Figure 7

Fenretinide treatment increases SPARC expression and secretion from mammary carcinoma cells. 4T1, 168FARN or 67NR cells were treated with various concentrations of fenretinide (1.25 μM, 2.5 μM and 5 μM) or left untreated (control) for 24 hrs. (A) Total RNA was isolated from different cell lines and SPARC mRNA level was analyzed using real-time RT-qPCR. Experiments were conducted in quadruplicate and normalized to GAPDH mRNA level. The relative SPARC mRNA level is represented as a fold of the SPARC mRNA level in untreated cells (data shown are mean ± SEM, n = 4). Compared with control, *P < 0.05, **P < 0.01, ***P < 0.001. (B) Total protein extracts were prepared from different cell lines and expression of SPARC protein levels were assessed using Western blot analysis. Expression of β-actin was used as an internal control. (C) Conditioned media were collected from 4T1 (left panel) and 67NR (right panel) cell lines and the levels of secreted SPARC protein were assessed by Western blot analysis. 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 untreated cells is set as 1. Data represented as mean ± SEM (n = 3). Compared with control, ***P < 0.001. (D) Effect of fenretinide treatment on the transcriptional activity of the SPARC promoter. 4T1, 168FARN and 67NR cells were transiently co-transfected with the luciferase reporter vector pREP4-SP-Luc and pRL-CMV. The pRL-CMV reporter was used as an internal control. 24 hrs after transfection of plasmids, cells were cultured for 24 hrs with or without different concentrations of fenretinide, and the luciferase activity was analyzed by the dual-luciferase reporter assay system. Luciferase relative activity is presented as a fold of the luciferase activity of the cells without fenretinide treatment. The data shown (mean ± SEM) represent the averages of three independent experiments performed in triplicate.