Figure 3

Increase in LD size upon LPCAT1/2 knock-down is independent of neutral lipid synthesis and accumulation. A) A431 cells were transfected with siRNA as described in Figure 1A. Seventy hours after siRNA transfection growth medium was exchanged to medium containing 10% delipidated FCS and 50 μM alkyne-oleate. After two hours cells were washed and lipids extracted. Extracts were subjected to quantitative click-analysis for the ratio of incorporation of alkyne fatty acid into TAG and PC. Data are mean ± StdDev, n = 3. Significances were calculated by unpaired two-sided T-test analysis relative to non-targeting siRNA (scrambled#5 + #6) and were found to be insignificant. B) A431 cells were transfected as described in Figure 1A. After 48 h, total lipid extracts were analyzed by mass spectrometry in duplicate samples, and species abundances were normalized to the corresponding internal standard. The molar contents of each species of the same class were summed up and normalized to the total content of all detectable lipids. C) HuH7 cells were transfected with siRNA as described in Figure 2A. 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, lipids extracted and extracts were subjected to quantitative click-analysis as in panel A. Data are mean ± StdDev, n = 3. Significances were calculated compared to non-targeting siRNAs (scrambled#5) and were found to be insignificant. D) HuH7 cells were transfected as described in Figure 2A. After 72 h, total lipid extracts were analyzed by mass spectrometry in duplicate samples. Species abundances were normalized as described for panel B.