(D) Confocal images of live GRAMD1 TKO HeLa cells that stably expressed either EGFP control or EGFP-GRAMD1b constructs as indicated. Cells were stained with recombinant mCherry-D4H proteins (10 mg/ml) (accessible PM cholesterol biosensor) for 15 min at room temperature before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note that expression of EGFP-GRAMD1b-WT, but not EGFP-GRAMD1b-R189W, reduced binding of mCherry-D4H to the PM, in GRAMD1 TKO HeLa cells. Further reduction in mCherry-D4H binding to the PM was observed in GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-G187L compared to GRAMD1 TKO HeLa cells that stably expressed EGFP-GRAMD1b-WT. Scale bars, 10 µm. (E) Values of mCherry-D4H signals at the PM after background subtraction, as assessed by confocal microscopy and line scan analysis of GRAMD1 TKO HeLa cells that stably expressed either EGFP or indicated EGFP-GRAMD1b constructs as shown in (D) [mean ± SEM, n = 30 cells for all the conditions; data are pooled from three independent experiments; Dunnett's multiple comparisons test, **P < 0.0001]. 