Figure 5. TRIM16 regulates stress-induced NRF2 response including ubiquitination of protein aggregates.

A Western blot analysis of HeLa and TRIM16^KO lysates of cells treated with increasing concentrations of H₂O₂ as specified and probed with antibodies as indicated.

B WB analysis of HeLa and TRIM16^KO 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 (in panel B) normalized with actin.

D WB analysis of HeLa and TRIM16^KO lysates of cells treated with 2.5 μM (2 h) of As₂O₃.

E–G RNA isolated from HeLa and TRIM16^KO cells, untreated or treated with MG132 (20 μM, 2 h), 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).

H–J 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 TRIM16^KO 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).

M Representative confocal images of control, Ubb, and Ube2n siRNA transfected cells treated with H₂O₂ (200 μM, 2 h) and IF analysis 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 (40×), n = 2, mean ± SD.

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

P Pictorial representation of results obtained in Figs 1–5.

Source data are available online for this figure.