Silencing of LPCAT1 and LPCAT2 by siRNA leads to enlarged lipid droplets in A431 cells. A) A431 cells were either left untreated (untreated, wt), mock transfected (mock), transfected with control siRNA (eg5 as transfection control, leads to cell death, or scrambled#5 + #6 as non-targeting siRNAs) or the four possible combinations of two sequences each against LPCAT1 or LPCAT2 as indicated. After 48 h cells were lysed and subjected to SDS-PAGE/Western blotting for LPCAT1, LPCAT2 and glycerol-3-phosphate dehydrogenase (GAPDH, as a load control). B) Confocal images of controls and LPCAT1/LPCAT2 double-knock-downs as described in panel A. Nuclei (blue), LDs (green), scalebar = 10 μm. C) Confocal images as described in panel B were quantified with Image J for LD size distribution as described in Methods. Data are mean LD size ± StdDev, calculated from >50 individual cells in 3 independent experiments. Significances relative to non-targeting siRNAs were calculated by unpaired two-sided T-test analysis (*** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). D) Confocal image as described in panel B were quantified with ImageJ. Data show mean lipid droplet number per frame, corrected for variations in cell density, calculated from >50 individual cells in 3 independent experiments. Control: scrambled#5 + #6, LPCAT siRNA: average of all siRNA treatments. Significance was calculated by unpaired two-sided T-test analysis (*** p ≤ 0.001).