TRIM16 controls assembly and degradation of protein aggregates by modulating the p62-NRF2 axis and autophagy (2018)
Figure 9

Figure 9. TRIM16 is protective against oxidative stress-induced cytotoxicity in vivo.

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

(C) Tumor volumes of HeLa and TRIM16KO tumors at indicated time points. As2O3 is injected when tumor volume of all groups was >100mm3 (day 0 in the graph). Tumor volumes were measured on an interval as indicated. Mean ±SE, n=6 (each group), *p < 0.05, **p < 0.005, ***p < 0.0005 (ANOVA).

(D) Graph shows the % tumor regression (Tumor volume on day 1 of As2O3 treatment / Tumor volume on day of sacrifice X 100). Mean ±SE, n=6 (each group), **p < 0.005 (Student's unpaired t-test).

(E) Representative images of HeLa and TRIM16KO tumors formed in the nude mice in the absence and presence of As2O3.

(F) Representative pictures of dissected tumors.

(G) Immunohistochemistry analysis performed with KI-67 cell proliferation marker and hematoxylin nuclear stain. The graph represents the % of cells with KI-67 staining. >400 cells were counted for this analysis from three different sections from different animals, mean ±SD, *p < 0.05 (Student's unpaired t-test). Scale bar: 50 µm.

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

(I) Graphical representation of work presented in this study.

Expanded View Figure Legends

Figure EV 1. TRIM16 is important for assembly of the protein aggregates.

(A) Confocal images of HeLa and TRIM16KO cells, untreated or treated with MG132 (20 µM, 2h), H2O2 (200 µM, 2h), and puromycin (5 µg/ml, 2h), and processed for immunofluorescence (IF) analysis with ubiquitin (FK2) antibody.

(B) HeLa and TRIM16KO cells were visualized, and cells with more than two visible aggresomes/ALIS were considered positive. Mean ±SD, n=3 **p < 0.005 (Student's unpaired t-test).

(C) Total numbers of visible Ub puncta's in each cell were counted and plotted. Data is from ≥10 microscopic fields for each condition, n=3, Mean ±SD, *p < 0.05 (Student's unpaired t-test).

(D) Representative confocal images of control siRNA and TRIM16 siRNA transfected cells treated with puromycin (5µg/ml, 2h), where IF analysis was conducted with Ub and p62 antibodies.

(E) The graph shows the average number of Ub-p62 co-localized aggregates per cell from panel D experiment. Mean ±SD, n=3, *p < 0.05 (Student's unpaired t-test).

(F) Representative confocal images of HeLa and TRIM16KO cells treated with As2O3 (2.5 µM, 2h), where IF analysis was conducted with Ub and p62 antibodies. Right panel: fluorescence intensity line is tracing corresponding to a white line in zoom panel.

(G, H) Representative confocal images of HeLa and TRIM16KO cells treated with (G) MG132 (20 µM, 2h) or (H) H2O2 (200 µM, 2h) and processed for IF analysis with Ub and LC3B antibody.

(I) WB analysis of LC3B levels in lysates of cells transfected with control vector or Flag-TRIM16.

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

(K) Representative confocal images HeLa and TRIM16KO cells treated with H2O2 (200 µM, 2h) and processed for IF analysis with Proteostat dye and p62 antibody.

Data information: Unless otherwise stated, scale bar: 10 µm.