Figure 4. Purmorphamine-mediated SMO activation recapitulates IGFBP-6 effects on HS5 cells. (A) qPCR results were obtained for SHH in HS5 cells exposed to 200 ng/mL of IGFBP-6 for 24h and 48h. Relative mRNA expression level normalized with β-actin by using a comparative 2-ΔΔCt method. **p <0.01; ***p < 0.001. (B) Immunofluorescence analysis were performed on HS5 cells treated with IGFBP-6 at the final concentration of 200 ng/mL, followed by fixing and staining with anti-Phalloidin (green) and anti-Gli1 (red). Nuclei were visualized using DAPI. Immunoreactivity was evaluated considering the signal-to-noise ratio of immunofluorescence (scale bar 20 μm). (C) z-score expression levels of Gli1, Gli2, GPATCH1, and SMO in healthy, JAK2 wild type, and JAK2V617F mutant PMF patients. (D–I) Multiplex immunobead assay technology on HS5 cells exposed or not to purmorphamine was performed on culture medium to determine concentrations of indicated cytokines. Histograms showed a significant increase of MMP9 (D), TIMP (E), CHI3L1 (F), BMP2 (G), OPG (H), and sRANKL (I) after purmorphamine stimulation, as compared to control. **p <0.01; ***p < 0.001.