Figure 5.

The MEK inhibitor U0126 or the glutamate release inhibitor Riluzole enhances or restores endocrine response in ILC cells. A, MEK inhibitor U0126 suppresses ERK phosphorylation. Cells were treated as indicated for 48 hours prior to Western blot analysis for phosphorylated ERK1/2 (MAPK1 gene = ERK2 protein), and β-actin as a loading control. Data shown are from a single representative experiment. B, Crystal violet cell proliferation assays conducted for 10 days (SUM44, LCCTam) or 6 days (MDA-MB-231) in the presence of the indicated concentrations of 4HT and/or U0126. Media were changed twice (SUM44, LCCTam) or once (MDA-MB-231) during the course of the experiment. Data are presented as mean relative cell number ± standard deviation for 5–6 technical replicates from an independent experiment performed at least twice. Data were analyzed by two-way ANOVA with post hoc Bonferroni correction. Relative index (RI) values = 1 are additive, > 1 are synergistic. C, Crystal violet proliferation assays conducted for 8 days in the presence of 1 μM Fulvestrant (ICI), 10 μM Riluzole (RIL), or the combination (Combo). Media were changed once during the course of the experiment. Data are presented as mean % survival for 5–6 technical replicates from an independent experiment performed three times. Data were analyzed by two-way ANOVA with post hoc Tukey’s test. Relative index (RI) values = 1 are additive, > 1 are synergistic. D, Expression of ER protein in SUM44 and LCCTam cells following 24 hours treatment with the indicated concentrations of Riluzole (RIL) alone or in combination with 1 μM Fulvestrant (ICI), with β-actin as a loading control. Data shown are from a single representative experiment performed independently three times.