Interfering the STAT1/STAT3 pathway with the small-molecule stattic hinders the IFN-γ-induced upregulation of PD-1 ligands in myeloid leukemia cells. ATRA- or D3-treated HL-60, THP-1, and U937 which displayed high capacity to express PD-1 ligands were used as representative myeloid leukemia cell lines. (a) Prior to induction with IFN-γ (for 15 min.), these cells were exposed to stattic and the change in pSTAT3 (Tyr705) and pSTAT1 (Tyr701) levels were assayed by Western-Blot. Please note that the blots from each cell line or patient sample are shown as cropped from different parts of the same gel. (b) The effect of stattic on the percentage PD-L1+ or PD-L2+ myeloid leukemia cells following 24 h stimulation with IFN-γ. (c) Representative flow cytometry dot plots are shown for the ATRA- or D3-treated HL-60 cells. The change in PD-L1 and PD-L2 mRNA levels in the IFN-γ-induced (d) ATRA-treated HL-60 cells (at 4, 8, 16, and 32 h) and (e) other myeloid leukemia cell lines (for PD-L1 at 32 h) with or without the stattic pretreatment was studied by real-time RT-PCR. (f,g) The effect of stattic on IFN-γ-induced PD-L1 expression (at 24 h) on the CD11b+ blasts from the control, ATRA- or D3-treated AML or MDS patent samples; (f) representative flow cytometry plots and (g) the percentage of PD-L1+ cells with or without stattic treatment are given. (*P < 0.05, **P < 0.01; for cell lines, n ≥ 3; patient samples, n = 7; ns, not significant).