Tgf β1

Tissue samples of the posterior joint capsule collected at 0, 1, 3, 7, and 14 days following injury or primary joint capsule fibroblasts treated with various drugs (MIF, TGF-β1, siRNA or inhibitors) were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with 1 mM PMSF (Beyotime). Protein concentration in the supernatant was quantified using the Protein Assay Kit II (Bio-Rad, Inc., California, USA) to ensure equal loading. Western blots were performed as described previously 21 (link). The relative intensities of protein bands were normalized to those of β-actin. The following antibodies were used: β-actin (ProteinTech, Wuhan, China, 1:5000), MIF (Abcam, 1:1000), TGF-β1 (Abcam, 1:1000), α-smooth muscle actin (α-SMA, Abcam, 1:1000), Collagen I (Abcam, 1:1000), CD74 (Santa Cruz, CA, 1:100), p-ERK/ERK (Cell Signaling Technology, Danvers, MA, 1:1000), p-P38/P38 (Cell Signaling Technology, 1:1000), p-JNK/JNK (Cell Signaling Technology, 1:1000). Secondary antibodies included Goat anti-Rabbit IgG (H+L) (DyLight 800 4X PEG) (Invitrogen, 1:20000) and Goat anti-Mouse IgG (H+L) (DyLight 680) (Invitrogen, 1:20000). The fluorescent signals were determined with an Odyssey imaging system (Li-Cor, Lincoln, NE, USA).

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).

AML cell lines KG-1a was ordered from the Shanghai cell bank of the Chinese Academy of Sciences and cultured in RPMI 1640 (Gibco) supplemented with 20% fetal bovine serum. Cells were maintained at 37 °C in a 5% CO₂ incubator. KG-1a was precultured with 10 ng/ml TGFβ1 (PeproTech) for 48 h and used for Cell Counting Kit-8 (CCK8) assay (Beyotime Institute of Biotechnology, China), apoptosis detection (Apoptosis Detection Kit, BestBio), cell cycle detection (COULTER DNA PREP Reagents Kit, Beckman). Cell cycle phase distributions were generated by fitting DNA content histograms to applicable models using ModFit LT software for Windows (Version 5.0). KG-1a cells were stained with Senescence‐associated‐β‐Galactosidase (SA‐β‐Gal) staining (Beyotime) according to the instructions after TGFβ1 treatment for 7 days, and cells that stained green‐blue were evaluated as positive senescent cells. Cell suspension (1 × 10⁵, 200 μl of serum-free medium) after TGFβ1 pretreatment was seeded onto 8-mm Pore Transwell Inserts (Corning) in 24-well plates with or without matrigel (BD biosciences) and photographed within 24 h.