Figure 1. Rab7 localizes to lipid droplets (LDs) and is activated upon starvation.

(a) LDs were purified from HuH7 cells under resting and starved conditions and the purity of the isolated fractions was tested using specific antibodies to various compartments. Note that Rab7 associates with the purified LDs under both conditions. (b) Hep3B cells expressing GFP-Rab7-wt, or the GTPase active (-Q67L) or inactive (-T22N) forms were loaded with 150 μM oleate overnight (“resting”, upper rows) and then starved for 24h in medium containing 0.1% FBS (“starved”, lower rows). LDs were visualized using Oil Red O (ORO). While no obvious changes in the Rab7 localization to LDs are observed upon starvation, cells expressing wt or active forms exhibit a marked association with LDs while the Rab7-T22N mutant does not. Scale bars represent 1 μm. (c) Rab7 activity increases 3 to 4 fold in Hep3B hepatoma cells, as assessed by a RILP pulldown assay. Cells were exposed to overnight loading with oleate followed by a 24h low serum starvation. (d) Quantitation of at least three independent experiments as described in (c). Data are presented as mean ± S.E., p value (**) = 0.001. (e) Activation of Rab7 in HuH7 cells exposed to 2h HBSS or 24h low serum starvation, as assessed by a RILP pulldown assay. (f) Quantitation of at least three independent experiments as described for (e). Data are presented as mean ± S.E., p values (*) ≤ 0.03. (g) A subpopulation of LD-associated Rab7 is activated upon 2h HBSS starvation in HuH7 cells. Cells were starved and the isolated LDs were subjected to a RILP pulldown assay. (h) Quantitation of three independent experiments as described in (g) reveals a 3-fold activation of Rab7 on LDs. Data are presented as mean ± S.E., p value (*) = 0.02. In all cases, total Rab7 was normalized to the actin loading control and active Rab7 was then normalized to the corrected total Rab7.