Tgf β2

We gathered 20 anterior lens capsules from 13 patients during the curvilinear capsulorhexis stage of pediatric cataract surgery. Modifying the procedure outlined by Wernecke [19 (link)], we established the following pLECs cultivation protocol:

The anterior lens capsules that adhered to pLECs obtained during pediatric cataract surgery, were laid as flat as possible in a 6 cm culture well and fixed at the bottom of the well with a glass coverslip. They were subsequently cultured in Dulbecco's Modified Eagle Medium (DMEM) with high glucose (Hyclone, Thermo Scientific, USA,CAT#C11995500BT), enriched with 20% fetal bovine serum (FBS, Gibco, USA,CAT#10099141) and 100 U/ml penicillin and 100 μg/mL streptomycin mixture(NCM Biotech,China,CAT#C100C5) at 37°C in a humidified atmosphere containing 5% CO₂.

The medium was replaced every three days once the cells began to migrate on the well’s bottom. Confluence was achieved at 7 to 15 days, observed as cells migrating on the bottom of the well and glass coverslip. pLECs were subcultured for 3 to 4 generations and seeded on 6-well plate.

Upon achieving a confluence of 70%, the pLECs were treated with 5 ng/mL TGF-β2 (Proteintech, USA,CAT#HZ-1092) for a period of 48 hours in a serum-free medium.

Gastric cell line (HGC-27) was purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured at 37°C in RPMI-1640 medium containing 10% 100 U/ml penicillin and 100 U/ml streptomycin. The active recombinant proteins TGFβ1 (Proteintech, HZ-1011), TGFβ2 (Proteintech, HZ-1092) and TGFβ3 (Proteintech, HZ-1090) were solubilized and added to normal medium to adjust the concentration of TGFβ factor to 10ng/mL. The gastric cancer cells cultured with TGFβ active protein were added as the TGFβ-treated group, and human serum albumin (HSA) of equal quality was added as the control group and incubated in the cell incubator for 48 hours.

According to the sequencing data and functional analysis, we analyzed the proteins associated with the transforming growth factor beta (TGF-β) signaling pathway in granulosa cells using the Western blot. Briefly, the whole protein samples were extracted, and their concentrations measured using the BCA Protein Assay Kit (GenStar, Beijing, China). Then, equal amounts of proteins (20 μg per lane) were loaded and separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel. Primary antibodies were used after proteins transferred to the 0.45-μm Immobilon-P PVDF Membrane, and GAPDH used as loading control. Signals were obtained in the linear range of detection and quantified with the Bio-Rad ChemiDoc Imaging System. The grey values of target bands were analyzed by ImageJ software and normalized to the reference band. The primary antibodies used were TGF-β2 (19,999–1-AP; 1:2000 diluted; Proteintech), TGF-βR2 (66,636–1-lg; 1:2000 diluted; Proteintech), Smad2 (12,570–1-AP; 1:2000 diluted; Proteintech), Smad3 (25,494–1-AP; 1:2000 diluted; Proteintech).

HFF-1 cells were used to prepare recombinant human TGF-β family treated lysates and supernatants for quantification. Briefly, cells for were seeded in 6-well plates at 200,000 cells/well. After treatment with either control (DMEM with vehicle supplemented with 2% FBS), TGF-β1 (R&D Systems), TGF-β2 (ProteinTech), or TGF-β3 (ProteinTech), cell lysates and supernatants were collected after 48 h for quantification by WB or FMOD ELISA (Abcam).

Human umbilical vein endothelial cells (HUVECs) were purchased from Cyagen (#HUVEC-2001). The HUVECs were cultured in a 1% gelatin coated plate in complete endothelial cell medium (ECM, #1001, ScienCell) containing 5% FBS, and endothelial growth factor and were maintained in a humidified chamber with 5% CO₂ at 37°C. Human umbilical vein smooth muscle cells (HVSMCs) were obtained from Otwo Biotech (#HTX2305) and maintained in DMEM supplemented with 10% FBS and 1% P/S. To establish the EndMT model, HUVECs were stimulated with 10 ng/ml TGF-β2 (#100-35B, Proteintech) in conditioned medium supplemented with 2.5% FBS for four consecutive days. For DSY application, HUVECs pretreated with TGF-β2 for 2 days were incubated with different doses of DSY for a further 2 days.