Methyl β cyclodextrin cholesterol complex

Cells (~7.5 ×10⁴) were seeded in wells of 24-well plates and maintained in culture for 30 h to reach confluence. Cells were then incubated for 24 h in serum-free DMEM/F-12 to induce cilia formation. Followed by 24 h treatment with 50 nM of Shh-N (R&D Systems) diluted in serum-free DMEM/F-12. For cholesterol supplementation, parallel RPE1 cell cultures, after cilia induction, were treated for 45 min with 1.5% methyl-β-cyclodextrin (MβCD, Sigma) in DMEM/F-12 to deplete cellular cholesterol. After extensive washing, cholesterol was delivered by incubating cells for 1 h with 100 μM of water-soluble methyl-β-cyclodextrin–cholesterol complex (Sigma-Aldrich) in DMEM/F-12. For LDL cholesterol complementation, cells were supplemented 0.034 mg/mL of LDL (MilliporeSigma) in DMEM/F-12, corresponding approximately to the amount of cholesterol supplemented with water-soluble methyl-β-cyclodextrin–cholesterol complex. Cholesterol depletion and supplementation assays were performed in the presence of both 40 μM pravastatin (Sigma-Aldrich) and 50 nM of Shh-N for 24 h. All samples were then processed for immunofluorescence to detect ciliary membrane components SMO and Arl13b.

To remove intracellular cholesterol, confluent cells were incubated in serum‐free DMEM for 24 h, after which they were treated for 45 min with 1.5% methyl‐β‐cyclodextrin (Sigma‐Aldrich) in DMEM. All subsequent incubations were performed in DMEM containing 40 μM pravastatin to attenuate cholesterol biosynthesis, with or without the indicated additives. For cholesterol complementation assays, cholesterol was delivered by incubating the cells for 1 h with 50 μM or 100 μM water‐soluble methyl‐β‐cyclodextrin–cholesterol complex (Sigma‐Aldrich) in DMEM supplemented with 40 μM pravastatin. Alternatively, cholesterol was compensated for by incubating the cells with 0.06 mg/ml LDL (Sigma‐Aldrich) in DMEM supplemented with 40 μM pravastatin and the desired agents. The cells were then treated for 24 h with the desired agents and subsequently processed for immunofluorescence analyses.