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. 2016 May 9;113(21):E2945–E2954. doi: 10.1073/pnas.1605691113

Fig. 3.

Fig. 3.

USP6 regulates WNT signaling at the level of the Wnt ligand and receptor complex. (A) Inhibition of USP6 induced Wnt signaling by XAV939. HEK293T cells with integrated Wnt/β-catenin reporter were transfected with USP6 expression plasmid. Following treatment with increasing concentrations of XAV939 for 16 h, luciferase activity was measured. β-catenin(SA) serves as a control; it is constitutively active and resistant to degradation by the destruction complex. Data represent mean ± SD. (B) USP6 does not potentiate Wnt/β-catenin signaling induced by GSK3 inhibitor. HEK293T cells stably expressing the Wnt/β-catenin reporter were transfected with control or USP6 expression plasmids. The cells were treated with 1 μM GSK inhibitor CT99021 for 16 h. Luciferase reporter activity as measured is expressed as mean ± SD (n = 3). ns, not significant. (C) Inhibition of USP6-induced Wnt signaling by DKK1. HEK293 cells with an integrated Wnt/β-catenin reporter were transfected with USP6 and/or WNT3A and incubated with varying amounts of DKK1. Data represent mean ± SD (n = 3). (D) LRP6 silencing decreases USP6-induced Wnt signaling. HEK293T cells were transfected with the indicated siRNAs. Forty-eight hours after siRNA transfection with two independent LRP6 siRNAs (siA and siB) or control siRNA (siCNT), plasmids expressing GFP, USP6, or β-catenin(SA) were transfected and cells were treated with control- or WNT3A-conditioned medium. Luciferase reporter activity is represented as mean ± SD (n = 4). (E) C59 inhibits USP6-induced Wnt signaling. HEK293 cells with an integrated Wnt/β-catenin reporter were transfected with USP6 and WNT3A expression plasmids and incubated with the indicated concentrations of C59. Data represent mean ± SD (n = 3). (F and G) USP6 expression increases LRP6 phosphorylation. HEK293T cells were transfected with control or plasmids expressing USP6 or its variants before treatment with control- or WNT3A-conditioned media for the indicated duration. Total cellular LRP6 levels and phosphorylated LRP6 (p-LRP6) levels were determined by Western blot analysis.