Figure 5.

Knockdown of Smad4 increases the proliferation of murine embryonic NSCs by enhancing the YAP/TEAD-mediated transactivation. A, qPCR analysis showed that Smad4 was effectively suppressed by Smad4 shRNA. Primary mouse NSCs were infected with control and Smad4 shRNA (ANOVA; n ≥ 3, p < 0.01). B–D, Murine NSCs infected with retroviral shSmad4 displayed an increase in primary neurosphere formation (B). The number of neurospheres was counted 1 week after mouse NSCs were infected. NSCs infected with shSmad4 retrovirus formed neurospheres at a higher frequency (D) and with a larger size (C) than those from control NSCs (ANOVA; n ≥ 3, p < 0.01). E, Murine NSCs infected with retroviral shSmad4 were transfected with 3xSD-luciferase reporter and Renilla plasmids. The cell lysates were subjected to dual luciferase assay (t test; n = 3, p < 0.01). Smad4 deficiency increases the YAP/TEAD-mediated transactivation (t test; n = 3, p < 0.01). F, Flag-immunoprecipitates from P19 cells transfected with HA-TEAD1 and Flag-YAP in Smad4 stable knockdown cells were immunoblotted with anti-HA or Flag antibody. As a control, 2% of the input was immunoblotted with anti-HA or Flag antibody. The interaction between YAP and TEAD1 increases upon knockdown of Smad4. G, Representative images of the subcellular localization of YAP in control and Smad4-knockdown cells. Scale bars: 100 μm. H, Quantification of the subcellular localization of YAP in control and Smad4-knockdown P19 cells (G) showing that Smad4 knockdown increases nuclear accumulation of YAP (t test; n = 3, p < 0.01).