Skip to main content
. Author manuscript; available in PMC: 2017 Sep 1.
Published in final edited form as: Mol Cancer Ther. 2017 Jan 30;16(3):516–528. doi: 10.1158/1535-7163.MCT-16-0552

Figure 2. Downregulation of Bim-EL and BMF contributes to HGF-mediated resistance to trametinib in UM cells.

Figure 2

(A) HGF inhibits trametinib-induced Bim-EL and Bmf expression in UM cells. UM001 cells (left) and UM004 cells (right) were treated with 50 nM trametinib, 10 ng/ml HGF, 2.5 µM MK2206, or combinations as indicated for 48 hours. Cell lysates were analyzed for expression of indicated BH3-only pro-apoptotic proteins (Bim-EL, Bmf, Bad, Bid, Noxa and Puma), multi-domain pro-apoptotic proteins (Bax and Bak) and anti-apoptotic proteins (Bcl-2, Bcl-w, Bcl-xl and Mcl-1). Actin was used as loading control. (B) Silencing of Bim-EL and Bmf in UM cells. UM004 cells were transfected with 20 nM control siRNA, Bim-EL siRNA, Bmf siRNA or Bim-EL/Bmf siRNA. Expression of Bim-EL and Bmf was examined by Western blotting with indicated antibodies. (C) Silencing of Bim-EL and Bmf renders UM cells resistant to trametinib-induced apoptosis. UM cells were transfected with siRNAs, as above. 48 hours post-transfection, cells were treated with DMSO or 50 nM trametinib for another 48 hours. Cell growth was determined by crystal violet staining. Representative images of the cells at 100× magnification are shown. The scale bar is equal to 100 µm. Quantitation of crystal violet staining is presented as mean of percentage crystal violet from triplicate experiments following normalization to siCtl. *P<0.05, **P<0.01, ***P<0.001.