Figure 4

Inhibition of de-novo PC synthesis results in increased LD size and TAG synthesis. A431 cells were left untreated (untreated), treated with transfection reagent (mock) or transfected with a non-targeting stealth siRNAs (non-targ.) or three different stealth siRNAs against CT alpha (siRNA1-siRNA3) for 72 h. A) Representative confocal images, stained with DAPI (blue) and LD540 (orange). Bar, 10 μm. B) Images were analyzed for LD size as described in Methods. Data are mean LD size ± StdDev, calculated from > 50 individual cells in 3 independent experiments. Significances were calculated by unpaired two-sided T-test analysis relative to non-targeting control (non-targ.) (*** p ≤ 0.001, ** p ≤ 0.01). C) Amount of CT alpha mRNA left after CTalpha knock-down relative to non targeting control, measured by real-time PCR. Standard deviations and significances were calculated by unpaired T-test analysis from 3 different experiments (*** p ≤ 0.001). D) Seventy hours after siRNA transfection as indicated, growth medium was exchanged to medium containing 10% delipidated FCS and 50 μM alkyne-oleate. After two hours cells were washed and lipids extracted and analyzed for alkyne labeled TAG and PC. Data are mean ± StdDev, n = 3. Significances were calculated by unpaired two-sided T-test analysis relative to non-targeting control (non-targ.) (*** p ≤ 0.001, * p ≤ 0.05). E) A431 cells were transfected as described for panel A. After 72 h, total lipid extracts were analyzed by mass spectrometry in triplicate samples. Species abundances were normalized to the corresponding internal standard. Species of each class were summed up and normalized to total detectable lipids. Significances for TAG, CE and PC O- were calculated by unpaired two-sided T-test analysis relative to non-targeting control (### p ≤ 0.001, # p ≤ 0.05) and relative to mock transfection (*** p ≤ 0.001).