Topro 3

Apoptotic cells were identified using an Annexin V-Cy3 kit according to the manufacturer’s instructions (BioVision, Mountain View, CA). Briefly, live cells were incubated in binding buffer supplied with the kit, along with Annexin V-Cy3 (1:100), propridium iodide (TOPRO-3, 1:1000) and Hoechst 33342 (1:2000, Molecular Probes). Samples were maintained in a heating block set to 37°C during analysis, and cells undergoing early (Annexin V⁺/TOPRO-3^-) or late (Annexin V⁺/TOPRO-3⁺) apoptosis were compared with the total number of cells (identified with Hoechst dye). In overexpression experiments where GFP could be used as a marker, only GFP⁺ cells were included in the analysis. Hoechst dye, GFP, Annexin V-Cy3 and TOPRO-3 were visualized using a Zeiss LSM 510 META confocal microscope, with excitation lasers set to 405 nm, 488 nm, 543 nm and 633 nm, respectively.

Human fibroblasts were cultured in 24‐well chambers on poly‐L‐lysine‐coated coverslips overnight prior to treatments. Cells were treated with 30 μM agonist in DMSO (to a final DMSO concentration of 0.3%) for 48 h, and the filipin staining was initiated: Cells were washed twice with ice‐cold PBS, and fixed in 4% PFA for 30 min. Fixed cells were again washed with cold PBS, and unesterified cholesterol was visualized by filipin staining (PBS with 0.05 mg/mL filipin, Sigma‐Aldrich, and 10% FBS) for 2 h at room temperature in a dark humid chamber. Cells were subsequently washed with ice‐cold PBS twice, and nuclei stained using TO‐PRO‐3 (1:500, Invitrogen). Cells were washed twice and mounted on microscope slides overnight for imaging. Images were captured using a Zeiss Confocal Microscope (LSM 880), using a 40X oil objective, at 405 nm (filipin), 560 nm (mCherry), and 633 nm (TO‐PRO‐3). For data quantification, we calculated average filipin intensity per cell using Harmony High‐Content Imaging and Analysis Software (PerkinElmer).