Fig. 4. Effects of ORP3 gene silencing on the actin cytoskeleton and HEK293 cell morphology. (A) Western blot analysis of ORP3 protein in HEK293 cells (NT) and cells treated with siORP3 or siC1 siRNAs twice over a 3-day period. (B) HEK293 cells transfected with the indicated siRNAs, or cotransfected with siORP3 and a resistant mutant ORP3 cDNA (ORP3mut), were trypsinized, replated on fibronectin-coated coverslips and allowed to spread for 20, 40, 60 and 120 minutes (as indicated) in the presence of normal culture medium. The F-actin was stained using OG488P. The insets in the siORP3 ORP3mut specimens, stained with anti-ORP3, verify expression of the ORP3 mutant cDNA in the imaged cells. (C) siORP3-transfected cells at the 120 minute time point. The arrow indicates a cell that expresses ORP3 protein at a higher level than the neighbouring cells. (D) R-Ras(38V)-transfected cell at the 120 minute time point of a spreading experiment. Scale bars: 20 µm.