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Fig. 1 Kinase profiling identifies GSK3-β as a biochemical effector of DIPQUO-mediated inhibition. A, to identify putative biochemical effector candidates for DIPQUO, in vitro FRET-based kinase profiling assays were performed using 10 μM DIPQUO. Sixty different kinases were tested in duplicate using Z'-LYTE (black spheres), LanthaScreen (blue spheres), and Adapta (purple spheres) assays. Kinases were scored for inhibition (positive scores) or activation (negative scores); for clarity and brevity, only positive scores are shown, with an arbitrary threshold of 70% inhibition (red dotted line) chosen for follow-up validation assays, with kinases achieving this threshold denoted by red spheres on graph x-axis, and in red print on the sidebar. Only GSK3-β (black arrow) displayed 100% inhibition, in both duplicate tests. Values are reported as the means ± SD. B, cellular thermal shift assay (CETSA) analysis of GSK3-β protein stability after 1 h of 10 μM DIPQUO treatment in C2C12 cells, compared with treatment with the inactive structural analog BT344 (also 10 μM). CETSA was performed across a broad temperature range (37–67 °C, upper panel) and a narrow range (50–60 °C, lower panel). Relative protein levels were quantified at each temperature gradient point from 50 to 60 °C in a 10-min thermal challenge and plotted on the graph at right in a decay curve to obtain a Tm (50 percent relative expression) of 53.1 °C for BT344 and 54.9 °C for DIPQUO. Values are reported as the means ± SD; ∗p < 0.05 and ∗∗p < 0.01 in unpaired two-tailed Student's t test. Two-way ANOVA yielded F = 3.805 and p = 0.0096. Inhibition of GSK3-β using indicated doses of the commercially available inhibitors CHIR99021 (CHIR) or AZD2858 (AZD) stimulated (C) ALP expression and (D) ALP enzymatic activity. Expression was detected using alkaline naphthol and hematoxylin staining, and activity was quantified using colorimetric analysis of pNPP substrate digestion, after 3-day treatment of C2C12 cells with 10 μM DIPQUO. Activity was normalized to the total protein content for each sample. Values are reported as the means ± S.D.; ∗∗p < 0.01 and ∗∗∗p < 0.001 in unpaired two-tailed Student's t test. Ordinary one-way ANOVA yielded F = 42.95 and p = 0.0003. The scale bar in panel C represents 200 μm. In all panels, representative images are shown for experiments performed in three biological replicates. ALP, alkaline phosphatase; GSK3-β, glycogen synthase kinase 3-beta; pNPP, p-nitrophenylphosphate; Tm, melting temperature.