Fig. 4.

Downregulation of JAK/STAT pathway decreases PD-L1 expression in ENKTL. NK-YS, SNK-6, and SNT-8 cells were treated with JAK2 inhibitor Fedratinib (TG-101348, 3 nM) alone, or GM-CSF (100 ng/ml) alone, or Fedratinib combined with GM-CSF for 12 h. a-c Protein expression of p-JAK2, JAK2, p-STAT5, STAT5, and PD-L1 of ENKTL cell lines NK-YS, SNK6, and SNT-8 were measured by Western blot. d-f And relative mRNA expression of PD-L1 was measured by quantitative polymerase chain reaction (qPCR). g-j SNK-6 and SNT-8 cells expressing shSTAT5 or control were evaluated for STAT5 and PD-L1 protein expression by Western blot, and mRNA expression by qPCR. k The − 620 to − 500 nucleotide sequence of the 5′-flanking region of PD-L1 is shown. Underlined sequences are putative STAT5A and STAT5B transcription factor binding sites, as predicted by JASPAR database and PROMO. And PD-L1 promoter fragments cloned into pGL3-Basic vector. l Analysis of PD-L1 promoter fragment A constructs in 293 T cells transiently transfected with STAT5A or STAT5B for 48 h. Relative luciferase activity was determined as described. Error bars represent the SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent the SD of three independent experiments