Fig. 3.

mTOR inhibition is necessary for the induction of syncytialization and macropinocytosis by FSK in BeWo cells. (A–D) Western blots (A) and corresponding semiquantification (B–D) of p-S6K, S6K, hCGβ,and Syncytin2 in BeWo cells exposed to 100 nM Rapa, with or without 20 μM FSK. (B–D) Semiquantification of p-S6K/S6K, hCGβ, and Syncytin2. (E and F) Representative immunostaining of E-cadherin (red) and DAPI (blue) in BeWo cells cultured in the indicated conditions in E. (Scale bar, 40 μm.) (F) Quantification of multinucleated BeWo cell. (E and G) Representative FITC-dextran (green) uptake in BeWo cells, cultured as detailed in E. (G) Quantification of FITC intensity. (H–J) Western blots (H) and corresponding quantification (I and J) of hCGβ and syncytin2 in BeWo cells cultured in AAS media, exposed to 20 μM FSK, with or without 10 μM MHY1485. (K and L) Representative immunostaining of E-cadherin (red) and DAPI (blue) in BeWo cells cultured as detailed in K. (Scale bar, 40 μm.) (L) Quantification of multinucleated BeWo cell. (K and M) Representative FITC-dextran (green) uptake experiment in BeWo cells cultured in indicated conditions in K. (M) Quantification of FITC intensity. The data are shown as mean ± SD and analyzed by one-way ANOVA test and Tukey–Kramer multiple comparison test based on at least three independent experiments. *P < 0.05; **P < 0.01.