Fig 2: Transfection with siRNA TSPAN14 for 24 h reduces TSPAN14 expression in NCI-460. (a) Quantitative real-time PCR analysis of the TSPAN14 expression normalized to HPRT as endogenous control, in non-transfected NCI-H460 cells (control), the cells transfected with non-coding siRNA (siRNA NC), and siRNA TSPAN14 transfected NCI-H460 cells; (b) Representative micrographics of specific fluorescent labeling of Tspan14 protein (green) with the ascribed histogram quantification in control, siRNA NC and siRNA TSPAN14 NCI-H460 cells. Cell nuclei are labeled with Hoechst 33342 (blue). The scale bar in the pictures marks a length of 100 μm. The histogram shows the Tspan14 expression normalized to the control. All results are from at least three independent experiments. p < 0.05 (*), p < 0.001 (***) and p < 0.0001 (****) indicate statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells relative to control, while p < 0.001 (###) and p < 0.0001 (####) indicates statistically significant difference between siRNA NC and siRNA TSPAN14.

Fig 3: Decreased expression of TSPAN14 in NSCLC patients is correlated with a lower survival rate. (a) TSPAN14 gene expression in tumor tissue and the surrounding normal lung tissue in NSCLC patients. TSPAN14 expression was normalized to HPRT as endogenous control. Normalized ΔCt values are presented in a logarithmic scale. p < 0.05 (*) indicate a statistically significant difference in TSPAN14 expression in tumor samples compared to normal samples. (b) Kaplan-Meier survival curves show the NSCLC patients with TSPAN14 decreased expression (“TSPAN14 low expression”) and the NSCLC patients with higher TSPAN14 expression (“TSPAN14 others”). Low TSPAN14 gene expression is indicated with a 3-fold decrease in the tumor compared to the matching normal tissue sample, and the differences were statistically significant (p < 0.05). (c) Kaplan-Meier plotter graph survival curves showing OS of NSCLC patients with TSPAN14 low expression and the NSCLC patients with high TSPAN14 expression (n = 1144); (d) Kaplan-Meier plotter graph showing PFS of NSCLC patients with TSPAN14 low expression and the high TSPAN14 expression (n = 591).

Fig 4: The qPCR analysis of matrix metalloproteinases MMP2 and MMP9 gene expression in control, the siRNA NC, and siRNA TSPAN14 transfected NCI-H460 cells. The gene expression of MMP2 and MMP9 was normalized to ACTB as endogenous control. All results are from four independent experiments. p < 0.01 (**) and p < 0.001 (***) indicate statistically significant differences between siRNA TSPAN14 transfected cells and control cells, while p < 0.001 (###) indicates a statistically significant difference between siRNA NC and siRNA TSPAN14 transfected cells.

Fig 5: NCI-H460 cells with silenced TSPAN14 expression demonstrate higher migratory and invasive potential. (a) Representative images of gelatin degradation by control, siRNA NC, and siRNA TSPAN14 transfected cells, attributed to histograms, show a degraded surface of gelatin under the cell per total surface of the cell. The scale bar in the figures marks a length of 50 μm. (b) Representative images of control, siRNA NC, and siRNA TSPAN14 cells that migrated through Matrigel® matrix to the other side of the porous membrane; the histograms represent an average number of cells per field, in 10 independent fields per membrane, from three independent experiments. The scale bar in the pictures marks a length of 100 μm. All results are from at least three independent experiments. p < 0.05 (*) indicates a statistically significant difference between siRNA TSPAN14 transfected cells and control cells.