Fig. 6
Ninj1 is a key modulator of the activation of Wnt/β-catenin pathway in NSCLC cells. a Real-time PCR analyses examining mRNA expression of Wnt ligands (WNT1-WNT8A), Frizzled receptors (FZD1-FZD10), LRP (LRP5 and LRP6), DVL (DVL2 and DVL3), and β-catenin (CTNNB1) in H1299-EV and H1299-Ninj1 cells. b, c Western blot analysis examining active β-catenin (β-cateninact) protein in the indicated NSCLC cells after treatment with cycloheximide (CHX, 100 μg/mL) for the indicated time-points (b) or in cells treated with MG132 (10 μM) (c). d Western blot analysis examining the expression of the indicated canonical Wnt/β-catenin signaling components and the phosphorylation of EGFR, IGF-1R, and ROR1 in H1299-Ninj1, PDX-siNinj1, and their control cells. e Western blot analysis examining the expression of the indicated canonical Wnt/β-catenin signaling components in H1299-EV and H1299-Ninj1 cells in response to treatment with IWP-2. f-h Western blot analysis of IgG, anti-Ninj1 (f, h), and anti-LRP6 (g, h) immunoprecipitates (IPs) for the indicated Wnt/β-catenin signaling proteins to determine interactions among Ninj1, LRP6, FZD2, and other Wnt/β-catenin signaling components in the indicated NSCLC cells. i Pull-down assays assessing the interaction between glutathione-agarose-bound full-length (FL) or N-terminal (NT) domain of GST-Ninj1 and the Wnt signaling components, including LRP6, FZD2, Dvl3, Axin1, and GSK-3β, in H1299 whole cell lysates (WCL). j Schematic model of the Wnt/β-catenin signaling pathway in the absence or presence of Ninj1. In the absence of Ninj1 (a resting state), Wnt signaling is inactivated by the destruction complex-mediated β-catenin destabilization. Ninj1 promotes the assembly of the LRP6 signalosome, thus leading to a weak activation of the Wnt/β-catenin signaling pathway. Wnt ligand fully activates the Wnt/β-catenin signaling pathway in the presence of Ninj1. All in vitro experiments were performed at least three times. The bars represent the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001, as determined by a two-tailed Student’s t-test