Figure 4. PLIN3 recruitment to LDs during nutrient starvation is dependent on RalA.

(A) Representative images of PLIN2 and LDs in MEFs during 0 h and 4 h of HBSS starvation. PLIN2 in magenta (antibody), LDs in green (Bodipy 493/503), and nuclei in blue (DAPI). Scale bar, 20 μm.

(B) Relative colocalization of PLIN2 and LDs during 0 h and 4 h of HBSS starvation performed by Pearson’s correlation coefficient. Each point on the graph represents a Pearson’s coefficient value for one cell. At least 25 cells were analyzed per time point. n = 2. mean ± SEM.

(C) Representative images of PLIN3 and LDs in MEFs during 0 h and 4 h of HBSS starvation. PLIN3 in magenta (antibody), LDs in green (Bodipy 493/503), and nuclei in blue (DAPI). Scale bar, 20 μm.

(D) Relative colocalization of Perilipin 3 and LDs during 0 h and 4 h of HBSS starvation performed by Pearson’s correlation coefficient. Each point on the graph represents a Pearson’s coefficient value for one cell. At least 75 cells were analyzed per time point. n = 3. mean ± SEM. ***p < 0.001.

(E) Western blot analysis of PLIN3 protein levels in MEFs incubated in HBSS media for 0 or 4 h.

(F) Western blot confirming knockdown of PLIN3 protein expression in MEFs.

(G) Representative images of PLIN3 (magenta) and LDs (green) in WT and RalA KO MEFs expressing either shScramble or shPLIN3 constructs during 0 h and 4 h of HBSS starvation. Scale bar, 20 μm.

(H) Quantification of LD area per cell from (G). At least 75 cells were analyzed for each time point. n = 3. mean ± SEM. **p < 0.01.

See also Figure S4.