Fig. 2
Y332D counteracted TGF-β1-induced inhibition of T cell proliferation and activation as well as epithelial-mesenchymal transition (EMT). a, b CCK-8 assays were performed to show the antagonistic effect of Y332D on TGF-β1-hampered proliferation in T cells. 1 × 103 CTLL-2 and HT-2 cells were seeded in 96-well plates. Then, 5 ng/ml TGF-β1 plus 106 pM antibodies or control were added. Cell viability was continuously monitored by CCK-8 reagent. c–g Multi-cytokine assay was performed to analyze the effect of Y332D on the alteration of TGF-β1-caused cytokine secretion during T cell activation. Murine T cells were obtained from the isolation of splenocytes from C57BL/6 mice. T cells (1 × 106/ml) supplemented with anti-CD28 (3 μg/ml), TGF-β1 (5 ng/ml) and 106 pM antibodies or control were cultured in 96 well flat-bottom plates precoated with anti-CD3 (3 μg/ml). After 4 days, the cellular supernatants were harvested to measure cytokines concentration. h, i Transwell migration/invasion assays were performed to demonstrate the antagonistic effect of Y332D on TGF-β-enhanced tumor cell motility. 4T1 and EMT-6 mammary tumor cells were cultured in RPIM-1640 with 1% FBS and treated with 5 ng/ml TGF-β1 plus 106 pM antibodies or untreated for 96 h. Then, about 5 × 104 cells in 100 µl RPIM-1640 supplemented with 1%FBS were seeded in the upper chambers. The lower chambers were added with 600 µl of RPIM-1640 containing 10% FBS. After incubation for 24 h, the migratory and invasive cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Bars, SDs; **p < 0.01, ***p < 0.001, and ****p < 0.0001 denote the significant difference relative to Y332D treatment. α-TGF-β: anti-TGF-β, α-VEGF: anti-VEGF