Figure 7: Plin1 restores the activity of CIDE-N dimerization-defective mutant.
From: Perilipin1 promotes unilocular lipid droplet formation through the activation of Fsp27 in adipocytes
(a) Representative images showing that Plin1 can restore the activity of QQN-Fsp27 in promoting LD enlargement in 3T3-L1 preadipocytes. Full-length CIDE-N homodimerization-defective Fsp27 (pCDNA-QQN-Fsp27) was co-expressed with HA-tagged Plin1, Plin1(233–405) or Plin1ΔAM in 3T3-L1 preadipocytes. Fsp27 was stained with antibody against Fsp27 (red) and LDs are stained with Bodipy 493/503 (green). Scale bars, 5 μm. (b) Plin1 restores the activity of QQN-Fsp27 in 3T3-L1 preadipocytes in promoting large LD formation. Large LD is defined as the LD with a diameter ≥2.5 μm in (a). One-way ANOVA Tukey test, ***P<0.001 and NS (no significant difference) represents statistical analysis between QQN-Fsp27 alone and its co-expression with Plin1 or Plin1 truncations. (c,d) No neutral lipid diffusion among LD clusters in 3T3-L1 preadipocytes expressing QQN-Fsp27-GFP. Neutral lipid signal (red) in the white circle was bleached (c), scale bars, 2 μm. (d) MOI in bleached region was plotted as the percentage of the initial intensity from four independent experiments (mean±s.d., lower panel). (e,f) Plin1 restores the lipid exchange activity of QQN-Fsp27 when co-expressed in 3T3-L1 preadipocytes. (e) Neutral lipid signal (red) in one LD was bleached (white circle). Scale bars, 2 μm. (f) MOI in bleached region (white circle) and unbleached region (blue circle) were plotted as the percentage of the initial intensity from four independent experiments (mean±s.d., lower panel). (g) Western blot showing similar QQN-Fsp27-GFP expression in the absence or presence of HA-Plin1. GAPDH was used as a loading control. (h) Representative time-lapse images showing that QQN-Fsp27-GFP cannot initiate lipid transfer. pEGFP-N1-QQN-Fsp27 was co-transfected with pmCherry-N1 into 3T3-L1 preadipocytes. Scale bars, 4 μm. (i,j) Representative time-lapse images showing that Plin1 but not Plin1ΔAM can restore the activity of QQN-Fsp27 in promoting lipid transfer when co-expressed in 3T3-L1 preadipocytes. Scale bars, 4 μm. (k) Western blot showing similar QQN-Fsp27-GFP expression and comparable protein levels of Plin1-Cherry and Plin1ΔAM-Cherry in h–j.
