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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Cancer Res. 2011 Feb 11;71(7):2750–2760. doi: 10.1158/0008-5472.CAN-10-2954

Figure 6. Dual PI3K/BRAF inhibition upregulates BIM and enhances apoptosis in PTEN− cells.

Figure 6

(A) (left) Western blot of 1205Lu cells treated with PLX4720 (3μM, 48 hrs), the PI3K inhibitor GDC-0941 (3μM, 48 hrs) or both drugs in combination (P+G). Right panel: Immunofluorescence staining of BIM (green) and DAPI (blue) in PTEN− cells following PLX4720 treatment (3μM, 48 hrs) the PI3K inhibitor LY294002 (10μM, 48 hrs) or both drugs in combination (PLX+LY). (B) Left panel: Immunofluorescence staining of PTEN−1205Lu following combined inhibition (3μM PLX4720 + 10μM LY294002, 48 hrs) increases nuclear localization of FOXO3a (green). DAPI is shown in blue. Magnification:X40. Right panel: Combined inhibition (3μM PLX4720 + 10μM LY294002, 48 hrs) increases PTEN− WM793 BIM mRNA levels to those observed with single BRAF inhibition (3μM PLX4720, 48 hrs) in the PTEN+ WM35. (C) PTEN− cells were treated with PLX4720 (3μM, 48 hrs), GDC-0941 (3μM, 48 hrs) or a combination of the two drugs (3P+3G) before Annexin-V staining was analyzed by flow cytometry (*indicates P<0.05 between the drug combination and each inhibitor alone). (D) Combined BRAF/PI3K inhibitor treatment blocks the escape of 1205Lu cells (PTEN−) from therapy. Spheroids of 1205Lu cells were treated with either PLX4720 alone (3 and 10μM: data shows 3μM), LY294002 (10μM) alone or a combination of the two drugs for 72hrs. In other studies, spheroids were treated with drugs for 72hrs and then allowed to recover for 120hrs. Micrograph shows viability staining (green=live cells, red=dead cells). Magnification x10.