Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( 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.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( 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.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Transwell Migration Assay, Expressing

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( 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.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Negative Control, Positive Control, Fluorescence, CyQUANT Assay

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) Calu-6 tumor growth in athymic (nu/nu) mice as expressed by relative tumor volume (mean ± SD; n = 4–5 mice). Relative tumor volumes for calu-6 tumors with rOA were significantly larger than calu-6 cells developed with PBS (calu-6; ** p < 0.01). ( B ) Tumor weights (mean ± SD; n = 4–5 mice) measured on day 34 post-tumor implantation in athymic (nu/nu) mice. Calu-6 tumors developed with rOA supplementation resulted in significantly bigger tumors (* p < 0.05) compared to calu-6 tumors without rOA.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Tumor Implantation

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A – B ) Representative tumor sections stained for cell proliferation based on for ki67 expression on day 34 post-tumor implantation in athymic nu/nu mice. The extent of ki67 staining was processed by ImmunoRatio for control calu-6 tumors (without exogenous rOA, with PBS) (A); and calu-6 tumors that received exogenous rOA supplementation (B). Top panel: original image. Bottom panel: original image with segmented staining components for quantification of Ki67 staining. ( C ) Quantification of Ki67 staining of tumor tissues based on percentage of positively stained nuclear area as processed with ImmunoRatio. *( p < 0.05, mean ± SD; n = 8 images) for calu-6 cells that were treated with exogenous rOA versus control calu-6 tumors.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Staining, Expressing, Tumor Implantation

Journal: Oncotarget

Article Title: Osteoactivin (GPNMB) ectodomain protein promotes growth and invasive behavior of human lung cancer cells

doi: 10.18632/oncotarget.7323

Figure Lengend Snippet: ( A ) Representative tumor sections stained for apoptosis using TUNEL assay on day 34 post-tumor implantation in athymic nu/nu mice. Arrows indicate TUNEL positive cells for control calu-6 tumors (PBS-treated) (A) and calu-6 tumors that received exogenous rOA supplementation ( B ). ( C ) Quantification of TUNEL staining of tumor tissues excised on day 34 from calu-6 tumor-bearing athymic (nu/nu) mice. Data-points are plotted as the percentage of apoptotic (TUNEL positive) cells (mean ± SD; n = 6 images). *( p < 0.05) for calu-6 cells that were treated with exogenous rOA versus control calu-6 tumors.

Article Snippet: Tumors were developed in female athymic nu/nu mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) by subcutaneous administration of calu-6 cells as we have previously reported [ ].

Techniques: Staining, TUNEL Assay, Tumor Implantation