(D) Left: Time course of normalized mCherry signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells stably expressing either EGFP or EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor mCherry-GRAM1b. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 31 cells (EGFP), n = 28 cells (EGFP-GRAMD1b WT), n = 23 cells (EGFP-GRAMD1b R189W), n = 27 cells [EGFP-GRAMD1b R189W/R191A (RW/RA)]; data are pooled from two independent experiments for each condition; Dunnett's multiple comparisons test, **P < 0.0001 (EGFP vs. WT and WT vs. R189W), **P = 0.0002 (WT vs. RW/RA)]. 