Figure 7. Characterisation of the consequences of the PLIN1 439fs mutation in pre-adipocytes co-expressing wild type perilipin-1.

3T3-L1 pre-adipocytes stably expressing pLXSN-Flag-PLIN1-WT (wild type) were infected with pBABEpuro EV or Myc-PLIN1 WT, PLIN1 439fs and PLIN1 398fs mutants as described in materials and methods. A) Immunoblot analysis of perilipin-1 expression using an anti-Myc or anit-Flag antibody. β-actin was assessed as a loading control. B) mRNA levels of total hPLIN1 normalized to mCyclophilinA expression. C) Stable cell lines were treated with 400 μM oleic acid for 48 hours, fixed and stained with anti-Myc and anti-mouse-Alexa488 for perilipin-1 (green), LipidTox DeepRed for lipid droplets (red) and DAPI for nuclei (blue); scale bar 10 μm. D) Lipid droplet (LD) volume was measured by taking the average of 10 Z-stacks at 63× magnification with 2-3 cells per focal plane, followed by analysis using Volosity 5 software. Results from three independent experiments are expressed as mean±SEM. E) Stable cell lines were loaded with ¹⁴C oleic acid for 16 hours in order to label the intracellular triglyceride pool. Release of incorporated radioactivity was measured over 4 hours in the presence of 6 μM Triascin C to prevent fatty acid re-esterifiation. Lipolysis is expressed as the percentage of released radioactivity in the media over the total radioactivity incorporated. Results from three independent experiments are expressed as mean as mean±SEM. In D and E, post hoc pairwise comparisons are shown versus EV, and between pairs, indicated with an overlying bar. * P<0.05; **P<0.01; *** P<0.001.