Figure 1: Identification of Plin1 as an adipocyte-specific activator of Fsp27.
From: Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes
(a) Upper panel: representative images showing that Plin1, but not ADRP/Plin2, can enhance Fsp27-mediated large LD formation; lower panel, neither Plin1 nor ADRP/Plin2 enhanced Cidea-mediated large LD formation. Empty vector, HA-tagged Plin1, or HA-tagged ADRP/Plin2 were co-expressed with Fsp27-GFP or Cidea-GFP in 3T3-L1 preadipocytes. HA-tagged proteins were stained with HA antibody (blue). LDs were labelled with BODYPI 558/568 C12 fatty acid (BODYPI-FA, red). DAPI (grey)-stained nuclei. Enlarged pictures for splitted channels were shown below and arrowheads point to the LDCSs. Scale bars, 5 μm. (b) Plin1 enhances Fsp27 but not Cidea’s activity in promoting large LD formation in 3T3-L1 preadipocytes. The diameters of the largest LDs in a were measured (mean±s.d.; n≥100 for each group; one-way ANOVA Tukey test, ***P<0.001; NS, no significant difference). (c) Western blot showing similar Fsp27 or Cidea expression levels when co-expressed with Plin1 or ADRP in panel a. GAPDH was used as a loading control. (d) LD size distribution in 3T3-L1 preadipocytes co-expressing Fsp27 and increasing amount of HA-Plin1. Data were collected from 30 GFP-positive cells in each group. The percentage of LDs with a diameter >6 μm increased, whereas the percentage of LDs with a diameter <2 μm decreased with increasing expression of Plin1. (e) Western blot showing protein expression levels in d. β-Actin was used as a loading control. (f) Western blot showing levels of Plin1 in 10-day differentiated 3T3-L1 adipocytes infected with lentivirus containing scramble siRNA (siControl) or siRNA specific for Plin1 (siPlin1). β-tubulin and FABP4 were used as a loading control and adipocyte differentiation efficiency control, respectively. (g,h) Representative repeated FRAP experiments showing lipid exchange between contacted LDs in 10-day differentiated siControl (g) or Plin1 knockdown (h) 3T3-L1 adipocytes. MOI in bleached (white circle) and unbleached (blue circle) areas were plotted as the percentage of the initial intensity (right panel). The starting points of photobleaching are indicated by black arrowheads. Scale bars, 5 μm. (i) Significantly lower lipid exchange rate (n=40, Mean±s.e.m., student t-test, ***P<0.001) in Plin1 knockdown 3T3-L1 adipocytes compared with that of control adipocytes.
