Ethidium homodimer ethd 1

N-Isopropylacrylamide (NiPAAm) (Tokyo Chemical Industry [TCI], Tokyo, Japan), hexane (Sigma-Aldrich), trimethylolpropane triacrylate (TMPTA) (Sigma-Aldrich), dimethylaminoethyl acrylate (DMAEA) (Sigma-Aldrich, Taufkirchen, Germany), 4-acryloylmorpholine (AMO) (Sigma-Aldrich), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (BAPO or Irgacure819) (Sigma-Aldrich), absolute ethanol (EtOH) (Sigma-Aldrich), tetrahydrofurfuryl alcohol polyethylene glycol ether (glycofurol or GF) (Sigma-Aldrich), Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich), calcein-AM (Thermo Fisher Scientific, Waltham, MA, USA), ethidium homodimer (EthD-1) (Thermo Fisher Scientific), low-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Merk), penicillin streptomycin (pen/strep) (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and fetal bovine serum (FBS) (Sigma-Aldrich) were purchased and used as received. L929 fibroblasts and C2C12 myoblasts were purchased from CLS Cell Lines Service GmbH (Eppelheim, Germany).
The NiPAAm was purified before use. In brief, the NiPAAm was dissolved in dried warm hexane under constant magnetic stirring. The solution was placed at −20 °C to encourage recrystallization of the NiPAAm. The recrystallized NiPAAm was then separated with a cylindrical funnel and washed again with cold hexane and dried under reduced atmospheric pressure for 72 h.

Filipin dye was used to stain unesterified cholesterol in cells [18 (link)]. Cells were seeded at 2000 cells/well in 100 μl of media in black, clear bottom, tissue culture-treated 96-well plates and cultured for 24 h. The test compounds were dissolved in media and then added at 100 μl /well; the cells were returned to incubation for 4 days. The cells were washed twice with DPBS and fixed with 100 μl/well of 4% paraformaldehyde (PFA) solution at room temperature for 30 min. After washing twice with DPBS, the cells were stained with 50 ng/mL Filipin solution (freshly dissolved in DMSO at 10 mg/mL and then diluted in DPBS) at room temperature for 1 h. The plates were stored in 100 μl/well DPBS at 4 °C after washing two times with DPBS before imaging analysis. On the day of the imaging, cell nuclei were stained with 100 μl/well of 4 μM ethidium homodimer (EthD-1) (Thermo Fisher Scientific) in DPBS at room temperature for 30 min. The plates were imaged using the INCell 2200 imaging system with a 20X or 40X objective lens. A DAPI filter set (excitation = 350 ± 50 nm, and emission = 455 ± 50 nm) and Cy3 filter set (excitation = 543 ± 22 nm, and emission = 604 ± 64 nm) were used to visualize Filipin and EthD-1 staining, respectively.