Fig 2: Characterization of GPNMB/OA expression in lung cancer cell lines(A) GPNMB/OA mRNA levels (mean ± SD; n = 4) in lung cancer cell lines as determined by qPCR (***p < 0.001 for SK-MES-1 and A549 cells versus calu-6). (B) The extent of GPNMB/OA ECD protein shedding (24 hr) into the conditioned media by SK-MES-1, A549 and calu-6 cells as determined by ELISA (mean ± SD; n = 6). The extent of GPNMB/OA ECD protein shedding in SK-MES-1 and A549 cells was significantly higher than calu-6 cells (***p < 0.001). (C) Knockdown of GPNMB/OA expression (mean ± SD; n = 4) resulted in a significant reduction (***p < 0.001) in GPNMB/OA ECD in conditioned media of SK-MES-1. #p > 0.05 when comparing scrambled siRNA-transfected cells versus control cells (-) GPNMB/OA-siRNA.

Fig 3: GPNMB/OA ECD protein promoted cell adhesion to fibronectin-coated or rOA-coated plates(A) Adhesion of SK-MES-1 and calu-6 cells (mean ± SD; n = 4–5) to fibronectin-coated plates at 37°C (***p < 0.001 for SK-MES-1 versus calu-6 cells). The extent of cell adhesion expressed as absorbance values was quantified using MTT dye. (B) Adhesion (mean ± SD; n = 6) of calu-6 cells to fibronectin-coated plates with or without rOA protein (50 ng/mL) treatment. (***p < 0.001; for calu-6 cells that were seeded with rOA versus control calu-6 cells. The extent of cell adhesion expressed as absorbance values was quantified using MTT dye. (C) Characterization (mean ± SD; n = 6) of calu-6 cell adhesion to culture plates coated with 1% BSA (negative control), fibronectin (8 µg/mL, positive control) and rOA (8 µg/mL, with or without RGD selective peptide). Cell adhesion to BSA-coated plates was significantly less than both fibronectin and rOA-coated plates. Cell treatment with a selective RGD peptide resulted in a significant reduction in cell adhesion to rOA-coated plates (*p < 0.05). The extent of cell adhesion expressed as fluorescence intensity was quantified by CyQuant assay.

Fig 4: Effects of GPNMB/OA on lung cancer cell invasion and migration(A) The percent healing rates (mean ± SD; n = 5–6) for SK-MES-1 and calu-6 cells at 24 hr after inflicting wound into the cell monolayers (***p < 0.001 versus calu-6 cells). (B) The number of migrated cells (24 hr, mean ± SD; n = 5–6) for SK-MES-1 and calu-6 cell lines using Transwell migration assay. (***p < 0.001 for SK-MES-1 cells versus calu-6 cells). (C) The number of migrated cells (24 hr, mean ± SD; n = 5–6) in transwell insert for calu-6 cells that were seeded with or without rOA (50 ng/mL). Calu-6 cells that were seeded with rOA showed a higher number of migrated cells versus control calu-6 cells (*p < 0.05). (D) The number of migrated cells (mean ± SD; n = 3–4) in transwell for SK-MES-1 with siRNA-mediated knockdown of GPNMB/OA was significantly reduced (*p < 0.05) compared to control cells with endogenous GPNMB/OA expression.