Inhibition of CLTC levels by SiRNA affects LD biogenesis. After silencing CLTC expression, biogenesis was examined by Nile Red (C, D) quantification and Oil Red O semi-quantitative determination (E, F). Experiments were performed in MALME-3M cells (C and E) and in MCF-7 cells (D and F). Cells were transiently transfected with CONT (Control) SiRNA or with CLTC SiRNA for 72 hours prior to the addition of the vehicle 24 hours prior to cell lysation. Cell lysates were then separated in a 4-12% Bis-Tris gel and probed by western blotting with antibodies against CLTC and Actin. The experiment was repeated three times and a representative western blot is shown for MALME-3M cells (A) and for MCF-7 cells (B). For the neutral lipid determinations, after transfection, cells were treated with either the vehicle or petroselinic acid (100 μg/ml) for 24 hours prior to the testing. Nile Red (C, D) and Oil Red O and hematoxylin (E, F) staining were performed as described in Materials and Methods. Quantification was assessed using the FlowJo software. Fold induction was estimated by calculating the ratio between treated conditions (Petroselinic acid) and the untreated condition (vehicle). The average values from three independent analyses are shown. *P <0.05 compared to each control in C and D. Representative photos are shown in E and F. The length of the shown size bars is 20 μm.