Figure 4: Fsp27–Plin1 interaction is important for giant LD formation in adipocytes.

(a) Western blot showing knockdown of endogenous Plin1 by lentivirus-mediated siPlin1 but not scramble sequence (siControl) and the expression levels of siRNA-resistant HA-tagged Plin1 and Plin1ΔAM in 10-day differentiated 3T3-L1 adipocytes. β-tubulin and FABP were used as a loading control and adipocyte differentiation efficiency control, respectively. (b) Basal and induced lipolysis rates of differentiated adipocytes in a. Mean±s.d. were obtained from three independent experiments. Iso, isoproterenol; IBMX, 3-isobutyl-1-methylxanthine (one-way ANOVA Tukey test; *P<0.05, **P<0.01, ***P<0.001; NS, no significant difference). (c) Relative TAG levels in differentiated adipocytes in a, measured by serum TAG determination kit. Mean±s.d. were obtained from three independent experiments (one-way ANOVA Tukey test; ***P<0.001; NS, no significant difference). (d) The diameters of the largest LDs were measured from at least 100 differentiated adipocytes in a in each group and the log2 (maximal LD diameter) values were shown as box-and-whisker diagram (one-way ANOVA Tukey test; **P<0.01, ***P<0.001; NS, no significant difference). (e) Plin1 controls LD size in adipocytes. Representative 3D reconstructed images of differentiated adipocytes in a and Fsp27 knockdown adipocytes (siFsp27) shown in depth-code colour (colour spectrum was shown on the bottom of the image). Lower panel, the LD diameter distribution histogram for each type of adipocytes. Data were collected from 10–15 GFP-positive cells in each group.