4 6 diamidino 2 phenylindole dapi c1002

Human-induced pluripotent stem cell-derived cardiomyocytes were separated and placed in 6-well plates (Corning, New York, USA). The combined staining of α-actinin (ab137346, Abcam), immunoglobulin G (IgG) H&L (Alexa Fluor® 488) (ab150077, Abcam), and propidium iodide (PI) 1 μg/ml (ST511, Beyotime, Shanghai, China) was used to detect cardiomyocyte death. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime, Shanghai China) and the dead cells were labeled with PI to pass through the damaged cell membrane. A Nikon A1R HD25 confocal microscope was used to capture images. The total number of cells in the PI-positive and five randomly selected fields was counted using ImageJ software by a researcher blinded to the treatment assignments. A manual pipeline (CellProfiler, Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard in Cambridge) was used to determine the cell surface area (22 (link)). Briefly, 5 to 6 random pictures were taken with 20X magnification and 150–200 cells/per condition were analyzed in order to determine cell size following the instructions of the software.

Cells (2×10³) were resuspended in 200μl of serum-free medium and seeded on the top chamber of the 8.0μm pore, 6.5mm polycarbonate transwell filters (Corning). The full medium (600μl) containing 10% FBS was added to the bottom chamber. The cells were allowed to migrate for 24h at 37°C in a humidified incubator with 5% CO2. The cells attached to the lower surface of membrane were fixed in 4% paraformaldehyde at room temperature for 30mins and stained with 4,6-diamidino-2-phenylindole (DAPI) (C1002, Beyotime Inst Biotech, China), and the number of cells on the lower surface of the filters was counted under the microscope. A total of 5 fields were counted for each transwell filter.

Porcine kidney epithelial cells (LLC-PK1) used in this study were cultured in modified Eagle's medium (MEM, Life Technologies, 11095098) with 10% fetal bovine serum (FBS, Gibco, 10,099,141) at 37°C in a humidified atmosphere of 5% CO₂. Swine testis epithelial cells (ST) and African green monkey kidney epithelial cells (MARC-145) were grown in Dulbecco' Modified Eagle's Medium nutrient (DMEM, Sigma-Aldrich, D6429) with 10% FBS at 37°C with 5% CO₂. Anti-STAT1 antibody (14994) and anti-Phospho-STAT1 antibody (9167) were purchased from Cell Signaling Technology (CST). Anti-ISG15 antibody (ab285367) was purchased from Abcam. Anti-β-actin antibody (60,008–1), Horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (SA00001-1), and HRP-conjugated anti-rabbit IgG antibody (SA00001-2) were obtained from Proteintech Group. The monoclonal antibody (Mab) against PEDV N protein was made in our laboratory (49 (link)). 4' 6-diamidino-2-phenylindole (DAPI, C1002) was purchased from Beyotime. poly(I:C) LMW (low molecular weight) was obtained from In vivoGen.

When the cells transfected with mimic control/miR-30c-5p mimic/inhibitor control/miR-30c-5p inhibitor were 70–80% confluent, they were fixed with 4% paraformaldehyde. Antibodies against SMαA (1:100), SM22α (1:250), SMMHC (1:50), and h1-calponin (1:150) were incubated with the fixed cells overnight at 4 °C followed by incubation with the fluorescent-labeled secondary antibody (SA00013-2/SA00013-4, Proteintech, Rosemont, IL, USA) for 1 h. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI: C1002, Beyotime, China) for 5 min before being photographed by immunofluorescence microscope and analyzed using Image J.

GT type A (V900863, 300 Bloom from porcine skin), PCL (440744, average M_w = 80,000 Da), acetic acid (HAc; 338826, ≥99.8%), and 2, 2, 2-trifluoroethanol (TFE; T63002, ≥99.0%) were purchased from Sigma Aldrich (United States) and were used as received without further purification. Dulbecco’s modified Eagle medium nutrient mix F12 (DMEM/F12, 11330033), fetal bovine serum (FBS; 10099-141C), penicillin-streptomycin (15140-122), and trypsin (25200-056) were purchased from Thermo Fisher Scientific (United States). Anti-cardiac troponin T (ab45932) and anti-vimentin (ab92547) primary antibodies were purchased from Abcam (United Kingdom). Anti-rabbit IgG fluorescent secondary antibody (4412S) was purchased from Cell Signaling Technology (United States). 4′, 6-Diamidino-2-phenylindole (DAPI; C1002) was purchased from Beyotime Biotechnology (China). The neonatal rat/mouse cardiomyocyte isolation kit (nc-6031) was purchased from Cellutron Life Technologies (United States). Live/dead cell viability assay kits (L3224) were purchased from Thermo Fisher Scientific (United States). Cell Counting Kit-8 (CCK-8; CK04) was purchased from Dojindo Laboratories (Kumamoto, Japan). A modified hematoxylin-eosin (H-E) staining kit (G1121) was purchased from Solarbio Technology (China).