Fig. 5

SCD-1 activity is important for 17β-estradiol induced MCF-7 cell proliferation. (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, 2 μM of the SCD-1 inhibitor A939572, a combination of both, or their vehicle controls (Ctrl). (a) Cellular lipids were extracted, hydrolyzed, transmethylated, and quantified by GC/FID and the indicated (MUFA/SFA) ratios were calculated. (b) Cells subjected to the different treatments were counted using a haemocytometer. (c-f) MCF-7 cells were starved for 5 days as above and were then incubated in starvation medium containing 2nM 17β-ED for 3 days (17β-ED), or were subjected to electroporation in the presence of a SCD1-targeting siRNA (17β-ED + SCD-1 siRNA) or a non-silencing duplex control (17β-ED + NS) and then incubated in starvation medium containing 2nM 17β-ED for 3 days. (c) Cellular proteins were separated by SDS-PAGE and immunoblot analysis of SCD-1 expression was performed using actin as loading control. The graphs show densitometry quantification of the SCD-1 blots. (d) RNA was extracted from cells and reverse transcribed into cDNA. Relative qPCR was performed using HPRT as reference gene. (e) Cellular lipids were extracted, hydrolyzed, transmethylated, and quantified by GC/FID and the indicated (MUFA/SFA) ratios were calculated. (f) Cells subjected to the indicated treatments were counted using a haemocytometer. Immunoblots are representative of 3 independent experiments. Data in (a, b, d-f) are means ± SEM n = 4. Data in (c) are means ± SEM n = 3. Values with a different superscript are significantly different (p < 0.05) as determined by one-way ANOVA test with subsequent Tukey’s adjustment