Recombinant human tgf β2

The human LEC line SRA01/04 was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Life Technologies Corporation, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin, and 100 µg/mL streptomycin (Gibco). LECs were treated with 20 ng/mL recombinant human TGF-β₂ (PeproTech, Cranbury, NJ, USA) for the specified duration. Stable cell lines with Smurf1 knockdown and overexpression were constructed using lentivirus. To develop lentivirus, transfection reagent lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific) and viral particles (psPAX2 packaging and pMD2.G envelope plasmids) with shSmurf1, control shRNA, pLVX-SMURF1-HA-Puro, or its mutant plasmid (Dahong Biotechnology Corporation, Guangdong, China) were transfected into HEK293T cells. After 48 hours, the supernatant of the culture medium, which contained lentivirus, was collected, and fresh media were added to the HEK293T cells. The filtered supernatant was transferred to each well of SRA01/04 cells. Positive cells were screened using 2 µg/mL puromycin. The pooled cells were harvested and their knockdown efficiency and overexpression level were confirmed by Western blot. These knockdown and overexpression cell lines were utilized for subsequent experiments and cultured in medium supplemented with 1.5 µg/mL puromycin.

Immortalized HLEB-3 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), placed in a humidified atmosphere containing 5% CO₂ at 37°C. The cells were washed with phosphate-buffered saline (PBS), and dissociated with 0.05% trypsin and 0.02% EDTA. As in our previous experiments (7 (link)), when the cell cultures reached a confluence of 80%, the cells were divided into three groups (1×10⁶ cells/ml each). In the first group, the cells were stimulated with 10 ng/ml recombinant human TGF-β₂ (Peprotech, Inc., Rocky Hill, NJ, USA) for 24 h in serum-free medium to induce EMT. This was termed the TGF-β₂ group. To determine the effect of PI3K inhibition on EMT, the cells were pretreated with LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA) at an appropriate concentration for 1 h prior to being co-treated with 10 ng/ml TGF-β₂ for 24 h. This was termed the LY294002+TGF-β₂ group. The control group consisted of cells, which were incubated under conventional conditions without the presence of either TGF-β₂ or LY294002 in the medium. Following treatment, the cells were collected for western blot analysis and confocal immunofluorescence assays.