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. 2022 Jul 8;13:3960. doi: 10.1038/s41467-022-31697-3

Fig. 4. Wnt induces osteogenesis by enhancing Mast4 stability through inhibition of GSK-3β.

Fig. 4

a Representative ALP staining results of osteogenic differentiated C3H10T1/2 cells obtained from at least three independent experiments. b MC3T3-E1 preosteoblasts were treated with Wnt3a conditioned medium for the indicated time. c Wild-type and Mast4-PDZ-overexpressing C3H10T1/2 cells were differentiated into osteoblasts for 10 days. d Mast4-PDZ and GSK-3β were transfected to C3H10T1/2 cells, followed by CHIR-99021 treatment (10 μM for 9 h). e Mast4-PDZ and GSK-3β were transfected to C3H10T1/2 cells, followed by immunoprecipitation assay. The bands, which were recognized by phosphoserine antibody, were later reprobed with HA antibody. f Smurf1, Mast4-PDZ and GSK-3β were transfected to C3H10T/12 cells treated with CHIR-99021 treatment (10 μM for 9 h), followed by immunoprecipitation assay. g Smurf1 and GSK-3β were transfected to wild-type and GSK-3β-depleted Mast4-PDZ-overexpressing C3H10T1/2 cells. h Mast4-PDZ WT, P628A/Y634A and Smurf1 were transfected to wild-type and GSK-3β-depleted C3H10T1/2 cells. i Various Mast4 kinase domain deletion mutants and GSK-3β were transfected to C3H10T1/2 cells. j Flag-Mast4-PDZ WT and ∆632-636 were co-transfected into C3H10T1/2 cells together with HA-Ubiquitin in the absence or presence of Smurf1 and GSK-3β. Cell lysates were immunoprecipitated with Flag antibody and immunoblotted with the indicated antibodies. k MC3T3-E1 cells were transfected with Mast4-PDZ WT and Δ632-636, followed by treatment of Wnt3a conditioned medium (2.5 h). l 6xOSE-Luc, Mast4-PDZ WT and ∆632-636 were transfected to C3H10T1/2 cells, followed by treatment of Wnt3a conditioned medium (18 h). Data are representative mean ± SD of three independent experiments, each conducted in triplicate (n = 3). Unpaired two-tailed Student’s t test (P < 0.05) with Benjamini-Hochberg correction for multiple tests was conducted for all statistical analyses. b–k The representative results were obtained from at least three independent experiments. TCL: total cell lysates. Uncropped blots in b–k are shown in Source Data file. Source data for l are provided as a Source Data file.