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. 2014 Nov 3;5(20):10127–10139. doi: 10.18632/oncotarget.2466

Figure 5. TRIM16 protein is increased with vemurafenib treatment and is required for the drug action.

Figure 5

(A) Melanoma (A375 and Mel-CV) cells were treated with vemurafenib at 0, 0.125, 0.25 or 0.5μM for 72 hours and whole cell lysates were used for immunoblotting against an anti-TRIM16 antibody. Anti-β-Actin was used as a loading control. (B) TRIM16 protein stability was assessed in melanoma (A375 and Mel-CV) cells following treatment with vemurafenib at 0.5 and 1.5μM respectively, and CHX at 100 μg/mL. At the specified time points, the cells were harvested and the protein was extracted for analysis by Western blotting against anti-TRIM16 or anti-β-actin. (C) Melanoma (A375) cells were transiently transfected with two different TRIM16 siRNA's or control siRNA, and then treated with 0.5 μM vemurafenib for 72 hours. Cell viability was measured using the Alamar blue assay. A statistically significant difference is indicated. (D) TRIM16 expression level was measured by immunohistochemistry in patient samples of pre-treatment (N=9), on vemurafenib (N=2) or dabrafenib (N=7) treatment, and progressed on BRAF inhibitor treatment (N=5). (E) Representative immunohistochemistry for two patients is shown.