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. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Mol Cancer Res. 2014 Jun 24;12(10):1509–1519. doi: 10.1158/1541-7786.MCR-14-0204

Figure 3. Knockdown of TBK1 enhances apoptosis in combination with AZD6244 specifically in mutant NRAS cell lines.

Figure 3

(A) Mutant NRAS cells were transfected with non-targeting or TBK1-targeting siRNA #1 and #2, and, after 72 hours in 2D conditions, were analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (B) Mutant NRAS cells were transfected with non-targeting or TBK1-targeting siRNA and plated at low density for a total of 7 days. Full-sized image, top, and 4× magnification, bottom. (C) Mutant NRAS cells were transfected as in (A), cultured in 3D collagen in the presence or absence of AZD6244 (3.3 μM). After 24 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (D) WM1366 cells were transfected as in (A) and cultured in 3D collagen as in (C). After 24 hours, cells were lysed and lysates analyzed by Western blotting. (E) WM1361A TR TBK1-myr pretreated with or without doxycycline, SKMel2 and SKMel2 TBK1-myr were cultured in 3D collagen in the presence or absence of AZD6244 (3.3 μM). After 48 hours, cells were released from the gel and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05). (F) WM3211, WM3211 transduced with NRASQ61K, and WM983A cells were transfected as in (A), then cultured and treated in 3D collagen as in (C). After 24 hours, cells were extracted and analyzed for annexin V staining by flow cytometry (n=3; errors bars, S.E.; *, p<0.05).