Fig. 4

ERα silencing and treatment with 4-OH Tamoxifen blocks the 17β-estradiol induction of SCD-1 levels and activity. (a, b) MCF-7 cells were incubated for 7 days in phenol red-free media supplemented with charcoal-stripped FBS (starved), followed by a 5 day incubation period in the same media that was supplemented with 2nM 17β-ED, 10nM of 4-OH tamoxifen (OH-Tam), a combination of both, or their vehicle controls (Ctrl). (a) Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SCD-1 expression was performed using actin as loading control. The graph shows densitometry quantification of the SCD-1 blots. (b) Cellular lipids were extracted, hydrolyzed, transmethylated, and quantified by GC/FID and the indicated (MUFA/SFA) ratios were calculated. (c) MCF-7 cells were starved for 5 days as above and were then subjected to electroporation in the presence of three different ERα-targeting siRNA (ERα-si1, ERα-si2, Erα-si3), or a non-targeting duplex (non-silencing) and were incubated in starvation medium for an additional 3 days. Cellular proteins were then separated by SDS-PAGE and immunoblot analysis of ERα was performed, using actin as loading control. The graph shows densitometry quantification of the ERα blots. (d) Starved cells were transfected with ERα-targeting siRNA or the non-silencing control as in (c) above, and were then incubated in starvation medium containing 2nM 17β-ED or its vehicle control (ctrl) for 3 days, as indicated. Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SCD-1 was performed, using actin as loading control. The graph shows densitometry quantification of the SCD-1 blots. All immunoblots are representative of 3 independent experiments. Data are means ± SEM, n = 3 independent experiments. Values with a different superscript are significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment