Cell culture
Atrial-derived HL-1 cardiomyocytes were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal bovine serum (FBS) and incubated in a humidified atmosphere containing 5% CO2 at 37°C. The cells were treated with DOX (Selleck, Shanghai, China) at indicated concentrations (5 µM) for 9 h.
LDH release assay
LDH release in cells or serum was assessed using LDH Assay Kit (Beyotime Biotechnology, China) according to the manufacturer's instructions[18].
Cell viability assay
HL1 cells were seeded into 96-well plates at a concentration of 5,000 cells/well. The next day, the cells were treated with DOX for 9 h. Cell viability was detected by the Cell Counting Kit-8 assay (CCK8, Bimake) according to the manufacturer’s instructions. We measured the Optical density (OD) values at 450 nm by an Infinite™ M200 Microplate reader (Tecan, Mannedorf, Switzerland).
Microscope imaging
To observe the morphology of pyroptotic cells, the cells were first incubated into a 6-well plate and then treated with DOX. Still bright-field images were taken with Nikon TE2000 microscope.
Western blot analysis
Protein from HL-1 cells were purified with RIPA Lysis Buffer System. In short, equal amounts (30 µg) of proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and blotted to Immobilon® PVDF Membranes (Merck KGaA, Darmstadt, Germany). Membranes were blocked in 5% non-fat milk for 1.5 h at room temperature and then incubated with the primary antibodies at 4°C overnight. After incubated with HRP-conjugated secondary antibodies (Goat anti-rabbit IgG, Proteintech, China) for 2 h at room temperature, the immune-complexes were visualized using the enhanced chemiluminescence (ECL) substrate (Cwbio, Beijing, China). The intensity of visualized protein bands was captured and analyzed by Image Lab™ software with tubulin as control for normalization (The antibodies manufacturers are shown in Table 1).
Cell transfection
The siRNA duplexes corresponding to HMGA1, SOX9 and negative control siRNA (Si-Ctrl) were purchased from RiboBio (Guangzhou, China). SOX9 expression vector, pcDNA3.1-SOX9 was constructed by cloning full-length wild-type SOX9 coding sequence into pcDNA3.1. HL-1 cardiomyocytes were transfected with siRNA or plasmids for 48 h with Lipofectamine 2000 reagent kit (Invitrogen, Carlsbad, CA). After 48 h, we treat the transfected cells with DOX.
Co-immunoprecipitation
HL-1 cells lysate was purified with ice-cold IP lysis buffer (Thermo Fisher Scientific, USA). The lysate was transferred into a microcentrifuge tube and centrifuged at 2500 rpm for 10 minutes. Then, we transferred the supernatant into a new microcentrifuge tube to determine the protein concentration and perform further analysis. Briefly, the protein A/G PLUS-Agarose (Santacruz Biotechnology, CA, USA), the target antibody and the lysate were mixed together and then incubated at 4°C overnight. Next day, we centrifuged the mixture at 2500 rpm for 10 minutes and washed the precipitated complex with phosphate buffer saline. Repeat this step at least five times. We used HMGA1 antibody as a bait antibody to capture SOX9 protein, and normal rabbit IgG (Cell Signaling Technology, USA) as a negative control. The control was processed in the same way as the Co-IP sample. Lysates from both control and DOX treated cells without immunoprecipitation were used as the positive control (input). After co-immunoprecipitation, the proteins pulled down by HMGA1 antibody were analyzed by Western blot.
Statistical analysis
The data were presented as means ± SD. Statistical analysis was performed by Graphpad Prism 6. The data were analyzed using one-way analysis of variance (ANOVA) and Student’s t-test. A value of P < 0.05 was considered to be statistically significant.