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. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Eur J Cancer. 2014 Sep 6;50(16):2866–2876. doi: 10.1016/j.ejca.2014.08.011

Figure 4. 4-OHT activated membrane-associated signaling in MCF-7:PF cells.

Figure 4

(A) Rapid activation of MAPK. MCF-7:PF cells were treated with E2 (10−9 mol/L) or 4-OHT (10−6 mol/L) for the time points indicated. p-MAPK was examined by Western blot. Total MAPK was measured as loading control. (B) Selective inhibition of 4-OHT-induced MAPK activation. MCF-7:PF cells were treated with 4-OHT (10−6 mol/L) alone or in the presence of specific inhibitors, PP2 (5×10−6 mol/L), AG1024 (10−5 mol/L), and ICI (10−6 mol/L) for 10 minutes. p-MAPK was examined by Western blot. (C) Activation of growth pathways by 4-OHT or E2. MCF-7:PF cells were treated with vehicle (0.1% EtOH), E2 (10−9 mol/L), or 4-OHT (10−6 mol/L) for 72 hours. p-Akt, p-MAPK, and p-STAT3 were determined by Western blot. Respective total proteins were examined as loading controls. (D) Activation of focal adhesion molecules by 4-OHT and E2. Cell lysates were the same as in (C). p-FAK and p-p130CAS were determined by Western blot. Respective total proteins were examined as loading controls. (E) The FAK inhibitor, PF573228, blocked the proliferation induced by 4-OHT. MCF-7:PF cells were seeded in 24-well plates as above. One day later, they were treated with vehicle (0.1% DMSO), 4-OHT (10−6 mol/L), PF573228 (10−6 mol/L), and PF573228 (10−6 mol/L) plus 4-OHT (10−6 mol/L) for 7 days. Total DNA was determined as above as a measure for cell proliferation. p<0.001, ** compared with control.