Blocking one hist

Cells were fixed in 4% paraformaldehyde (pH 7.4) for 10 min at room temperature and rinsed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by rinsing with PBS. Nonspecific binding was blocked with Blocking One HIST (Nacalai, Kyoto, Japan) for 10 min at room temperature. Cells were incubated with primary antibodies overnight at 4 °C, and then labeled with appropriate fluorescent-tagged secondary antibodies. DAPI (Thermo Fisher Scientific) was used to label nuclei. Acquisition of fluorescence images and quantification were performed using In Cell Analyzer 6500 (GE Healthcare). The following primary antibodies were used in this assay: SREBP2 (Abcam, Emeryville, CA, 1:50).

Activation of the mitochondria-dependent cell death pathway was assessed by detection of cytochrome c release from mitochondria. This was done by immunofluorescence analysis using antibodies against cytochrome c and ATP synthase subunit C, which serves as a mitochondrial marker. Briefly, NRVMs were exposed to 1% CSE in serum-free DMEM for 1 h. After PBS washing, cells were fixed with 4% paraformaldehyde (10°C, 10 min) and permeabilized with 0.2% Triton-X 100 (room temperature, 1 min). After blocking with Blocking One Hist (Nacalai Tesque, Kyoto, Japan) for 10 min, NRVMs were treated with primary antibodies (anti-cytochrome c: mouse, 1/200, ab110325, and anti-ATP synthase subunit c: rabbit, 1/200, ab181243, abcam, Cambridge, UK) and incubated overnight at 4 °C. Secondary antibodies (Anti-mouse IgG Alexa fluor 488: 1/200, and Anti-rabbit IgG Alexa fluor 594, 1/200, Invitrogen) were then added, and nuclei were further stained with Hoechst 33342 (Invitrogen). Fluorescence images were acquired using LSM900 confocal microscopy and ZEN3 imaging software (Carl Zwiss). Colocalization of cytochrome C and ATP synthase subunit C in the myocytes treated with 1% CSE and Tyrode were analyzed by Pearson’s correlation using Image J.