Fig. 6: STAT3 is activated in MF fibrocytes and induces expression of GLI1.

A Representative micrographs of phosphotyrosine (p) STAT3 and IL-6 expression in BM fibrocytes. BM tissue sections from MF patients (n = 6) and NCs (n = 4) were analyzed by mfIHC using pSTAT3 Tyr705 (Opal 480) and IL-6 antibodies (Opal 520) (pseudo-colored red) in combination with fibrocyte-specific antibodies CD45 (Opal 690), CD68 (Opal 620), and procollagen-I (Opal 570) (pseudo-colored green). Shown are composites of each fibrocyte marker with pSTAT3 (left panel) and IL-6 (right panel). As shown, pSTAT3 was detected in CD45⁺/CD68⁺/procollagen-I⁺ cells (fibrocytes) which are abundant in the BM of MF patients, but not NCs. IL-6 exhibited mostly extracellular localization and did not significantly differ between the two groups. DAPI (pseudo-colored blue) was used as a nuclear counterstain. All micrographs were adjusted for brightness and contrast linearly and equally between groups. The scale bar represents 100 µm. B Quantitation of STAT3, GLI1, MMP2, MMP9, and procollagen-I (COL1A1) mRNA levels in MF fibrocytes after STAT3 silencing. Cultured fibrocytes from MF patients (n = 4) were transfected with STAT3-siRNA. qRT-PCR analysis was conducted 48 h after transfection. All four analyzed genes were significantly downregulated following transfection with STAT3-siRNA as compared with transfection with Ctrl-siRNA. The panel depicts relative expression of mRNA normalized to mean mRNA levels in NC fibrocytes. Matched values were compared using the paired t-test. The PPIA gene was used as an internal control. C Protein levels of STAT3, GLI1, MMP2, and MMP9 in fibrocytes of MF patients (n = 4) and an NC after STAT3 silencing as analyzed by western immunoblotting. A significant decrease in all 4 analyzed proteins was detected in MF fibrocytes but was not detected in GLI1-target genes from NC fibrocytes. D Protein levels of pSTAT3 (Tyr705), STAT3, GLI1, MMP2, and MMP9 in MF fibrocytes after treatment with JAK1/2 inhibitor ruxolitinib (Ruxo) and IL-6. BM fibrocytes cultured from MF patients (n = 3) or NCs (n = 2) were incubated with 0.3 μM ruxolitinib, 100 ng/mL IL-6, or left untreated (Ctrl) for 24 h. Protein levels were then analyzed by western immunoblotting. Treatment with ruxolitinib reduced levels of pSTAT3, GLI1, and MMPs in most MF and NC fibrocyte cultures. In contrast, the addition of IL-6 enhanced the phosphorylation of STAT3 and increased the expression of GLI1 and its target genes. All target proteins were detected by sequential probing of the same blot. β-actin was used as the loading control. *P < 0.05; **P < 0.01, respectively.