Figure 2.

Role of VLA-4 integrin in PD-L1 expression downregulation by SAS. (A) B16/F10 melanoma cells or (B) D122 cells were cultured on vascular cell adhesion molecule-1 (VCAM-1)- or bovine serum albumin (BSA)-coated plates with or without SAS for 1 h. The cells were washed twice. The percentage of attached cells, representing VLA-4 (very late antigen-4) activity, was determined by the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) viability test relative to the control PBS. *p<0.001 vs. BSA; #p<0.01 vs. PBS; **p<0.001 vs. PBS. Significance was calculated via one-way ANOVA. The results are presented as the mean+SE of 3 experiments. (C) Human VLA-4-negative AML cells isolated from AML patients were cultured on FN-coated plates for 24 hours. The cells were collected and stained with PE-conjugated anti-mouse PD-L1. (D) B16/F10 melanoma cells were transfected with either control or (E) VLA-4 shRNA (short hairpin RNA) and cultured with or without different concentrations of SAS. The cells were collected and stained with either PE-conjugated anti-mouse PD-L1 antibodies or isotype-matched controls. The results show one representative of 3 experiments. Figs 2C, D and E are accompanied by quantitative data from flow cytometry analysis. # p<0.001 vs. the isotype control; *p<0.05 vs. the PBS group. **p<0.01 vs. PBS.