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. 2017 Jun 1;8(6):e2844. doi: 10.1038/cddis.2017.226

Figure 9.

Figure 9

BAD phosphorylation is essential for VIP-induced cytoprotection in CSCs. (a) Tumorospheres were infected with lentiviral vectors expressing wt-BAD or mutant-BAD. After 24 h, spheres were placed in supplement-free DMEM containing indicated inhibitors and VIP for 3 h. Phase-contrast images of spheres are shown. Scale bar=100 μm. (b) CSCs were purified by FAC-sorting from spheres that express wt-BAD or mutant-BAD and luciferase. CSCs were placed in supplement-free DMEM containing indicated inhibitors and VIP for 3 h. Luciferase activity was measured from cell lysates to judge the CSC survival. (c) Caspase activity was measured from whole-cell lysates as collected in b. (d) Sphere-forming assay was performed using 100 cells in b, and the number of spheres formed was counted. (e) Expression levels of wt-BAD and mutant-BAD are shown. (f) Cell lysates in a were probed with indicated antibodies. Note that mutant-BAD behaves like dephosphorylated BAD as S112-BAD antibodies do not recognize phosphorylation on S112 of mutant-BAD. The error bars represent S.D. of the biologic triplicates. All experiments presented in this figure are representative of three independent experiments