Figure 6—figure supplement 2—source data 1

(A) HepG2 cells were treated with 10 μM MG132 or 5 μM lactacystin for 24 hr (without oleic acid), and stained with BODIPY-C₁₂ 558/568 for 1 hr. Cells were fixed and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 1 μm in the inset. The ratio of APOB- or ADRP-positive neutral lipid particles was quantified. Solid bars indicate median, boxes the interquartile range (25th to 75th percentile), and whiskers 1.5 times the interquartile range. The outliers are plotted individually. Differences were determined by one-way ANOVA with Dunnett test (n ≥ 39 cells). (B and C) HepG2 cells were treated with 2 ng/ml tunicamycin, 100 nM thapsigargin (B), or 10 μM MTTP inhibitor (CP-346086) (C) for 24 hr, stained with BODIPY-C₁₂ 558/568 for 1 hr, fixed, and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 1 μm in the inset. (D) HepG2 cells were treated with siRNA oligonucleotides against luciferase (Luc) or FITM2. The relative expression level of FITM2 was quantified by real-time PCR using ACTB as an internal control. Data represent the mean ± standard error of the mean performed in triplicate. (E) HepG2 cells were treated as in (D), cultured in regular medium in the presence of 200 nM oleic acid for 24 hr, stained with BODIPY-C₁₂ 558/568 for 1 hr, fixed, and stained with anti-APOB and anti-ADRP antibodies. Scale bars, 10 μm and 1 μm in the inset. Total pixel area of neutral lipids per cell was quantified using ImageJ software. Solid bars indicate median, boxes the interquartile range (25th to 75th percentile), and whiskers 1.5 times the interquartile range. The outliers are plotted individually. Differences were determined by two-tailed Welch’s t-test (**, p<0.01; n ≥ 35 cells).

10.7554/eLife.48834.019Figure 6—figure supplement 2—source data 1.