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. 2013 Feb 28;8(2):e58193. doi: 10.1371/journal.pone.0058193

Figure 3. Activation of Cdk4 enhances PAX3-FOXO1-mediated gene expression.

Figure 3

A) NIH3T3 cells were co-transfected with wild-type GFP-PAX3-FOXO1 (left panel) or GFP-PAX3-FOXO1 S430A (right panel), 6 X PRS9 reporter, TK-renilla (PRL-TK), and one of the plasmids as indicated. Comparisons were made between other samples and a sample transfected with pcDNA3 (set as 100 relative luciferase units, or RLU). Cdk4, pCMV-Cdk4 (wild-type); Cdk4DN, pCMV-Cdk4DN (inactive Cdk4); cyclin D1, pCMV-cyclin D1. B) Protein expression in A) was confirmed by Western blot analysis using anti-GFP antibody (IB: GFP), anti-Cdk4 antibody (IB: Cdk4), anti-cycin D1 (IB: cyclin D1), and anti-actin (IB: actin). Actin was used as a loading control. GFP-PF, GFP-PAX3-FOXO1. C) NIH3T3 cells were transfected with 6 X PRS9 reporter, TK-renilla, and one of the plasmids indicated. PF WT, wild-type GFP-PAX3-FOXO1; PFS430A, GFP-PAX3-FOXO1S430A; PFS430D, GFP-PAX3-FOXO1S430D. Comparisons were made between other samples and a sample transfected with wild-type GFP-PAX3-FOXO1 (set as 100 RLUs). D) Protein expression in B) was confirmed by Western blot analysis. Actin was used as a loading control. Luciferase activity was measured 24 h after transfection. Firefly luciferase activity was normalized using Renilla luciferase activity. * p<0.05; *** p<0.001.