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. Author manuscript; available in PMC: 2010 Sep 22.
Published in final edited form as: Oncogene. 2009 Jan 12;28(8):1089–1098. doi: 10.1038/onc.2008.456

Figure 3. Knockdown of Id1 and Id2 gene expression caused apoptosis.

Figure 3

RIE-1 cells were transfected with rat Id1, Id2 and Id3 siRNA (50 nmol/L) using DharmaFECT3 in DMEM supplemented with 5% dFBS for 20 h and then changed fresh medium supplemented with 0.5% dFBS for 4 h after siRNA transfection. Scrambled siRNA was used as non-specific control (NS). A. Total RNA was prepared and converted to cDNA for real-time quantitative PCR analysis of Id1, Id2 and Id3 mRNA expression. Id mRNA levels were represented as fold of NS control. B. Total cell lysates were prepared and Western blotting was applied to detect Id1, Id2 and Id3 protein expression level using specific anti-Id1, anti-Id2 and anti-Id3 antibodies. The lysates of Cos-1 cells transfected with Id1, Id2 and Id3 expression plasmids were used as positive control (PC). C. RIE-1 and RIE-1/Smad3 cells were harvested and stained with Annexin V-FITC and propidium iodide. The percentage of apoptotic cells stained with Annexin V-FITC only was expressed as mean±SEM. The percentage of apoptotic cells stained with Annexin V-FITC only was analyzed in RIE-1 cells treated with and without TGF-β1. *p<0.05 compared to cells with NS transfection.