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. 2018 Jul 24;26(5):902–917. doi: 10.1038/s41418-018-0169-5

Fig. 6.

Fig. 6

TRIM17 invalidation induces BCL2A1 degradation and sensitizes melanoma cells to BRAF-targeted therapy. a Method outline depicting generation of TRIM17-depleted SK-MEL-28 cells by inducible CRISPR/Cas9 method and workflow of subsequent steps of analysis. b SK-MEL-28 cells expressing Cas9 and inducible sgRNAs against TRIM17 were treated for 72 h with 1 μg/ml doxycycline. Genomic DNA of cells was amplified by PCR, around the Cas9 cleavage sites targeted by the two sgTRIM17, and PCR products were analyzed by a T7 Endonuclease I assay. The presence of InDels was visualized by the digestion of the PCR products at the Cas9 cleavage sites by T7EI. c PLA was performed in SK-MEL-28 cells expressing DOX-inducible sgRNA against TRIM17, using anti-TRIM28 and anti-BCL2A1 antibodies. Note that the number of green bright spots indicating the very close proximity between endogenous TRIM28 and BCL2A1 proteins increases when expression of endogenous TRIM17 is inhibited by doxycycline treatment. Negative control was obtained by omitting the anti-BCL2A1 antibody. Scale bars, 10 μm. d The protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody in total protein extracts from non-transduced SK-MEL-28 cells left untreated or treated with 20 μM MG132 for 6 h, or from transduced cells expressing Cas9 alone or Cas9 together with inducible sgRNAs against BCL2A1 or mouse bim (negative control) and treated or not with 1 μg/ml doxycycline for 72 h, as indicated. e Two sgRNAs against TRIM17 were induced in SK-MEL-28 cells by doxycycline treatment for 72 h and protein level of endogenous BCL2A1 was assessed by immunoblot using anti-BCL2A1 antibody. f SK-MEL-28 cells expressing Cas9 and the indicated sgRNAs were treated with 1 μg/ml doxycycline for 72 h and subsequently treated with 20 μM PLX4720 (or DMSO control) for 48 h. PLX4720-specific induced apoptosis (SIA) was assessed by annexin V staining and flow cytometry. Data are the means ± SEM of three independent experiments. ****p < 0.0001; **p = 0.0093 (sgTRIM17#1), and p = 0.0032 (sgTRIM17#2) significantly different from negative control (two-way ANOVA followed by Sidak’s multiple comparison test)