(A) Schematic illustrating the final steps of post-squalene cholesterol synthesis. Structural alterations of sterol precursors relative to cholesterol are indicated in red. Small molecules used in this study are noted in blue.
(B) Sterol profiles of HEK293T hCLTAEN-mTq2 cells cultured in cholesterol-replete FBS or 7.5% delipidated (LPDS) serum ± various treatments (mean ± SD).
(C) FBS prevents AY9944-mediated CME trafficking defects. Scale bar, 10 μm.
(D) Transferrin (Tfn) uptake under sterol-depleted conditions (mean ± SD). One-way ANOVA (F(4,40) = 38.57, p < 0.0001) and Dunnett’s test versus LPDS control (n = 9 biological replicates from 3 independent experiments, 2,000–4,000 cells per replicate).
(E) Sterol depletion results in the accumulation of clathrin at the cell periphery and functional impairment of Tfn internalization in HEK293T hCLTAEN-mTq2 cells. Scale bar, 10 μm.
(F and G) Tfn uptake correlates with sterol content by GC-MS analysis (mean ± SD). n = 3 biological replicates for Tfn uptake and n = 2 biological replicates for sterol analysis performed in parallel.