Figure 3 | Scientific Reports

Figure 3

From: Molecular hydrogen suppresses activated Wnt/β-catenin signaling

Figure 3

H2 facilitates β-catenin phosphorylation.

(a) L cells were treated with 10 μM MG132 in the presence or absence of 2 μM BIO with 10% H2 or 10% N2 gas for 6 h. Cell lysates were then immunoprecipitated (IP) with an antibody against β-catenin or mouse control IgG (IgG). Blots were immunostained with an antibody against phospho-β-catenin (at Ser45), phospho-β-catenin (at Ser33/Ser37/Thr41) and β-catenin. Representative Western blots of IP samples and input samples were shown in the upper panels and lower panels, respectively. Arrows indicate mono-ubiquitinated phospho-β-catenin. (b) L cells were exposed to indicated concentrations of BIO with 10% H2 or 10% N2 gas for 24 h. Representative Western blots are shown with densitometry of β-catenin/β-actin (n = 4). *P < 0.05 by Student’s t-test with Bonferroni correction. (c,d) HCT-116 cells (c) and HepG2 cells (d) were incubated with control CM (Cont.), Wnt3a CM, or 2 μM BIO with 10% H2 or 10% N2 for 24 h. HCT-116 cells carry Ser45-deleted β-catenin in one allele. HepG2 cells carry truncated β-catenin in one allele. Representative Western blots are shown with densitometry of β-catenin/β-actin (n = 3). Wild-type and Ser45-deleted β-catenin were analyzed together for HCT-116 cells (c), whereas wild-type and truncated β-catenin were individually analyzed for HepG2 cells (d). *P < 0.05 by Student’s t-test. (e) L cells were transfected with wild-type (HA-WT-β-catenin) or mutant forms of HA-β-catenin (HA-ΔN-β-catenin and HA-S37A-β-catenin). Cell were then treated with 10% H2 or 10% N2 gas for 24 h. Wnt/β-catenin signaling activity was measured by Topflash luciferase reporter assay (n = 4). *P < 0.05 by Student’s t-test. (f) L cells were pretreated with 30 nM okadaic acid for 30 min. Cells were then added with 2 μM BIO with 10% H2 or 10% N2 gas for 12 h. Representative Western blots are shown with densitometry of β-catenin/β-actin (n = 4). *P < 0.05 by Student’s t-test.

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