Fig. 4

Reconstitution of 32D-BCR-ABL and 32D-JAK2V617F STAT1ko and STAT2ko cells. 32D-BCR-ABL and 32D-JAK2V617F cells (depicted as WT, respectively), which passed through the CRISPR STAT KO process but showed no knockout, were used as control cell lines. 32D-BCR-ABL (a) and 32D-JAK2V617F (b) STAT1ko or STAT2ko cells reconstituted with wt-STAT1, wt-STAT2, STAT1Y701F (Y/F), or STAT2Y689F (Y/F) were applied in a MTT assay and treated with the indicated concentrations of IFNa (0–10⁴ U/ml) for 72 h. MTT assays have been performed four times in independent experiments, and untreated controls were analyzed with one-way ANOVA and Dunn’s multiple comparison test. Further statistical analysis can be found in Additional file 9: Figure S7A, B. c Western blot analysis of the 32D cell lines depicted in a and b treated for 4 h with 100 U/ml IFNa or left untreated. Phosphorylation of STAT1, STAT2, and STAT3 was analyzed. GAPDH served as the loading control. d mRNA expression of interferon-stimulated genes in the indicated cell lines. Stat2 qPCR primer detected the ectopically expressed Stat2 mRNA, explaining the strong upregulation, and endogenous Stat2 can thus not be evaluated in the reconstituted experiments. Gene expression was calculated as a percentage of Gapdh, and the mean values ± SD are depicted. *p < 0.05, **p < 0.01, ***p < 0.001. The respective 32D cells were grown in IL3-source-free medium 24 h before application into the experiment