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. 2007 Aug 29;2(8):e802. doi: 10.1371/journal.pone.0000802

Figure 2. TrpC3 is necessary for hypertrophic gene expression.

Figure 2

(A) Real-time RT-PCR measurement of TrpC3 mRNA in myocytes infected with lentiviruses expressing shRNAs targeting either LacZ or TrpC3. Data is presented as percent decrease in expression compared to the mean expression of myocytes expressing LacZ-shRNA#1. Asterisks indicate significant difference compared to LacZ-shRNA#1. n = 4 per condition. (B) Real time RT-PCR measurements of BNP mRNA levels in myocytes infected with the same lentiviruses as in A. TrpC3 shRNAs reduce the expression of endogenous BNP mRNA. n = 4 per condition. (C) Luciferase activity in myocytes transfected with BNP-Luc and plasmids expressing shRNAs targeting either TrpC3 or LacZ. Cells were stimulated for 60 hours with 20 µM phenylephrine (PE), 100 nM endothelin-1 (ET1) or 150 ng/mL epidermal growth factor (EGF) to induce hypertrophy. n = 4 biological replicates per condition. (D) Luciferase expression in myocytes transfected with ANP-Luc and untreated or stimulated with 20 µM PE. n = 6 biological replicates per condition. (E) Myocytes transfected with plasmids expressing shRNAs targeting either LacZ or TrpC3 were stimulated with 20 µM PE for 60 hours and then fixed and immunostained with an anti-ANP antibody. TrpC3 shRNA expression caused a decrease in ANP staining. (F) Quantification of immunostained cells treated as in E. The integrated ANP-immunofluorescence intensity across the two-dimensional projection of transfected cells was measured. Average n = 31 cells per condition. In all panels, cells not expressing TrpC3-shRNA were expressing a control shRNA against LacZ. * indicates statistically significant difference compared to control, P<0.01. ** indicates P<0.05. For all bar graphs, data are represented as mean±SEM.