Fig. 5.

Syncytin-1 and -2 do not play a role in ferroptosis in BeWo cells. (A,B) The expression of syncytin-1 (Syn-1) and syncytin-2 (Syn-2) in various cell types used in our study. Expression was determined using RT-qPCR as detailed in Materials and Methods, and all relative expression values were compared to RNA expression in BeWo cells, defined as 1. Values are shown as mean±s.d. **P<0.01 (one-way-ANOVA with Tukey's method for multiple comparisons). (C) The effect of forskolin (60 µM for 48 h, and RSL at various concentrations), which stimulates BeWo cell fusion, on ferroptosis, measured by LDH release assay as described in Materials and Methods. Values are shown as mean±s.d. **P<0.01 (unpaired two-tailed t-test). (D–F) The effect of knockdown of Syn-1 (D), Syn-2 (E) or both Syn-1 and Syn-2 (F) on cell susceptibility to RSL3-induced (8 h) ferroptosis. Silencing of Syn-1 and -2 and assessment of ferroptosis, measured using the LDH release assay, were performed as detailed in Materials and Methods. Fer-1 was used to block ferroptosis, as a control. Values are shown as mean±s.d. Data were analyzed using unpaired two-tailed t-test (no significant differences were found between control shRNA shSyn-1, shSyn-2 or both shSyn1+2). (G) The effect of KD of Syn-1, Syn-2 or both on BeWo blebbing, in the absence or presence of RSL3 (200 nM for 8 h). CellMask was added in the lower panels to enhanced visualization of blebs, also highlighted using white arrows. Scale bars: 50 μm. (H) Bleb formation in BeWo cells, based on the paradigms shown in G. Values are the number of counted blebs per total cells in the field (%), as detailed in Materials and Methods, shown as means±s.d., compared to control. Data were analyzed by one-way-ANOVA with Tukey's method for multiple comparisons test (no significant differences were found between control shRNA and shSyn-1, shSyn-2 or both shSyn1+2). The data represent three independent experiments for A-C,G,H and six independent experiments for D-F.