Fig 1: (A) Fluorescence of adenocarcinoma lysate and QA-2OMeSiR in the presence of various concentrations of sitagliptin and puromycin in combination. Puromycin at concentrations above 1 µM (above the IC50) suppressed the increase in fluorescence. When both sitagliptin and puromycin were used at concentrations around the IC50 values ("sitagliptin 10 nM, puromycin 1 µM"; light blue line) and above, the inhibitory effect was increased. Error bars indicate standard errors. Triplicate measurements were conducted using the same lysate and inhibitors. S sitagliptin, P puromycin. (B) Assay of purified enzymes and QA-2OMeSiR. The fluorescence intensity of purified DPP4 and PSA (0.114 µM) mixed with QA-2OMeSiR increases with time. (C) Plot of the initial velocities of the enzymatic reaction with DPP4 and PSA against concentration of QA-2OMeSiR, and the kinetic parameters obtained using the Michaelis–Menten equation, V0 = Vmax × [S]/(Km + [S]). The catalyst efficiency, kcat/Km, was about 1.1 times higher for DPP4 than for PSA. Error bars indicate standard errors. Triplicate measurements were conducted using enzymes with the same lot numbers.
Fig 2: (A) Images taken 10 min after addition of QA-2OMeSiR to cell lines at 2 days after siRNA transfection. A549, H441, and H226 cells transfected with DPP4 siRNA or PSA siRNA show a suppressed increase of fluorescence compared to the Control siRNA-transfected cell lines. The scale bar represents 50 µm. (B) Increase in fluorescence intensity at 10 min after addition of QA-2OMeSiR, averaged over 15 cells. (C) Mean values of 15 measurements for each cell line and results of statistical comparison (t-test) of the effects of the siRNAs used.
Fig 3: Pathological confirmation of the presence of DPP4 and PSA by HE staining and immunohistochemical staining of adenocarcinoma, squamous cell carcinoma and normal lung tissue. Both adenocarcinoma and squamous cell carcinoma are positive for DPP4 and PSA staining. In normal lung tissue, some macrophages and alveolar epithelium were positive for DPP4, but others were negative. PSA was negative in all normal tissues.
Fig 4: Quantitative PCR analysis of DPP4 and PSA expression in lung cancer. In adenocarcinoma, both DPP4 and PSA were more highly expressed in the tumor than in normal lung tissue (n = 3, #11, #14, #17). In squamous cell carcinoma, DPP4 was rarely observed in either normal or tumor tissues, but PSA was frequently observed in tumor tissues (n = 3, #22, #23, #24).
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