Fig. 2. Stomatin over-expression promoted LD growth and fusion in adipocyte-like cells.
a Mouse 3T3-L1 cells over-expressing human stomatin conjugated with RFP (hSTOM-RFP), or RFP as a control, were induced to differentiate. Lipid contents of the resulting adipocyte-like cells were stained and quantified. Mean ± s.d. for four experiments. Bar = 50 µm. b The protein of hSTOM, PPARγ, C/EBPα, and perilipin were examined by Western blotting. c The numbers of LDs were counted and plotted as a function of LD sizes by the histogram. Large LDs (>80 µm2) were more frequently found in hSTOM-RFP cells than control cells which contained mostly small (<40 µm2) LDs. Mean ± s.e.m. for three experiments. *P < 0.05 by two-sided paired t-test. Bar = 100 µm. d The time-lapse recording of a representative LD-LD fusion event (black arrow) when a small LD (circled green) fused with a large LD (circled red). Image tiles in hr:min. Bar = 10 µm. e FRAP experiments were done on cells transfected with hSTOM-RFP or RFP. After pre-treating cells with Bodipy-FL-C12, fluorescence of a LD was photobleached (dashed box). Recovery of fluorescence, quantified as % of original intensity, was recorded at a 30-s interval. Mean ± s.e.m of LD. for three experiments. Bar = 10 µm. f In vitro assay for LD fusions. LDs isolated from adipocyte-like cells were pre-treated with either Bodipy-FL-C16 or Bodipy-558/568-C12. Two LD suspension individually labeled red and green, were mixed in vitro in the presence of conditioned-medium (CM) prepared from 293 T cells over-expressing hSTOM-Flag (hSTOM), or empty control (Ctl). Excreted stomatin in CM was depleted by anti-hSTOM antibodies. g Most isolated LDs exhibited green- or red-fluorescence (arrowheads) alone; some LDs, however, contained both (arrows) as a result of LD fusions. Stomatin-containing CM increased LD fusion, while depletion of stomatin (hSTOM+Ab) inhibited fusion. Data represent six experiments. *P < 0.05 by two-sided paired student t-test. Source data are provided as a Source data file.