Fig. 5.

TRIM17 and TRIM28 in BCL2A1-dependent chemoresistant melanoma cells. a Total RNA was extracted from SK-MEL-5 and SK-MEL-28 melanoma cells and mRNA levels of indicated genes were estimated by quantitative RT-PCR. b SK-MEL-28 cells were transfected with a control siRNA (siLUC) or with a specific siRNA against BCL2A1 for 24 h. Total RNAs were collected and the mRNA levels of BCL2A1 were estimated by quantitative RT-PCR. The data are the means ± SD of triplicate samples from a representative experiment. c SK-MEL-28 cells were transfected with a control siRNA (siLUC) or with a specific siRNA against BCL2A1 for 24 h. Then, cells were treated with 20 μM PLX4720 for 24 h and apoptosis was estimated by flow cytometry using AnnexinV staining. d SK-MEL-28 cells were transfected with GFP or GFP-tagged TRIM proteins, as indicated, for 24 h. Total protein lysates were analyzed by western blot for the expression of endogenous BCL2A1 and overexpressed proteins. Note that a part of SK-MEL-28 cells was not transfected and thus BCL2A1 variations were certainly underestimated. e SK-MEL-28 cells were transfected with GFP-tagged TRIM28 or TRIM28(C65A/C68A) for 24 h and subsequently treated with 20 μM PLX4720 for 24 h. Apoptosis was estimated in the GFP-positive cell population by flow cytometry using AnnexinV (APC) staining. Data are presented as % of specific induced apoptosis (SIA, see Methods) and are the means ± SEM of four independent experiments. ***p = 0.0001 significantly different from GFP-transfected cells, ns p = 0.1025 non-significantly different from GFP-transfected cells (one-way ANOVA followed by Dunnett’s multiple comparison test). f SK-MEL-28 cells were treated or not (NT) with 20 μM PLX4720 for 24 h. Total RNA was extracted and mRNA level of TRIM17 was assessed by quantitative RT-PCR. g SK-MEL-28 cells were treated or not (NT) with 20 μM PLX4720 for 24 and 48 h. BCL2A1 protein level was assessed by immunoblot