Fig. 3

DIPQUO can synergize with other known GSK3-β inhibitors to promote osteogenic differentiation. C2C12 cells were treated for 3 days with (A) subthreshold (5 μM) DIPQUO, (5 μM) CHIR, or a combination thereof or (B) subthreshold DIPQUO, (500 nM) AZD, or a combination thereof and analyzed by staining for ALP expression and by colorimetric substrate assay for ALP activity. C, ALP expression and activation normally promoted by the full dose of DIPQUO was attenuated by cotreating C2C12 cells with 10 μM XAV-939. The scale bar in panels A–C represents 200 μm. Values in ALP activity graphs are reported as the means ± SD; ∗∗∗∗p < 0.0001 in unpaired two-tailed Student's t test. Ordinary one-way ANOVA yielded F = 1868 and p < 0.0001 in panel A, F = 579.6 and p < 0.0001 in panel B, and F = 858.3 and p < 0.0001 in panel C. D, differentiating human skeletal muscle satellite cells were cotreated for 3 days with low-dose DIPQUO and CHIR (5 μM each) and then analyzed by quantitative RT-PCR for expression of transcripts associated with osteoblast specification, compared with treatment alone. Relative expression levels compared with DMSO-treated samples and normalized to glyceraldehyde 3-phosphate dehydrogenase are reported as the means ± SD; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 in unpaired two-tailed Student's t test. Ordinary one-way ANOVA yielded F = 29.08 and p = 0.0008 for hDLX5 (DISTAL-LESS HOMEOBOX 5); F = 9.654 and p = 0.0133 for hOSX (OSTERIX); F = 81.15 and p < 0.0001 for hOPN (OSTEOPONTIN); and F = 110.1 and p < 0.0001 for hGPNMB (OSTEOACTIVIN). E, synergistic activation of p38 MAPK was analyzed by Western blotting after 2.5 h treatment with subthreshold concentrations of AZD (100 nM) and DIPQUO (2.5 μM), compared with cotreatment using the same concentrations. Relative phospho-p38 MAPK levels are reported as the means ± SD; ∗p < 0.05. Ordinary one-way ANOVA yielded F = 3.521 and p = 0.0686. F, C2C12 cells were either mock-transfected or transfected for 24 h with hemagglutinin-tagged GSK3-β containing a serine-to-alanine mutation (S9A), treated with DIPQUO or DMSO vehicle, and analyzed by Western blotting for construct expression. G, equivalent samples were analyzed for ALP activity using colorimetric assay for pNPP substrate after 3 days. Activity levels are normalized to the total protein and reported as the mean ± SD; ∗p < 0.05 and ∗∗p < 0.01 in unpaired two-tailed Student's t test. Ordinary one-way ANOVA yielded F = 25.60 and p = 0.0002. In all figure panels, representative images are shown from at least three biological replicates. ALP, alkaline phosphatase; AZD, AstraZeneca GSK3-β-specific inhibitor AZD2858; CHIR, Wnt signaling activator CHIR99021; GSK3-β, glycogen synthase kinase 3-beta; pNPP, p-nitrophenylphosphate.