Figure 5

LD size is unaffected by knock-down of LPCAT3 and LPCAT4. A) A431 cells were either left untreated, mock transfected (mock) or transfected with a non-targeting stealth siRNAs (non-targ.) or with the two different stealth siRNA sequences against LPCAT3 (siRNA3-1, siRNA3-2) or LPCAT4 (siRNA4-1, siRNA4-2) or combinations thereof as indicated. After 72 h incubation cells were fixed, stained with DAPI and LD540 and imaged. LD size was quantified with ImageJ. Standard deviations and significances were calculated from 3 experiments by unpaired T-test analysis compared to non-targeting control and were found to be insignificant. B) mRNA was measured by real-time PCR after single or double knock-down of LPCAT3 and LPCAT4 as described for panel A. Values are shown relative to the non-targeting control. Standard deviations and significances were calculated as above (*** p ≤ 0.001, ** p ≤0.01). C) A431 cells were either transfected with non-targeting stealth siRNA or with stealth siRNA sequence against LPCAT3 (siRNA3-2) or LPCAT4 (siRNA4-1) or combination (siRNA3-2;4-1). 48 h after transfection 100 μM oleate was added to the medium to induce formation of LDs. 72 h after transfection cells were lysed and lipid droplets were purified using sucrose gradient and subjected to a LPCAT activity assay. Values are normalized to the amount of the LD protein NSDHL as determined by western blotting. Standard deviations and significances were calculated as above and were found to be insignificant. D) A431 cells were treated with stealth siRNA and supplemented with oleate as described for panel C. 72 h after transfection cells were lysed and lysate was subjected to a LPCAT activity assay using equal amounts of total protein. Standard deviations and significances were calculated from three experiments as above (*** p ≤ 0.001, * p ≤0.05) compared to non-targeting control.