Ang 2

The siRNA sequences targeting the mice Slit2 gene (si‐Slit2) and the mice TGF‐β1 gene (si‐TGF‐β1) and the scrambled siRNA sequence (si‐Scramble) were designed and synthesized by RiboBio (Guangzhou, China). Passage 3 CFs were plated in a 6 well plate (1 × 10⁵ cells/well) in 10% fetal bovine serum‐containing medium and at 80% confluence were starved by incubation in serum‐free medium for 24 h. siRNAs (50 nM) were transfected into CFs with Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer's protocol. Cells in the 1st–6th wells received the following treatments for 24: (i) 50 nM si‐Scramble, (ii) 50 nM si‐Scramble and 100 nM Ang II (Sigma, North St. Indianapolis, IN, USA), (iii) 50 nM si‐TGF‐β1, (iv) 50 nM si‐TGF‐β1 and 100 nM Ang II, (v) 50 nM si‐Slit2, and (vi) 50 nM si‐Slit2 and 100 nM Ang II. The second dose of Ang II, at the same concentration as the first dose, was added to the media after 12 h.

To illustrate the effects of AngII on Angptl2 expression, AngII (Sigma-Aldrich Chemie GmbH, Hamburg, Germany) was added to the culture medium in six-well plates containing cardiomyocytes (5×10⁵ cells/ml) at the following final concentrations: 0, 50, 100 and 200 nmol/l for 24 h. In addition, cells were incubated with 100 nmol/l AngII for 0, 6, 24 or 48 h. The culture medium from each condition was collected. Total protein was extracted from the rat cardiomyocytes using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Beijing, China), which contained 0.1 M phenylmethylsulfonyl fluoride. After washing with phosphate-buffered saline (PBS), grinding, lysis and centrifugation (at 4°C, 13,000 × g, 15 min), the supernatant was collected. Protein concentrations were measured using the bicinchoninic acid assay (Protein Assay kit; Beyotime Institute of Biotechnology), in which bovine serum albumin was used as a standard. After adding 5× loading buffer [sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) Loading Buffer/Lane Markers; Beyotime Institute of Biotechnology], the mixture was boiled in water at 100°C for 8 min, and was stored at −80°C.

Primary VSMCs were isolated from rat thoracic aortas as previously performed [29 (link)]. The obtained VSMCs were maintained in Dulbecco's modified Eagle's medium (DMEM, YT8231; YITA Bio) containing 10% fetal bovine serum (FBS, 76294-180; AVANTOR), 1% penicillin-streptomycin (15140-122; Gibco) at 37°C in the presence of 5% CO₂ and 95% O₂. VSMCs were treated with Ang II (100 nM; Sigma) alone for 6, 12, and 24 h, coincubated with Ang II (100 nM) and wogonin (0.1, 1, 3, 10, and 20 μM) for 24 h, or co-incubated with Ang II (100 nM) and wogonin (10 μM) for 24 h.