Fig 2: KLK7 overexpression in M74 cells results in altered cell morphology and modulates expression of cell adhesion molecules. (A) Representative phase-contrast images of KLK7-overexpressing cells: both the clonal M74-D6 and the batch-transfected M74-H KLK7 overexpressing cell lines reveal an altered cell morphology compared with M74-mock vector control cells. Cells overexpressing KLK7 display a spindle-shaped morphology with membrane protrusions. Scale bar = 50 µm. (B) Representative immunofluorescence images (green) show a strong decrease in E-cadherin (upper panel) and increase in MCAM/CD146 (lower panel) staining in KLK7-overexpressing M74-D6 and M74-H cells compared to M74-mock cells. Nuclear staining was performed with DAPI (blue). Scale bar = 100 µm.

Fig 3: Detection of KLK7 on formalin-fixed, paraffin-embedded sections of primary and metastatic melanomas by immunohistochemistry. (A) Representative images of the KLK7 expression pattern in human melanocytic lesions obtained by immunohistochemical staining. (a) Nevi with negative staining (intensity: 0); (b, c) primary melanoma with weak KLK7 staining (intensity: +1); (d, f) metastatic melanoma with moderate to strong KLK7 staining (intensity: +2 and +3); (e) primary melanoma with strong KLK7 staining (intensity: +3). Note that there is no staining in the nevi, but a significantly increased staining in primary (PM) and metastatic melanoma (MM). Immunohistochemical staining generally exhibits a heterogeneous cytoplasmic KLK7 expression regardless of the stage (compare b, c, and e versus d and f). As expected, normal-appearing epidermal cell layers adjacent to nevi (a) or to primary melanoma (b) show the highest KLK7 levels in the stratum corneum layers (white stars) (Yousef et al., 2000). Black arrows in (b), (d), (e), and (f) point to positive KLK7 immunoreactivity in the cytoplasmic compartment of the cells. The gray arrow in (a) points to a normal melanocyte with negative staining. Black stars in (c) indicate KLK7 staining in the vessels. Scale bar = 200 µm. (B) Staining intensity of KLK 7 was categorized into intensity ranges from 0 to 3+. The average percentage of cells in each group was determined and is shown in a stacked graph for each tissue type (n = 38). N, nevi; PM, primary melanoma; MM, metastatic melanoma.

Fig 4: Quantification of KLK7 mRNA and measurement of KLK7 protein secretion in melanoma M74-derived cell lines. Melanoma M74 cells were stably transfected with the KLK7 expression plasmid pRcRSV-KLK7 or with the pRcRSV vector alone. M74-D6 represents a selected clone overexpressing KLK7, and M74-H is a batch-transfected M74-derived KLK7-overexpressing cell line. M74-mock, M74 cells, stably transfected with the empty vector only. (A) Total RNA from either M74-D6, M74-H, or M74-mock cells was reverse-transcribed and subsequently analyzed by SYBR Green fluorescence-based QPCR to quantify the KLK7 mRNA expression levels. GAPDH was used as the endogenous reference gene for normalization of KLK7 expression. (B) Conditioned medium was collected from either M74-D6, M74-H, or M74-mock cells after 24 h, and KLK7 antigen levels were assessed by ELISA. The inset depicts the KLK7 antigen levels measured in cell culture supernatants of HaCaT cells. Data represent the mean protein values ± SEM of three independent experiments.

Fig 5: KLK7 overexpression but not KLK7 mutant induces melanoma cell migration and invasion. (A) M74-D6, M74-H, and M74-mock cells were plated on 8-µm pore-size Transwell® inserts. After 24 h, cells were stained with crystal violet/methanol and migrating cells were counted using an optical imaging system. Migrated cells from six filter fields were calculated with the image j software. **P < 0.01; ***P < 0.001. (B) M74-cells overexpressing wild-type KLK7 (M74-H), its active site mutant (M74-KLK7-S/A), or M74-vector cells (M74-mock) were plated and analyzed as described in (A). M74-H versus M74-mock, ***P < 0.001; M74-KLK7-S/A versus M74-mock. NS (P > 0.05). (C) Transwell invasion assay: M74-D6, M74-H, and M74-mock cells were plated on 8-µm pore-size Transwell® inserts precoated with 10 µg of Matrigel™ (5 × 104 per well) and invasion of the cells analyzed as described in (A). ***P < 0.001.