Pre-LDs are restricted and stable ER microdomains. (A and B) Starved cells cotransfected with OFP-HPos and GFP-HNeu (A) or OFP-HPos and GFP-HydER3 (an HPos peptide with the positive sequence of ALDI; B) were fixed and analyzed by confocal microscopy. Bars: (left) 25 µm; (right) 2 µm; (insets) 0.5 µm. (C) Starved GFP-HPos–transfected cells were fixed, and distribution of endogenous Plin3 was analyzed with a specific antibody (red). Red arrows indicate the pre-LDs selected for the high-magnification panel. Bars: (main panels) 2 µm; (insets) 0.5 µm. (D) Starved GFP-HPos–transfected cells were photobleached in tubules (black circles) or in pre-LDs (red circles). Fluorescence recovery was quantified. (E and F) Starved cells (stv) were treated for 10 min (E) or 24 h (F) with OA and then submitted to a second starvation for an additional 16 h (16 h stv). Neutral lipids were labeled with Nile red and quantified by flow cytometry. (G–I) Cells treated as in E were stained with Nile red and analyzed by microscopy. Bars: (top) 25 µm; (bottom) 2 µm. (J) Cells submitted to the second starvation period as in E (16 h stv) were fixed and labeled with Nile red (red) and ACSL3 antibody (green). Bars (main panel) 25 µm; (insets) 0.5 µm. Error bars indicate the standard deviation of five (D) or three (E and F) independent experiments.