Fig 2: Expression of EN2 and PSA in prostate cancer‐ and normal cells. (a) Confocal microscopy images of prostate normal (RWPE‐1 [1 × 105 cells/dish]) and cancer cells (DU‐145 [1 × 105 cells/dish] and PC‐3 [1 × 105 cells/dish]) treated with PE‐conjugated anti‐PSA antibodies (scale bar: 10 μm). (b) Relative fluorescence intensities of (a). (c) Results of immunoblotting of prostate normal cell (RWPE‐1) and cancer cell lysates (DU‐145 and PC‐3) using anti‐PSA antibodies. (d) Confocal microscopy images of prostate normal (RWPE‐1 [1 × 105 cells/dish]) and cancer cells (DU‐145 [1 × 105 cells/dish] and PC‐3 [1 × 105 cells/dish]) treated with FITC‐conjugated anti‐EN2 antibodies (scale bar: 10 μm). (e) Relative fluorescence intensities of (d). (f) Results of immunoblotting of prostate normal cell (RWPE‐1) and cancer cell lysates (DU‐145 and PC‐3) using anti‐EN2 antibodies. In (b) and (d), relative fluorescence intensities were presented by regarding the intensity of RWPE‐1 as 1.0. In (c) and (f), β‐actin is a constitutively expressed protein in all types of cells (positive control). EN2, engrailed‐2 protein; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PSA, prostate‐specific antigen

Fig 3: PCa diagnosis through detecting EN2 and PSA in PCa patient urine. (a) Results of 3p3 immunoassay‐based PCa diagnosis through EN2 detection using the urine of 30 PCa patients and 30 healthy individuals, presented in box and dot plots. (b) Results of ELISA‐based PCa diagnosis through EN2 detection using the urine of 30 PCa patients and 30 healthy individuals. COV represent cut‐off‐value, calculated as per instructions from ELISA kit supplier. False‐positive and ‐negative signals are marked by red and green bars, respectively. (c) Results of 3p3 immunoassay‐based PCa diagnosis through PSA detection using the urine of 30 PCa patients and 30 healthy individuals, presented in box and dot plots. (d) Correlation between urinary EN2 and PSA levels, indicating a very low Spearman's rank correlation coefficient (ρ = 0.176). 3p3, 3D‐plus‐3D; EN2, engrailed‐2 protein; PCa, prostate cancer; PSA, prostate‐specific antigen