LE cholesterol alters the conformation of ORP1L. (A) Intramolecular FRET for mRFP-ORP1L-GFP. GFP and mRFP are attached to the same molecule, allowing FRET from donor GFP to acceptor mRFP. FRET depends on distance and orientation and thus indicates conformational changes. FRET can be detected by sensitized emission. GFP is excited by 488-nm light, and then, after energy transfer, 582–675-nm light emission by mRFP is detected. (B) Cholesterol effects on ORP1L conformation detected by sensitized emission. Mel JuSo cells expressing mRFP-ORP1L-GFP were cultured under control (FCS) or cholesterol-depleting (statin) or -enhancing (U-18666A) conditions before imaging by CLSM for FRET determination. Panels show the GFP signal, the mRFP signal, calculated FRET, and FRET related to donor fluorophore input: the donor FRET efficiency (E_D). The color LUT visualizes the differences in the E_D panels. (right) quantification of the donor FRET efficiency detected for mRFP-ORP1L-GFP under the different conditions of cholesterol manipulation. The mean and SD from two experiments (>10 cells analyzed) are shown (*, P = 0.05; **, P = 0.03). (C) ORP1L controls LE positioning in NPC1-silenced cells. Mel JuSo cells were transfected with mRFP-ORP1L, -ΔORD, or -ΔORDPHDPHD and siRNA for NPC1 and analyzed by CLSM. n > 100. (D) Sensitized emission and ORP1L conformation in NPC1-deficient cells. mRFP-ORP1L-GFP–expressing MelJuSo cells were transfected with control (siCTRL) or NPC1 (siNPC1) siRNAs before imaging by confocal FRET. (right) Donor FRET efficiencies determined in >10 control siRNA– or NPC1 siRNA–transfected cells. The mean ± SD is shown (**, P = 5.1 × 10⁻⁶). (E) NPC1, cholesterol, and LE clustering. Mel JuSo cells were transfected with control or NPC1 siRNA and then mRFP-ΔORD before staining with filipin for cholesterol. Pixel analyses are shown in Fig. S2 E. n > 50 for each condition. Bars, 10 µm.