Fig 1: Enhanced recruitment of Rab5 to lipid droplets (LDs) upon lipophagy stimulation. A, Western blot analysis of Rab5 and EEA1 GTP-pulldown in regular medium (CT) or 4 h HBSS starvation in Hep3B cells. B, quantification of the fold change of GTP-bound/total Rab5 as well as EEA1 from n = 4 independent experiments. C, quantification of total Rab5–β-actin confirming that starvation does not change overall Rab5 expression. D, Western blot analysis of LDs isolated from Hep3B cells by density gradient centrifugation showing abundant levels of Rab5 in isolated LDs following 4 h of HBSS starvation compared with the regular medium (CT). E, quantification of Rab5–Plin2 in the biochemically isolated LDs. F, fluorescence micrograph showing immunofluorescence staining of Hep3B cells expressing Myc-tagged Rab5 (green). Cells were treated with oleic acid for 16 h and then either maintained in regular medium (control) or subjected to 4 h of HBSS starvation before being fixed and stained with Oil Red O (ORO; red) to label LDs. Note the marked increase in Rab5 staining around LDs in HBSS-starved cells versus control (yellow arrowheads). Graphs represent mean ± SD from n = 3 to 4 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. CT, control; EEA1, early endosome antigen 1; HBSS, Hank’s balanced salt solution; Plin2, perilipin-2.