Cholesterol-dependent ORP1L-mediated contacts between LEs and the ER. (A) ΔORD recruits endogenous VAP-A. Mel JuSo cells expressing mRFP-ΔORD, -ORP1L, or -ΔORDPHDPHD were stained for VAP-A. The merge panels show an overlay of the two channels, and the boxes indicate the zoomed-in areas. Pixel analyses of the zoomed-in areas for fluorescence distribution of the mRFP-ORP1L variants and VAP-A are shown. n > 100. (B) Mel JuSo cells expressing TAP1-GFP (red) were exposed to different cholesterol manipulating conditions, as indicated. Cells were stained with antibodies for CD63 (green). Colocalization of the markers was determined by computational pixel analysis and visualized in blue. (right) Quantification of the colocalizing surface of the CD63-positive area. The mean ± SEM for >20 cells analyzed per condition is shown (*, P = 0.068; **, P = 1.55 × 10−6; ***, P = 0.0003). Bars: (A) 10 µm; (B) 5 µm.
