Figure 2

Silencing of LPCAT1 by siRNA leads to enlarged lipid droplets and reduced lipoprotein secretion in HuH7 cells. A) HuH7 cells were either left untreated (untreated, wt), mock transfected (mock), transfected with control siRNA (scrambled#5 or scrambled#6 as non-targeting siRNAs) or the two different siRNA sequences against LPCAT1 as indicated. After 72 h cells were subjected to a LPCAT activity assay or Western blotting (WB) for LPCAT1 using GAPDH, as load control. B) Confocal images of controls and LPCAT1 knock-downs as described in panel A. Nuclei (blue), LDs (green), scale bar = 10 μm C + D) Confocal images as described in panel B were quantified for LD size distribution as described in Methods. Data represented 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 siRNA (scrambled#5) (*** p ≤ 0.001). For analysis of LD size distribution (panel D), LDs were grouped into size classes, and the distribution displayed as percentage of total LDs per size class. Controls (black, light and dark grey and white), siRNAs against LPCAT1 (red, blue). E) Confocal images as described in panel B were quantified with Image J. Data show mean lipid droplet number per frame, corrected for variations in cell density, calculated from >50 individual cells in 3 independent experiments. Control: average of scrambled#5 and scrambled#6, LPCAT siRNA: average of both siRNA treatments. Significance was calculated by unpaired two-sided T-test analysis (*** p ≤ 0.001). F) HuH7 cells were transfected with non-targeting siRNA (control) or different siRNA sequences targeting LPCAT1 as indicated. ApoB secretion was measured by ELISA (dark grey, data represent mean ± StdDev, n = 4) or by Western blotting (light grey, data represent mean ± StdDev, n = 5). [3H]lipid secretion after labeling with 1 μCi [3H]oleate was calculated as % of total radioactivity recovered (supernatant + cells) and normalized to control. Data represent mean ± StdDev, n = 4. p-Values were obtained by unpaired T-test relative to the respective control (** p ≤ 0.01, * p ≤ 0.05).