C11995500

Human PDLSCs (8 × 10⁴ cells/scaffold) were seeded on aligned and random scaffolds (15 mm × 7.5 mm × 0.5 mm, L × W × H) in a nonadherent 24-well plate. The constructs were cultured in high glucose DMEM (Gibco, C11995500) supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin and 50 μg/ml L-ascorbic acid 2-phosphate (A2-P; Sigma-Aldrich, A8960). After 7 days of culture, samples were collected, fixed, permeabilized and blocked. Subsequently, samples were incubated with rabbit anti-collagen type I (COL I) primary antibody (Proteintech, 14695-1-AP) at 4 °C overnight. After washing, samples were incubated with 488-conjugated goat anti-rabbit IgG (Proteintech, SA00013-2) for 1 h at room temperature. Finally, 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, C1002) was used to reveal the nuclei of the cells.

NRK-52E and NRK-49F cells (obtained from the National Infrastructure of Cell Line Resource, Chinese Academy of Medical Sciences) were cultured in DMEM (C11995500, Gibco, China) with 5% or 10% fetal bovine serum (10099–141, Thermo Fisher, USA) and 1% penicillin-streptomycin and incubated at 37°C and 5% CO₂. When the cell density reached 50%, the cells were cultured in a serum-free medium for 12 h and then treated with different concentrations of SSR according to the results of the cell counting kit-8 (CCK8) assay: NRK-52E: SSL (200 mg/L), SSM (400 mg/L), and SSH (800 mg/L) and NRK-49F: SSL (200 mg/L) and SSH (400 mg/L). Simultaneously, other stimuli were used in this study: NRK-52E cells were subjected to hypoxia (1% oxygen, 94% nitrogen, and 5% carbon dioxide), whereas NRK-49F cells were treated with IL-1β (10 ng/mL; PeproTech EC Ltd., UK).

The virus titration of BA.5 followed this protocol. Endpoint titration in Vero E6 cells was employed for virus titrations. Vero E6 cells were seeded in a 96-well plate and incubated overnight at 37 °C. Subsequently, the cells were inoculated with 10-fold serial dilutions of the virus. One-hour post-inoculation, the inoculum was aspirated and replaced with 100 μL of DMEM (C11995500, Gibco, USA) supplemented with 2% FBS (10091148, Gibco) and 100 IU/mL penicillin–streptomycin (15140122, Gibco). After 4–7 days of incubation, the cytopathic effect was assessed, and the TCID₅₀ was calculated.
The titration of the Beta virus for the novel coronavirus variants was determined utilizing the Focus Forming Assay (FFA). This method involves immunostaining Vero E6 cells infected with the virus, utilizing an anti-SARS-CoV-2 N-protein antibody along with an HRP-conjugated secondary antibody. Subsequently, the stained spots on cell plates were quantified and analyzed using an enzyme-linked immunospot analyzer (ELISPOT device). The titer (FFU/mL) was calculated using the formula: titer (FFU/mL) = number of spots × dilution factor / volume, with the detection limit determined when one spot was observed in the undiluted experimental sample.

The Marc-145 cell line derived from African green monkey kidneys and the HEK-293 T cell line derived from human embryonic kidneys were cultured in Dulbecco’s modified Eagle’s medium (Gibco, C11995500) supplemented with 10% foetal bovine serum (VivaCell Biosciences, 2238253). The cells were cultured under controlled conditions in an incubator at 37 °C with 5% CO₂. The PRRSV strains XH-GD (GenBank accession number: EU624117.1), GM2-like (lineage 3) and NADC30-like (lineage 1) were preserved in our laboratory. The PRRSV JXA1 strain (GenBank accession no. EF112445.1) was obtained from Prof. Kegong Tian.

Primary cultures of neonatal rat ventricular myocytes (passage 1) were incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, as previously described (Jin et al., 2018 (link)). After 16 h of serum starvation, the cardiomyocytes were treated with matrine (5 nM). Cardiomyocytes in the control group were cultured in 5.5 mmol/L standard glucose medium (control group), while those in the in vitro hyperglycemia injury model group were cultured in 25 mmol/L high-glucose DMEM for 12 h. Both types of medium were supplemented with 10% fetal bovine serum (Gibco, C11995500), 100 IU/ml penicillin and 100 μg/ml streptomycin. The cells were incubated in 95% air and 5% CO₂ (Wang et al., 2020a (link)).

Cells were incubated with a final concentration of 250 μM palmitic acid-16,16,16-d³ (615951, Sigma, USA) in serum-free DMEM (C11995500, Gibco, China) for 24 h according to a previously described method 53 (link). Briefly, palmitic acid was dissolved in ethanol and combined with fatty acid-free bovine serum albumin (BSA) at a molar ratio of 10:1 (PA:BSA) in a serum-free medium.