Fig. 4: Phosphorylation of vimentin at S339, T327, and S83 accelerates tumorigenesis by increasing the expression of PD-L1 in metastatic cancer.

NCI-H460 cells expressing RFP-tagged empty vector (Mock), wild-type (WT) vimentin and S339A, S339E, T327A, T327E, S83A, and S83E mutants were injected intravenously into the tail-veins of four-week-old BALB/c nude mice, and the tumorigenic and metastatic properties were evaluated after 8 weeks. a Representative lung tumors from the mouse model. b The number of metastatic lung tumors was counted and plotted. (n = 5). Data are presented as mean ± SD. c Representative H&E staining (upper panel) and Ki-67 staining (lower panel) were performed using lung tissue from the mice. d The relative density of H&E staining was analysed and plotted. *p < 0.05; **p < 0.01; ***p < 0.001; (n = 3). Data are presented as mean ± SD of three independent experiments (significantly different as compared with experimental control). e The populations of Ki-67 positive cells were analysed and plotted (n > at least 400 cells in each experiment). *p < 0.05; **p < 0.01; ***p < 0.001; (n = 5). Data are presented as mean ± SD of three independent experiments (significantly different as compared with experimental control). f Immunoblotting was performed using lung tissue lysates from each mouse model. E-Cadherin, N-cadherin, RFP, PD-L1, PD-L2, and β-actin were detected using specific antibodies. g The relative intensity value of PD-L1 was normalized to that of β-actin and plotted. *p < 0.05; **p < 0.01; ***p < 0.001; (n = 3). All experiments were performed at least three independent experiments. n.s. no significant.