CALR regulates IL-6/JAK1/STAT3 signaling through interaction with IL-6 and gp130. (A) UT7/mpl cells were incubated with TPO (100 ng/mL) or without for 7 days and the percentage of CD41⁺/CD61⁺ cells was measured by flow cytometry. Data shown are the mean plus SD of at least 3 individual experiments and represent fold change compared with WT cells incubated in the same conditions. (B) Total cell extracts of CALR WT, DEL, and KO UT7/mpl cells, incubated with or without TPO, were subjected to western blotting and immunodetection for p-STAT3, STAT3, p-STAT5, STAT5, p-JAK1, JAK1, p-JAK2, and JAK2; GAPDH was used as the loading control. (C-D) Densitometric analysis of 3 individual immunoblots (cells without TPO [C]) and (cells incubated with TPO [D]). LIF, OSM, and IL-6 mRNA levels were measured by qRT-PCR in CALR DEL and KO UT7/mpl cells and expressed relative to WT cells (dashed line). (E) RNAseP mRNA levels were used for relative quantity (RQ) calculation. (F) The IL-6 mRNA levels were also measured in CALR WT (reference fixed to relative level 1, dashed line), DEL and KO UT7/mpl cells that were cultured in the presence or absence of TPO in culture medium. (G) The quantification by ELISA of the levels of IL-6 in the culture medium of CALR DEL and KO UT7/mpl cells, expressed relative to parental cells (dashed line). (H) The expression of IL-6R and gp130 on the cell membrane of CALR WT, DEL, and KO UT7/mpl cells was assessed by confocal microscopy. Carboxyfluorescein succinimidyl ester (CSFE) dye was used to identify living cells; original magnification ×200. (I) In these experiments, CALR KO UT7/mpl cells were transfected with CALR WT-GFP– and CALR DEL-GFP–expressing plasmids (pl.), then they were challenged with antibodies (red fluorescence) against membrane-associated gp130 (left panels) and IL-6R (right panels). In the “merge” fields, green fluorescence points to cells that were successfully transfected with the expression plasmid; cells labeled with antibody only (red fluorescence) and lacking green fluorescence denote baseline expression of either gp130 or IL-6R in KO cells that were not transfected. Representative cells in confocal microscopy fields are presented to illustrate changes of receptor expression on the membrane of GFP-transfected KO cells; original magnification ×200. (J-K) The levels of gp130 and IL-6R mRNA (J; expressed relative to WT cells, dashed line) and protein in total cell extracts (K) were assessed by qRT-PCR and western blot, respectively, in CALR DEL and KO UT7/mpl cells; GAPDH was used for loading normalization. (L) In the coimmunoprecipitation experiment shown (1 representative of at least 3 for each experimental condition), whole-cell extracts of CALR WT, DEL, and KO cells were immunoprecipitated with gp130 (top) and IL-6R (bottom) antibodies and revealed with respective antibodies and CALR N-terminal antibody. Input panels represent the quantification of respective proteins in whole extracts before immunoprecipitation. (M) ChIP assay was performed in extracts of CALR WT, DEL, and KO UT7/mpl cells using STAT3 antibody, or normal rabbit serum (immunoglobulin G [IgG]) as negative control, with the primers amplifying the indicated regions of the IL-6 promoter. (N) CALR WT, DEL, and KO UT7/mpl cells were seeded at 2 × 10⁵ cells per milliliter, in cytokine-free medium, and living cells were counted daily by trypan blue dye exclusion; data shown are at day 3 of culture and are expressed as percent change compared with respective control cell cultures in the absence of drug. TCZ (1 μg/mL), a monoclonal anti–IL-6R antibody, SC144 (5 μM), a small molecule inhibitor of gp130, and anti–IL-6 antibody (1 μg/mL), were added at the initiation of culture (chosen drug concentrations were predetermined in dose-response curves). Data are expressed as 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).