Analyses of SPARC expression and secretion from mammary tumor cell lines with different metastatic capacities. Cells were seeded into 6-well plates at a density of 1 × 105 cells (4T1 or 168FARN) or 1.5 × 105 cells (67NR) per well and cultured at 37°C. Cell-free medium served as a control. Seventy-two hrs after culture, cell media were collected, and total RNA as well as protein extracts were prepared from the three cell lines. (A) Real-time qRT-PCR was used to analyze the relative expression of SPARC mRNA level. The SPARC mRNA was normalized by GAPDH expression and the relative expression level is represented as a fold of the SPARC mRNA level in 4T1 cells. (Data represented as mean ± SEM, n = 4). *P < 0.01, compared with 4T1; **P < 0.01, compared with 168FARN. (B) Cellular SPARC protein expression was assessed using Western blot analysis. The illustrated result is a representative of three independent experiments. (C) The levels of secreted SPARC protein in the culture media were assessed by Western blot analysis. Cell-free medium (Med) was used as a control. Densitometric quantitation was performed and results were adjusted for total protein content of cell lysate (densitometry/μg cell total protein). The relative level of secreted SPARC in the medium of 4T1 cells is set as 1. Data represented as mean ± SEM (n = 3). *P < 0.01, compared with 4T1; **P < 0.01, compared with 168FARN.