Fig. 2. The mutation JAK2^V617F promotes de novo PD-L1 gene transcription in human cells.

(A) The histograms show the MFI for PD-L1 on K562 cells (transfected with empty vector or JAK2^V617F vector). One representative experiment of three experiments with a comparable pattern is shown. The analysis was done on GFP⁺ sorted cells within 3 days after transfection.

(B) The bar diagram displays the fold change of PD-L1 expression (flow cytometry) on K562 cells transfected with empty vector or with JAK2^V617F. The data are pooled from 4 independent experiments (n=12 per group).

(C) The bar diagram displays the fold change of PD-L1 expression (flow cytometry) on K562 JAK2^V617F cells that were exposed to different concentrations of the JAK2 inhibitor SD-1029. Pooled data from two independent experiments (n=6 at each concentration).

(D) The bar diagram displays the fold change of PD-L1 expression (flow cytometry) for the JAK2^V617F-positive cell line UKE-1 treated with the JAK2 inhibitor SD-1029 (n=7 at each concentration).

(E) The Western blots display STAT3 total protein, β-actin and phospho-STAT3 in UKE-1 cells being treated with the JAK2 inhibitor SD-1029. The blots are representative of three independent experiments.

(F) The bar diagram indicates the ratio of pSTAT3/STAT3/β-actin (normalized to 1 in the condition without JAK2 inhibitor) for the cells described in (E). Pooled data from three replicates (n=3 for each concentration).

(G) The bar diagram displays the fold change of PD-L1 expression (flow cytometry) for JAK2^V617F positive cell line SET-2 treated with ruxolitinib. Pooled data from three independent experiments (concentration 0 – 0.5 μM: n=12, concentration 1 and 2 μM: n=6).

(H) The Western blots display STAT3, β-actin, and phospho-STAT3 in SET-2 cells being treated with ruxolitinib. The blots are representative of three independent experiments.

(I) The bar diagram indicates the pSTAT3/STAT3/β-actin ratio for cells described in (H). Pooled data from three independent experiments (n=3 for each concentration).

(J) The bar diagram displays the fold change of PD-L1 expression (flow cytometry) for JAK2^V617F positive cell line MUTZ-8 that was treated with the JAK2 inhibitor SD-1029 (n=3 for each concentration).

(K) The Western blots display STAT3 total protein, β-actin and phospho-STAT3 in MUTZ-8 cells being treated with the JAK2 inhibitor SD-1029. The blots are representative of three independent experiments.

(L) The bar diagram indicates the ratio of pSTAT3/STAT3/β-actin for cells described in (K). Pooled data from three independent experiments (n=3 for each concentration).

(M) The bar diagram indicates the relative luminescence activity of K562 cells (containing empty vector or JAK2^V617F) which were left untransfected or transfected with either a promoterless luciferase reporter-vector pgl4.13 or a reporter vector containing the PD-L1 promoter. Pooled data from three technical replicates (n=6 for each condition).

(N) The bar diagram indicates the relative luciferase activity of K562 JAK2^V617F cells transfected with either the promoterless luciferase reporter vector pgl4.13 or the reporter vector containing the PD-L1 promoter and treated with the JAK2-inhibitor SD-1029. Pooled data from three independent experiments (n=6 for each condition).

(O) The bar diagram indicates the relative luminescence activity of K562 JAK2^V617F cells transfected with either the promoterless luciferase reporter vector pgl4.13 or the reporter vector containing the PD-L1 promoter and treated with the STAT3 inhibitor S3I-201. Pooled data from three independent experiments (n=6 for each condition).