Smad7

After culturing in 6-well plates and 21-day induction using an osteogenic medium, we rinsed MC3T3-E1 cells twice with PBS, followed by lysis with RIPA lysis buffer (Solarbio, Beijing, China) supplemented with 1 mM PMSF (Sigma-Aldrich, Shanghai, China) to inhibit protein degradation [18 (link)]. We detected the total protein concentration after centrifugation using a BCA kit (CWBIO, Beijing, China). For the WB assay, we separated proteins (20 μg) using 12% SDS-PAGE, followed by transfer onto PVDF membranes. Thereafter, the membranes were blocked using 5% nonfat milk at 4°C overnight and rinsed three times with TBST. Next, we used primary antibodies (Abcam, Cambridge, UK) to incubate the PVDF membranes for 2 h, including Runx2 (1:1500), OCN (1:1000), Col1A1 (1:2500), ALP (1:1000), and GAPDH (1:3000). The primary antibodies against Smad proteins, including Smad7 (1: 1500), Smad2 (1:2000), Smad3 (1:1000), phospho-Smad2 (1:1500), and phospho-Smad3 (1:2000), were obtained from Beyotime (Shanghai, China). After rinsing thrice with TBST, the membranes were incubated with HPR-labeled secondary antibody (1:6000; CWBIO, Beijing, China). ECL substrate (CWBIO, Beijing, China) and ChampChemi Professional (Sage, Beijing, China) were used to visualize the bands on the membranes. ImageJ software was used to quantify protein levels through densitometry, with GAPDH as the endogenous control.

Metformin (Met, purity >98%), SA (CAS No.: 530‐59‐6, purity >98%), carboxymethyl cellulose (CMC), and letrozole were received from Topscience Co. Ltd. (Shanghai, China). Goat antirabbit immunoglobulin G (IgG) and other primary antibodies (Smad2, connective tissue growth factor (CTGF), p‐Smad2, TGF‐β1, Smad7, Smad3, and p‐Smad3) were received from Beyotime Biotech Inc (Shanghai, China). Papanicolaou staining solution and primary antibodies (collagen I, PPAR‐γ, β‐actin, α‐smooth muscle actin (α‐SMA), Smad4, and TGF‐βR1) were provided by Servicebio Technology Co., Ltd. (Wuhan, China).