Fig. 7

17β-estradiol induces SREBP-1C expression and activation which depend on the IGF-1 pathway. MCF-7 cells were incubated for 7 days in phenol red-free media supplemented with charcoal-stripped FBS (starved). (a and b) Starved cells were then incubated in the same media that was supplemented with 50 ng/ml of IGF-1, 2nM of 17β-ED, a combination of 17β-ED, and 10 μM of the IGF-1R antagonist Ag1024, or their vehicle controls (Ctrl) for 5 days. (a) RNA was extracted from cells and reverse transcribed into cDNA. Relative qPCR for SREBP-1C was performed using HPRT as reference gene. (b) Cellular proteins were separated by SDS-PAGE and immunoblot analysis of the precursor (P) and mature (M) SREBP-1 was performed, using actin as loading control. The graph show densitometry quantification of the SREBP-1 (M) blots. (c) Starved cells were then transfected with anti-SREBP-1 siRNA or its non-silencing control, and incubated with 17β-ED, or its vehicle control as indicated 3 days. Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SREBP-1 (P) and SCD-1 were performed, using actin as loading control. The graphs show densitometry quantification of the SREBP-1 and SCD-1 blots. All immunoblots are representative of 3 independent experiments. Data are means ± SEM, n = 3 or 4 independent experiments. Values that have a different superscript are significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment