Fig. 5

LCN2 regulated lactic acid-mediated cancer stem-like cells. (a) 5637 and MC-T2 cells were cultured in 2D and 3D sphere conditions. Representative images of 2D cells and 3D spheres are shown (200x magnification, scale bar: 100 μm). (b) qPCR analysis of stemness-related markers (Nanog, Oct-4, SOX2 and SOX5) in 2D cells and 3D spheres. Relative gene expression was normalized to GAPDH level and expressed as mean ± SEM (*p < 0.05, **p < 0.01). (c) Cell lysates of 2D and 3D spheres were collected, LCN2 expression and STAT3 activity (pY705) were examined by western blot analysis. (d) Western blot results confirmed the efficient knockdown of LCN2 expression in LCN2-silenced cells compared to scrambled control cells. β-actin was used as a loading control to ensure equal protein loading. (e) Quantitative PCR (qPCR) analysis was performed to evaluate the expression levels of stemness-related markers, including Nanog, HOXA4, Oct-4, and SOX5, in sphere-forming scrambled control, STAT3-silenced, and LCN2-silenced 5637 cells. Gene expression levels were normalized to GAPDH and are presented as mean ± SEM from at least three independent experiments. Statistical significance was determined using an unpaired two-tailed t-test (*p < 0.05, **p < 0.01). (f) Stemness-related markers (Nanog, HOXA4, Oct-4, and SOX2) expression was examined in sphere-forming scrambled control, STAT3-silenced, and LCN2-silenced MC-T2 cells by qPCR analysis (*p < 0.05, **p < 0.01). (g) Representative images of 3D spheres formed by scrambled control, and lactic acid-treated scrambled control, STAT3-silenced, and LCN2-silenced 5637 cells (LA 10 mM) were captured at 100x magnification (scale bar: 100 μm) to assess sphere formation efficiency among the different treatment groups (left panel). Additionally, qPCR analysis was performed to evaluate the expression levels of stemness-related markers (Nanog, HOXA4, Oct-4, and SOX2) in sphere-forming cells. Gene expression was normalized to GAPDH and presented as mean ± SEM (*p < 0.05, **p < 0.01). (h) Evaluation of the role of lactic acid and LCN2 in chemoresistance to gemcitabine. Scrambled control and LCN2-silenced 5637 cells were pre-treated with 10 mM lactic acid before exposure to gemcitabine (0, 7.8, 15.625, 31.25, 125, 500, 1000, 2000 nM). Cell viability was measured using the MTT assay, and the IC50 values were calculated.