Fig. 2.

UBXD8-mediated recruitment of p97/VCP to LDs inhibits LD turnover. (A and B) HeLa cells transfected with the indicated constructs were analyzed by immunofluorescence microscopy and BODIPY 493/503 staining (A), and the size and number of LDs were quantified (B). Cells are outlined in black (untransfected) or red (transfected). (C) UBAC2 depletion increases total LD area under basal conditions. LD content in HeLa cells expressing control or UBAC2 shRNA was analyzed by BODIPY 493/503 staining, and the area per cell was quantified. (D) UBXD8 recruits p97/VCP to LDs via its UBX domain. The distribution of endogenous p97/VCP (red) was analyzed in the presence of the indicated UBXD8-S constructs (green) in HeLa cells. Nuclei were stained with DAPI (blue). (E) p97/VCP ATPase function is required for LD homeostasis. U2OS cells expressing inducible p97/VCP(WT) or p97/VCP(EQ) were incubated in the presence or absence of 200 µM oleate and doxycycline, BODIPY 493/503-stained LDs were analyzed by immunofluorescence microscopy, and the area per cell was quantified. Representative images are shown in Fig. S4B. All graphical data are quantified as mean ± SEM. An asterisk indicates a significant difference (P < 0.05, t test) from the control based on n = 500–600 droplets from each of three independent biological replicates. In the micrographs, white boxes indicate the magnified regions. (Scale bars: 10 μm.)