Fig. 1: GLI1 levels in BM fibrocytes and MSCs of MF patients in situ and in vitro.

A Representative micrographs of fibrocytes (left panel) and MSCs (right panel) in BM biopsy sections of an MF patient were analyzed using mfIHC. In addition to GLI1 (Opal 520, pseudo-colored red), fibrocytes were stained using CD45 (Opal 690), CD68 (Opal 620), and procollagen-I (Opal 570) antibodies and MSCs were stained using CD90 (Opal 650) and CD105 (Opal 540) antibodies (pseudo-colored green). Shown in zoomed-in panels are a representative fibrocyte (arrows) and an MSC (asterisks). Fibrocytes are typified by a granular staining pattern inside the BM cavity, whereas MSCs by a linear staining pattern within the endosteal or perivascular space. GLI1 co-localized with the cell nuclei (DAPI, pseudo-colored blue) in both fibrocytes and MSCs. The right-most panel depicts combined CD45 (hematopoietic) and CD90 (MSC) markers (pseudo-colored white and green, respectively) together with GLI1 (pseudo-colored red). B Quantitative analysis of GLI1 signal intensities in BM biopsy sections of MF patients (n = 7) and NCs (n = 4) by mfIHC. Whole BM tissue sections were imaged at ×400 resolution and individual fibrocytes (CD45⁺/CD68⁺/procollagen-I⁺) and MSCs (CD90⁺/CD105⁺) were detected using an unsupervised tissue and cell segmentation algorithm. GLI1 signal intensity was quantitated in each cell and mean signal intensity of all detected fibrocytes and MSCs was calculated for each BM biopsy. Horizontal lines denote the mean. The statistical significance of the difference between groups was calculated using the one-way ANOVA followed by Tukey’s post hoc tests. C Representative micrographs of cultured fibrocytes from an MF patient were analyzed using immunofluorescence. BM LDCs aspirated from 11 MF patients were cultured in conditions favoring differentiation to fibrocytes and co-stained for GLI1 (Alexa Fluor 594, pseudo-colored red) and F-actin (Alexa Fluor 488, pseudo-colored green). Like in MF BM biopsies GLI1 is highly expressed in cultured MF fibrocytes and co-localizes with the cells’ nuclei (DAPI, pseudo-colored blue). D Representative micrographs of cultured fibrocytes of an MF patient were analyzed using multiplexed fluorescence RNA in situ hybridization. BM LDCs from three MF patients were cultured in the fibrocyte-forming assay and hybridized using GLI1 (Opal 520, pseudo-colored red), MMP2 (Opal 620, pseudo-colored green), MMP9 (Opal 570, pseudo-colored cyan), and procollagen-I (COL1A1; Opal 690, pseudo-colored magenta) probes. mRNA of all 4 analyzed genes was detected in MF fibrocytes. DAPI (pseudo-colored blue) was the nuclear counterstain. E Representative micrographs of cultured MSCs of an MF patient were analyzed using fluorescence immunostaining. BM LDCs aspirated from five MF patients were cultured in conditions favoring differentiation to MSCs and co-stained for GLI1 (Alexa Fluor 594, pseudo-colored red) and F-actin (Alexa Fluor 488, pseudo-colored green). DAPI (pseudo-colored blue) was the nuclear counterstain. F Quantitation of mRNA expression of GLI1 and the GLI1-target genes MMP2 and MMP9 in cultured fibrocytes or MSCs of MF patients and NCs as analyzed by qRT-PCR. mRNA levels in MF fibrocytes (n = 18) were compared to MF MSCs (n = 9) and NC fibrocytes and MSCs (n = 8 each). Horizontal lines denote mean. The statistical significance of differences between groups was calculated by one-way ANOVA followed by Tukey’s post hoc tests. G Protein levels of GLI1, MMP2, and MMP9 in cultured fibrocytes and MSCs of MF patients (n = 3) and an NC were assessed by using western immunoblotting. GLI1, MMP2, and MMP9 proteins were detected only in MF fibrocytes. All target proteins were detected by sequential probing of the same blot and β-actin was used as a loading control. All micrographs were adjusted for brightness and contrast linearly and equally between groups. Scale bars represent 100 µm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, respectively.