D3922

To visualize the effect of 1,25(OH)₂D on neutral lipid accumulation, MCF10C1a cells were plated onto 8 well Permanox^® chamber slides (Sigma-Aldrich, ST. Louis, MO) and treated for 5 days with either vehicle or 1,25(OH)₂D. After 5 days of treatment, cells were fixed using 10% neutral buffered formalin for 15 minutes at room temperature. Neutral lipid droplets were next stained with BODIPY® 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene), a fluorescent dye with specificity for neutral lipids (D-3922, ThermoFisher Scientific, Waltham, MA) at a concentration of 10µg/ml in PBS containing 1 % BSA, 0.1 % saponin, and 0.02 % Sodium azide for one hour. A DNA stain was further applied as a nuclear counterstain (DAPI (4′,6-diamidino-2-phenylindole), D21490, ThermoFisher Scientific, Waltham, MA). Imaging was performed using an A1 RMP multiphoton confocal microscope and then analyzed using FIJI, an ImageJ based image processing software available under the General Public License (fiji.asc). For quantification, cell images were taken 1µm apart across the whole cell and stacked onto one image. BODIPY signal per each stacked image was then quantified using FIJI and normalized to the number of stained nuclei in each image.

Cells were stained with LipidTOX kit or BODIPY staining following the manufacturer’s protocol (Thermo Fisher #H34475 or #D3922, respectively). Briefly, after indicated treatment, NRVCs grown on coverslips were fixed in 4% paraformaldehyde at room temperature for 10 min and subjected to LipidTOX labeling (1: 1000 in PBS, RT for 30 min) or BODIPY labeling (1: 5000 in PBS, RT for 30 min). The cells were counterstained with cardiac Troponin-T (TnT)/Phalloidin (Thermo Fisher) and DAPI (diamidino-2-phenylindole, Thermo Fisher) to label cardiomyocytes and nuclei, respectively. Confocal images were quantified by ImageJ. LipidTOX/BODIPY intensity was normalized with DAPI intensity. Eight views/sample, 3 samples/group were quantified.