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Fig. 3 | Journal of Experimental & Clinical Cancer Research

Fig. 3

From: Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/β-Catenin signaling through Frizzled2-LRP6 assembly

Fig. 3

Ninj1 mediates the acquisition of CSC phenotypes in NSCLC cell lines. a Western blot analysis examining Ninjurin1 (Ninj1) expression in the indicated NSCLC cells with an enforced or knocked down Ninj1 expression via stable transfection with a mammalian expression vector or shRNA, respectively. b-d NSCLC cells that achieved upregulation (H1299-Ninj1 and H226Br-Ninj1) or downregulation (A549-shNinj1 and H460-shNinj1) of Ninj1 were subjected to cell counting (b), hanging drop assays for cell-to-cell cohesion (c; left), cell adhesion assays for cell adhesion to the extracellular matrix (type I collagen [Col] and fibronectin [Fn]) adhesion (c; right), and anchorage-independent (AID) colony-forming assays (d). e-l Analyses of the indicated NSCLC cell lines with manipulation of Ninj1 expression via anchorage-dependent (AD) (e) and -independent (AID) (f) colony formation under hypoxia (1% O2), serum starvation (0% FBS), glucose deprivation (1 mM 2-deoxy-L-glucose [2-DG]), and exposure to paclitaxel (10 nM) and cisplatin (10 μM) in combination (Pc/Cs); Western blot analysis revealing pyroptotic or apoptotic cell death by treatment with Pc/Cs (g); Western blot (h) and real-time PCR (i) analyses on the levels of protein and mRNA expression of CSC markers; flow cytometric ALDH assay (j); sphere formation analysis (k); limiting dilution assay examining tumorigenic potential (l). Tumor initiating cell frequency was determined using ELDA (l). m Growth of xenograft tumors from the indicated NSCLC cell lines with stable overexpression/or knockdown of Ninj1 expression. 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 or Mann-Whitney test by comparison to the indicated group. Cl-Cas3: cleaved caspase 3; Cl-Cas1: cleaved caspase 1; Cas1: caspase 1

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