Fig. 6: Cited1 interacts with Bmpr2 to activate BMP signaling and induce trophoblast differentiation.

a Protein levels of pSmad1/5 and pSmad2 upon Cited1 overexpression in E14T ESCs at the times as indicated. Smad5, Smad2/3, and α-Tubulin were used as loading controls. b Protein levels of pSmad1/5 and pSmad2 upon Cited1 overexpression and inhibitor treatment for 3 days in E14T ESCs. Smad5, Smad2/3, and α-Tubulin were used as loading controls. c Bright field images of Cited1-overexpressing or control ESCs after treatment with DMSO or different inhibitors for 3 days. LDN193189 (0.1 μM), Noggin (400 ng/mL) and K02288 (10 μM) are all BMP signaling inhibitors. SB431542 (10 μM) is a TGF-β signaling inhibitor. d qRT-PCR analysis of transcript levels of trophoblast markers after transfection and treatment with inhibitors for 3 days. The average mRNA level in cells transfected with control vector pPy and treated with DMSO was set at 1.0. Data are shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. e qRT-PCR analysis of mRNA levels for ligands of the BMP pathway induced by Cited1 overexpression in E14T ESCs for 3 days. The average mRNA level in cells transfected with control vector pPy was set at 1.0. Data are shown as mean ± SD (n = 3). *p < 0.05. f The representative western blot result from Co-IP (immunoprecipitation) assays showing that ectopically expressed Flag-Cited1 binds to endogenous Bmpr2 specifically in E14T ESCs. Whole-cell lysates (WCL) were immunoprecipitated with anti-Flag M2 beads and analyzed by western blotting with antibodies as indicated. Five percent of WCL was loaded as the input. g qRT-PCR analysis of mRNA levels of trophoblast specific markers in parental WT ESCs or Bmpr2 knockout (KO) cells upon Cited1 overexpression for 3 days. The average mRNA level in WT cells transfected with empty vector pPy was set at 1.0. Data are shown as mean ± SD (n = 3). ***p < 0.001