Figure 3.

SAS inhibits both PD-L1 expression and VLA-4 activity in U937 myelomonocytic human leukemic cells. (A) U937 human cells were cultured in the presence or absence of SAS. The cells were collected and stained with either PE-conjugated anti-human PD-L1 antibodies or their respective isotype-matched controls. The results show one representative experiment of 3 performed. (B) U937 cells were cultured on VCAM-1- or BSA-coated plates with or without SAS for 1 h. The cells were subsequently washed twice. The percentage of attached cells (representing VLA-4 activity) was determined by the XTT viability test relative to the control PBS. * p<0.001 vs. BSA **p<0.001 vs. PBS. Significance was calculated via one-way ANOVA. The results represent the mean +SE from 3 experiments (B). (C) The VLA-4 conformational structure was detected by FRET. VLA-4 activation involves the separation of the fluorescently tagged cytoplasmic ends of the α- and β-subunits of the integrin, resulting in a reduction in the FRET signal. U937 cells were transfected with α4-mCFP and β1-mYFP. After 48 hours, the cells were activated and fixed. FRET of the molecular interaction between the cytoplasmic domains of α4 and β1 was determined as described in the Materials and Methods. (D) Results for FRET positive controls (cells expressing CFP and YFP encoded on the same plasmid (i.e., maximal FRET obtained in this system) and negative controls (CFP and YFP expressed by different plasmids and undergoing minimal FRET, i.e., only that produced by random colocalizations). *p<0.01 vs. the negative control. Significance was calculated via a two-tailed t test. PLL (poly-L-lysine). FN (Fibronectin).