Figure 3

Effects of ω 3PUFAs on HMGCoAR transcription, phosphorylation and ubiquitination in colon cancer cells. HT29 and HT29-dx cells were incubated for 24 h in the absence (CTRL) or in the presence of 50 μM arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA). (A) Total RNA was extracted, reverse-transcribed and subjected to qRT-PCR for HMGCoAR gene. Measurements were performed in triplicate and data are presented as means ± SD (n = 3). Versus CTRL HT29: * p < 0.005. (B) Cells were subjected to ultracentrifugation to isolate the microsomal fraction. Left panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-phosphoserine (pSer) antibody, anti-ubiquitin antibody or anti-HMGCoAR antibody, to detect the amount of the immunoprecipitated (IP) enzyme. No Ab: extracts in the absence of the anti-HMGCoAR antibody, a condition used as internal control. Right panel: extracts of microsomal fraction were immunoprecipitated with an anti-HMGCoAR antibody, then probed with anti-ubiquitin antibody. As a control of proteasome involvement in HMGCoAR degradation, the proteasome inhibitor MG-132 (10 μM, MG) was added for 16 h, alone or during the last 16 h of the incubation with DHA. The figures are representative of three experiments with similar results. The 95 kDa band corresponding to native HMGCoAR protein is indicated by the arrow.