Figure 1: ERK sustains proliferation of TAM-treated ER-Src cells at 12 h.

(a) Western blots on protein extracts from ER-Src cells treated with EtOH or TAM for 4, 12, 24 or 36 h, blotted with anti-pSrc, which reveals ER-pSrc or endogenous pSrc or anti-GAPDH. (b) Ratio of ER-pSrc levels between TAM- and EtOH-treated ER-Src cells for the same time points, normalized to GAPDH. (c) Images by phase contrast microscopy of ER-Src cells, treated with TAM or EtOH for 4, 12, 24 or 36 h. Scale bars, 50 μm. (d) Percentage of ER-Src cells in S-phase after treatment with EtOH (grey bars) or TAM (orange bars) for 4, 12, 24 or 36 h in the absence of EGF. (e) Western blots on protein extracts from ER-Src cells treated with EtOH or TAM for 12 h, blotted with anti-pERK or anti-ERK, and quantification of the ratio of pERK over total ERK levels, normalized to GAPDH for the corresponding lane on western blots. EtOH treatment (grey bar), TAM treatment (orange bar). (f) Cyclin D1 mRNA levels on extracts from ER-Src cells treated with EtOH (grey bar) or TAM (orange bar) for 12 h. (g) Western blots on protein extracts from ER-Src cells treated with EtOH or TAM and DMSO or MEKi for 12 h, blotted with anti-pERK or anti-ERK, and quantification of the ratio of pERK over total ERK levels, normalized to GAPDH for the corresponding lane on western blots. (h) Cyclin D1 mRNA levels on protein extracts from ER-Src cells treated with EtOH or TAM and DMSO or MEKi. (i) Number of ER-Src cells in S-phase treated with EtOH or TAM and DMSO or MEKi. DMSO treatment (grey bars), MEKi treatment (blue bars). Quantifications are from (a) six or (c–h) three biological replicates. Error bars indicate s.d.; NS indicates non-significant; *P<0.05; **P<0.001; ***P<0.0001. Statistical significance was calculated using (d) two-way ANOVA or (e,f) impaired t-test or (b,g–i) one-way ANOVA. See also Supplementary Figs 1–3 and 7, and Supplementary Movies 1–4.