Fig. 4

Ninj1 mediates the acquisition of CSC phenotypes in NSCLC cells in patient tumors. a Schematic diagram presenting the procedure for isolating primary tumor cells form patient-derived xenograft (PDX) tumors. b, d Immunofluorescence (IF) (b) and real-time PCR (d) analyses of Ninjurin1 (Ninj1) expression in the ALDH^low and ALDH^high populations from three different PDX tumors. Ninj1⁺ cells were identified using ImageJ software. Scale bars: 20 μm. c, e Western blot (c) and real-time PCR (e) analyses examining the levels of Ninj1 and CSC marker genes, respectively, in primary cultured patient-derived tumor cells grown in monolayer (M) or sphere-forming conditions (S). f Sphere formation and real-time PCR analyses assessing sphere formation and the mRNA expression of CSC markers and NINJ1 in the Ninj1^low and Ninj1^high population of three PDX-derived primary cultured cancer cells. g The Spearman correlation coefficient detailing the relationship between NINJ1 expression and the expression of stemness markers (POU5F1, NANOG, and SOX2). The correlation was determined by analyzing a GSE77803 dataset. h, i Real-time PCR (h) and flow cytometric ALDH (i) analyses used to determine mRNA expression of NINJ1 and stemness markers (h) and ALDH activity (i) in primary cultured patient-derived tumor cells after siRNA-mediated knockdown of Ninj1 expression. j Schematic diagram detailing the procedure for analyzing residual PDX tumors after chemotherapy. k Changes in the growth of three lung PDX tumors after treatment with a combination of paclitaxel and cisplatin (Pc/Cs; at a dose of 20 mg/kg of paclitaxel and 3 mg/kg of cisplatin, once a week) at the end of the treatment (PDX #1 and PDX #2: 30 days after the start of the treatment; PDX #3: 45 days after the start of the treatment). Con: vehicle-treated control. l IF analysis examining the levels of Ninj1 and Nanog expression and their correlation in three PDX tumors treated with Pc/Cs. The Ninj1⁺ cells were identified using ImageJ software. The significance of the correlation was determined by the Spearman rank correlation test. Representative images are presented in Fig. S3g. 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. Scr: scrambled siRNA