Bs1530

SiO₂, 80% of which had particle diameters of <5 μm, was purchased from Sigma (S5631), selected via sedimentation according to Stokes’ law, acid-hydrolyzed, and baked overnight (200 °C, 16 h)^{32 (link)}. The silica samples for the cell experiments were suspended in phosphate-buffered saline (PBS) at a concentration of 5 mg/ml, and the volume of administration was 20 μl/well in 24-well plates at a dosage of 50 µg/cm². Fetal bovine serum (FBS), normal goat serum and Dulbecco’s modified Eagle’s medium (DMEM; #1200-046) were purchased from Life Technologies. GlutaMax Supplement (35050-061) was obtained from Gibco. Lentiviral vectors carrying circHIPK2-siRNA were obtained from HANBIO. Control siRNA (sc-37007) was purchased from Sigma-Aldrich. Antibodies against σ-1R were obtained from Invitrogen. Santa Cruz Biotechnology, Inc. The antibody against α-SMA (SAB5500002) was purchased from Sigma, Inc. The antibody against COL1A2 (BS1530) and COL3A1 (BS1531) were purchased from BioWorld (St. Louis Park, MN, USA).

Mouse heart tissue were lysed by RIPA lysis buffer (Keygen Biotech) complemented with phenylmethylsulfonyl fluoride (PMSF). BCA Protein Assay (TaKaRa) was adopted to determine protein concentrations. After equal quantities, total proteins were separated in SDS-PAGE gel (10–12%). Using wet tank transfer method, protein bands were transferred onto PVDF membranes (PALL). After blocked with 5% BSA and washed with TBST, protein bands were blotted with primary antibodies at 4 °C overnight as follows: Bax (Abclonal, A12009, 1:1000 dilution), Bcl₂ (affbiotech, AF6139, 1:1000 dilution), Caspase3 (Abclonal, A2156, 1:1000 dilution), COL1A2 (Bioworld, BS1530, 1:1000 dilution), NRF2 (Abclonal, A0674, 1:1000 dilution), and β-actin (Bioworld, AP0060, 1:10,000 dilution). After washing with TBST, protein bands were blotted with HRP conjugated secondary antibody and monitored using ECL buffer (Tanon). Quantifications of Western Blots were measured with ImageJ.