Fig. 1

VISTA is highly expressed in AML cells and associated with poor prognosis of AML patients. A Correlation analysis between mRNA expression levels of immune-modulating molecules and the overall survival in AML patients (n = 132, divided into two groups based on gene expression) in GDC-TCGA database (https://xena.ucsc.edu) by Kaplan–Meier long-rank test. B Kaplan–Meier long-rank test of AML patients from GDC-TCGA database (n = 132, Low, n = 68; High, n = 64) with VISTA genes high or low expression levels. C VISTA expression changes in the CD34⁺ or CD34⁻ bone marrow-derived mononuclear cells from individual primary AML patient samples. D T cell-mediated cytotoxicity in co-culture system containing MOLM-13-EGFP/Luc cells and different amount of Jurkat cells in the presence of VISTA mAb (5 μg/mL). E Kaplan–Meier survival curve of intravenous C1498-EGFP/Luc mice (n = 7) treated with VISTA mAb. F, G VISTA levels and pY705-STAT3 levels in the CD34⁺ bone marrow-derived mononuclear cells of individual primary AML patient samples. H STAT3 were knockdown by specific shRNA for 48 h and the RT-PCR analysis of STAT3 and VISTA were measured in MOLM-13 cells. I STAT3 were knockdown by specific shRNA for 72 h or inhibited by W1046 for 24 h and the immunoblotting analysis of STAT3 and VISTA were measured in MOLM-13 cells. J Binding sites of STAT3 on VISTA gene were obtained from ChIP-seq data in GEO database (https://www.ncbi.nlm.nih.gov/gds/?term, GSM935276). K ChIP-qPCR analysis was performed to certify the binding between STAT3 and VISTA gene in MOLM-13 cells treated with DMSO or W1046 (3 μM) for 48 h. P1 represented the fragment from VISTA promoter, P2 represented the fragment from the first intron of VISTA gene. L The schematic of recombinant luciferase reporter construct containing 1000 bp of VISTA promoter or 1256 bp of the first intron of VISTA gene. M Relative luciferase activity of the P1 or P2 changed after STAT3 activation or treated with STAT3 inhibitor W1046 for 24 h in 293T cells. N Chemical structures of W1046. O Cell proliferation and IC₅₀ values of MOLM-13 and Mv4-11with aberrantly active STAT3 and MCF-7 cells with low-active STAT3. P Cell viability of MOLM-13 cells with STAT3-WT or STAT3-KO treated with W1046 at different concentrations. Q melt curves of CETSA depicted degradation of STAT3 protein in MOLM-13 cells treated with W1046 or DMSO after being heated in the indicated temperature points. R Western blot was used to detect expression of pY705-STAT3, T-STAT3, c-Myc, and Bcl-XL after being treated with W1046 for 24 h in MOLM-13 cells. S The transcriptional activity measured by dual-luciferase reporter assay in 293T cells treated with W1046 at different concentration for 24 h. All data were presented as means ± SEM, n = 3. * P < 0.05, ** P < 0.01, *** P < 0.001