YAP promotes proliferation and survival in irradiated CGNPs
(A) Western blot analysis of proliferation (cyclin D2) and apoptosis (cleaved caspase 3) in GFP- or YAP-transduced Shh-treated CGNPs untreated (NT) and 20 hours after γ-irradiation (IR) with 10 Gy. Panel to the right shows over-exposed image. Similar infection efficiencies were obtained with both retroviruses (Supplementary Figure 2).
(B) Immunofluorescence analysis and quantification of cleaved caspase 3 and the proliferation markers Ki67 and phosphorylated histone H3 (P-Hist3) in GFP- or YAP-transduced CGNPs 20 hours post-irradiation.
(C) Left, immunofluorescence analysis of BrdU incorporation (1-hour pulse) untreated (NT; top panels) and 18 hours after irradiation (IR; middle panels). The S-phase ratios for GFP and YAP-transduced CGNPs (%BrdU-positive of treated / %BrdU-positive untreated) are shown at the bottom. Irradiated GFP-transduced cells were normalized to non-irradiated GFP-transduced CGNPs; irradiated YAP-transduced CGNPs were normalized to non-irradiated YAP-transduced CGNPs. The higher S-phase ratio indicates that YAP-over-expressing CGNPs present a defect in the G1/S checkpoint. Right, immunofluorescence analysis of phosphorylated histone H3 before (NT; top panels) and 1 hour after radiation (IR; middle panels) in GFP- or YAP-transduced CGNPs. YAP-expressing CGNPs showed a higher mitotic ratio (%P-Hist3 positive of treated / % P-Hist3 positive untreated) (bottom right), indicating failure of the G2/M checkpoint in these cells. Again, irradiated GFP-transduced cells were normalized to non-irradiated GFP-transduced CGNPs; irradiated YAP-transduced CGNPs were normalized to non-irradiated YAP-transduced CGNPs.
Statistically significant differences are indicated as (*) P < 0.05; (***) P < 0.001.