Tgf β

BMDMs were flushed from the tibiae and femurs of 6-8 weeks old C57BL/6 mice with PBS. The cell suspension was filtered through a 70 µm cell strainer and subjected to red blood cell lysis (Solarbio, #R1010). Cells were cultured in 1640 medium containing macrophage-stimulating factor (M-CSF, Peprotech, USA) for 7 days before subsequent experiments. M1 phenotype was induced with 1 µg/ml LPS (Sigma-Aldrich, USA), and M2 phenotype with Interleukin-4 (IL-4, Peprotech, USA). For exploring Tgf-β induced macrophage phenotype transition, cells were stimulated with 1 µg/ml LPS for 24 h, followed by 5 µg/ml Tgf-β (SinoBiological, China) for another 24 h. For MMT, mature induced BMDMs were treated with 5 µg/ml Tgf-β for 5 days before subsequent experiments.

Mouse retinal 661W cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA). The cells were maintained at 37 °C in a humidified 5% CO2 incubator.
661W cells were divided into 5 groups: (1) UV group, in which a control vehicle was added before UV exposure (30 J/m², 254 nm, irradiated for 40 s); (2) Tgf-β group, in which Tgf-β (Sinobiological, Beijing, China, final concentration at 1 ng/mL [19 (link)]) was added 1 h prior to UV exposure; (3) LSKL group, in which LSKL (MCE, HY-P0299, final concentration at 10 μg/mL) was added 24 h prior to UV exposure; (4) Tgf-β + LSKL, in which a pretreatment with Tgf-β for 1 h and LSKL for 24 h followed by UV exposure was added; and (5) Sham, in which there was no UV exposure. For UV exposure, the cell medium was removed and replaced with PBS, and after 40 s of UV radiation, the cells were reincubated with a fresh cell culture medium and allowed to repair in the dark.

The human umbilical vein endothelial cell line EA.hy926 was donated by the research group of Professor Qin Ling of the Chinese University of Hong Kong. anti-MMP-2 (Cat. no.40994S), Anti-p-JAK2 (Cat. no.3771S), anti-p-STAT3 (Cat. no.9145S), anti-p-SRC (Cat. no.6943T), anti-p-ERK (Cat. no.4370S), anti-p-p38 (Cat. no.9216S), anti-p-FAK (Cat. no.8556T), anti-p-AKT (Cat. no.9275S), anti-STAT3 (Cat. no.9139), anti-p-SMAD2 (Cat. no.8828S) and HRP-linked secondary antibody (cat. no.#7074) were purchased from CST. CCK-8 (Cat. no.CK04-3000T) was purchased from DOJINDO. Anti-VEGF (Cat. no.ab46154) and Anti-TGF-β (Cat. no.ab217402) antibodies were obtained from Abcam. Inhibitors of STAT3 (WP1066), ERK (Selumetinib), SRC (Dasatinib), FAK (PF-562271), P38 (SB203580), AKT (MK-2206), EGFR (EGFR-IN-12), VEGFR (SU5204), PDGFR (AG1296), as well as Pirfenidone (Cat. no.S2907) were purchased from Selleckchem. Matrigel (Cat. no.354234) and Transwell (Cat. no.3422) were purchased from Corning. TGF-β (Cat. no.10804-HNAC) was Purchased from Sinobiological. anti-MMP-9 (Cat.no.10375-2-AP) and GAPDH (Cat.no.60004-1-Ig) primary Antibodies were obtained from Proteintech (Wuhan, China). Total RNA miniprep kit (Cat. no. AP-MN-MS- -RNA-250) was Purchased from Axygen. PrimeScript™ RT Master Mix (Cat. no.RR036A) and TB Green Premix Ex Taq II (Cat. no.RR820B) were purchased from Takara.