Dynamics of protein incorporation into emerging LDs. (A and B) Starved cells (top) cotransfected with OFP-HPos and tagged forms of DGAT2, Plin2, Plin3, and ACSL3 (green), were additionally treated with OA for 7.5, 15 (bottom), or 30 min and analyzed by microscopy. Arrows indicate the structures selected for the high-magnification panels. Bars: (main panels) 2 µm; (insets) 0.5 µm. (B) The percentage of HPos-positive pre- and emerging LDs that accumulated the cotransfected proteins. Error bars indicate the standard deviation of three independent experiments. (C) Starved OFP-HPos and GFP-Plin2 cotransfected cells were loaded with OA and followed for 15 min by video microscopy. After formation (t = 0), HPos-positive emerging LDs require a mean time of 4.5 ± 1.8 min to be recognized by Plin2 (blue and green arrows). Arrows mark a representative emerging LD, and the color of the arrow indicates the degree of colocalization between HPos and Plin2. Bar, 1 µm.