Abnormal IL-6 pathway activation is independent of MPL. (A) The IL-6 mRNA levels were measured by qRT-PCR in CALR DEL and KO UT7 cells grown in the presence or absence of GM-CSF and expressed relative to CALR WT cells (dashed line). RNAseP mRNA levels were used for relative quantity (RQ) calculation. The levels of IL-6 in the culture medium of CALR DEL and KO UT7 cells were quantified by ELISA and expressed as fold change relative to parental cells (dashed line; panel B). (C) ChIP assay was performed in extracts of CALR WT, DEL, and KO UT7 cells using STAT3 antibody, or normal rabbit serum (IgG) as negative control, with the primers amplifying the indicated regions of the IL-6 promoter. (D) CALR WT, DEL, and KO UT7 cells were seeded at 1 × 10⁵ cells per milliliter, in the presence of TCZ (1 μg/mL), SC144 (5 μM), and anti–IL-6 antibody (1 μg/mL), and living cells were counted daily by trypan blue dye exclusion; data shown represent percent changes compared with respective control cells maintained in the absence of drugs, for 3 days of culture, and are the mean plus or minus SD of 3 independent experiments. All P values were determined by Student t test (*P < .05; **P < .01; ***P < .01).