Effect of 14-3-3 tau protein on differentiation in BeWo choriocarcinoma cells

Expression of 14-3-3 tau protein in human placentas. Shown are examples of the first trimester villi (A)–(B) and term placenta (C)–(D) that were immunostained to determine 14-3-3 tau expression. (B) and (D): Negative control. Representative images were taken at ×40 magnification. CTB, cytotrophoblasts; STB, syncytiotrophoblasts; SC, stromal cells.

Effects of forskolin-induced differentiation on 14-3-3 tau expression in BeWo cells. (A) Forskolin treated BeWo cells for 0, 24, 48 and 72 h were electrophoresed on 12% SDS-PAGE gel and probed with rabbit polyclonal antibody to 14-3-3 tau. (B) The relative abundance of syncytin transcript, 14-3-3 tau transcript were analyzed by RT-PCR.

Effects of 14-3-3 tau knockdown on forskolin-induced differentiation of BeWo cells. BeWo cells transfected with negative control or 14-3-3 tau siRNA for 24 h and then cultured without or with forskolin (20 μM) for 2 days. (A) Knockdown effect of 14-3-3 tau siRNA. a: RT-PCR. b:Western blot. 14-3-3 tau siRNA had a maximal inhibition effect on both transcript and protein level at 72 h after transfection. (B) Total RNA was subjected to semi-quantitative RT-PCR to determine 14-3-3 tau, β-hCG, syncytin and ACTB expression. ACTB served as an internal control. (C) β-hCG secretion in the culture media was detected using an enzyme immunoassay kit and normalized to culture media protein. *P < 0.05, vs. negative control without foskolin; #P < 0.05, vs. negative control with foskolin. (D) a: BeWo cells were stained with DAPI (blue) and anti-E-cadherin protein (green) to indicate cell fusion. Representative pictures are shown. b: The number of multinuclear cells in five randomly selected areas was counted and is represented as ratios relative to the control [Negative control/forskolin (−)]. The data from three independent experiments are shown as mean ± S.D. *P < 0.05, vs. negative control without foskolin; #P < 0.05, vs. negative control with foskolin.

Effect of 14-3-3 tau over expression on forskolin-induced differentiation of BeWo cells. BeWo cells transfected with vector control or 14-3-3 tau plasmid were cultured without or with forskolin (20 μM) for 2 days. (A) Over expression effect of 14-3-3 tau plasmid. a: RT-PCR. b:Western blot. The HA tagged 14-3-3 tau and the endogenously expressed 14-3-3 tau bands could easily be distinguished, since the HA tagged protein migrated slightly slower than the endogenous protein. (B) Total RNA was subjected to semi-quantitative RT-PCR to determine 14-3-3 tau, β-hCG, syncytin and ACTB expression. ACTB served as an internal control. (C) β-hCG secretion in the culture media was detected using an enzyme immunoassay kit and normalized to culture media protein. *P < 0.05, vs. vector control without foskolin; #P < 0.05, vs. vector control with foskolin. (D) a: BeWo cells were stained with DAPI (blue) and anti-E-cadherin protein (green) to indicate cell fusion. Representative pictures are shown. b: The number of multinuclear cells in five randomly selected areas was counted and is represented as ratios relative to the control [vector control/forskolin (−)]. The data from three independent experiments are shown as mean ± S.D. *P < 0.05, vs. vector control without foskolin; #P < 0.05, vs. vector control with foskolin.

Effect of 14-3-3 tau on BeWo proliferation. BeWo were treated with negative control or 14-3-3 tau siRNA cultured for 48, 72 or 96 h. (A) Cell viability was evaluated by using the WST-1 assay. The data from three independent experiments are represented as ratios of the control levels and are mean ± S.D. *P < 0.05, vs. negative control. (B) 14-3-3 tau, PCNA, p27kip1 and ERK1/2 protein level after 72 h treatment was analyzed by Western Blot. The results presented are from a representative experiment.