Fig. 2

Ninj1 is tightly associated with the survival capacity and CSC-like traits of NSCLC cells. a Western blot analysis of Ninjurin1 (Ninj1) expression in the indicated NSCLC and normal HBE cell lines. b, c Real-time PCR (b) and Western blot (c) analyses of the levels of Ninj1 mRNA and protein expression, respectively, under culture conditions of hypoxia (1% O₂), serum starvation (0% FBS), glucose deprivation (1 mM 2-deoxy-L-glucose, 2-DG), or chemotherapy treatment (10 nM paclitaxel and 10 μM cisplatin in combination; Pc/Cs). d Schematic diagram and gating strategy of flow cytometry sorting for Ninj1^low and Ninj1^high populations. e-g, j, k, l The basal Ki67 positivity (e) and the anchorage-dependent (AD) (f) and -independent (AID) colony formation (g), mRNA expression of NINJ1 and CSC markers (POU5F1, NANOG, and SOX2) (j), sphere formation (k), and tumorigenicity (l) of Ninj1^low and Ninj1^high subpopulation of H460 (e-g, j, k) and A549 (e-g, j, k, l) cells. Scale bars: 50 μm (e). Tumor initiating cell frequency was determined according to ELDA (l). h, i Anchorage-dependent (AD) (h) and -independent (AID) colony formation (i) of Ninj1^high cells under hypoxia (1% O₂), serum starvation, glucose deprivation, and exposure to chemotherapy (Pc/Cs) compared to those of Ninj1^low cells. m, n Western blot (m) and/or real-time PCR (n) analyses of the levels of Ninj1 protein and mRNA expression and also of the CSC marker genes in the indicated NSCLC cells grown in monolayer (M) or sphere-forming conditions (S). o Gating strategy to isolate ALDH^high and ALDH^low populations. p, q Immunofluorescence (p) and real-time PCR (q) analyses assessing the levels of Ninj1 protein and mRNA expression, respectively, in the ALDH^low and ALDH^high populations in the indicated NSCLC cell lines. The Ninj1⁺ cells were determined using ZEN software. Scale bar: 20 μm (o). All 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