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Figure 4 | Molecular Cancer

Figure 4

From: Brg-1 mediates the constitutive and fenretinide-induced expression of SPARC in mammary carcinoma cells via its interaction with transcription factor Sp1

Figure 4

Brg-1 is required for SPARC gene expression. RNA interference- mediated depletion of Brg-1 reduces SPARC expression. 4T1 cells were transfected with mock, siRNA control or different concentrations of siRNA targeting Brg-1[0(mock)-50 nM] and were then cultured for another 48 hrs prior to harvesting the cells. (A) Total RNA was isolated from transfected 4T1 cells and mRNA levels of Brg-1 and SPARC were analyzed using real-time RT-qPCR. The relative mRNA level of Brg-1 or SPARC was represented as a percentage of the Brg-1 or SPARC mRNA level in mock-treated 4T1 cells (data shown are mean ± SEM, n = 4). (B) Total cell extracts were prepared from siRNA-transfected (30 nM) or mock-transfected 4T1 cells. Western blot analysis was performed to assess the protein expression levels of Brg-1, SPARC, Sp1 and β-actin (upper panel). Quantification of the protein expression of Brg-1, SPARC and Sp1 (data shown are mean ± SEM, n = 3) (bottom panel). **P < 0.001, compared with mock-treated cells. (C) Conditioned media were collected from siRNA-transfected (30 nM) or mock-transfected 4T1 cells, and the levels of secreted SPARC 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 mock-transfected 4T1 cells is set as 100%. Data represented as mean ± SEM (n = 3). (D) Overexpression of Brg-1 enhances the SPARC promoter-driven reporter gene transcription. 4T1 cells were co-transfected with the luciferase reporter vector pREP4-SP-Luc and expression plasmids encoding Brg-1 or its mutant (Brg-1K798R) or empty plasmids (control). Cell lysates were prepared 48 hrs after transfection and assayed for luciferase activity. Luciferase relative activity is expressed as a fold of the luciferase activity of the cells untransfected with Brg-1. The data shown (mean ± SEM) are the averages of three independent experiments performed in triplicate.

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