co‐IP analysis of the interaction between TRIM16 and KEAP1 in HEK293T lysates of cells expressing vector control or Flag‐TRIM16 and GFP‐KEAP1.IP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates in absence and presence of MG132. *The panel (H) here, Fig 2J, and Appendix Fig S3H are part of same blots; hence, the input for TRIM16 is same. Source data are available online for this figure.

Fig 5: TRIM16 regulates the p62‐KEAP1‐NRF2 system AWestern blot analysis of HeLa and TRIM16KO cell lysates probed with indicated antibodies.BWestern blot analysis of lysates from HeLa and TRIM16KO cells treated with or without MG132 and probed with indicated antibodies.CLeft 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).HWB analysis of HeLa and TRIM16KO lysates of cells treated with cycloheximide (100 μg/ml) 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.IQuantification of NRF2, p62, and KEAP1 band intensities relative to actin. n = 3, **P < 0.005, *P < 0.05 (Student's unpaired t‐test).JImmunoprecipitation (IP) analysis of interaction between endogenous TRIM16 and endogenous p62 in HeLa cell lysates in absence and presence of MG132 (10 μM, 4 h). *The panel (J) here, Fig EV2H, and Appendix Fig S3H are part of same blots; hence, the input for TRIM16 is same.KThe domain organization map of TRIM16 and deletion constructs cloned as Myc‐tagged proteins.L, MIP analysis to map the interaction between p62 and TRIM16 domains in HEK293T cell lysates in (L) absence and (M) presence of MG132 (10 μM, 4 h).NIP analysis of the interaction between endogenous TRIM16 and endogenous NRF2 in HeLa cell lysates. Mr, molecular weight marker.OCo‐IP analysis to map the interaction between NRF2 and TRIM16 domains in HEK293T cell lysates.PIP analysis of the interaction between endogenous TRIM16 and endogenous KEAP1 in HeLa cell lysates. Source data are available online for this figure.