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. Author manuscript; available in PMC: 2010 Aug 1.
Published in final edited form as: Mol Cancer Res. 2009 Aug 11;7(8):1285–1293. doi: 10.1158/1541-7786.MCR-08-0508

Figure 2. Knockdown of UNG by RNAi suppress uracil excision activity and induces DNA damage.

Figure 2

(A) The UNG enzyme activities were analyzed by uracil cleavage assay using the siMM- and siUNG-transfected prostate cell lines LNCaP, DU145 and PC3. Cells were transfected with siUNG (200 nM) or siMM (200 nM) for 24 h. Cell extracts were incubated with a 34-mer double-stranded oligonucleotide containing uracil in the 5′-32P-end labeled strand. Purified UNG and BSA served as positive (+) and negative (−) controls, respectively. Reaction products were run on a denaturing PAGE and detected by autoradiograph. The upper band represents the non-cleaved DNA probe and the lower band represents the cleaved probe.

(B) Human prostate cancer cell lines LNCaP, DU145 and PC3 were transfected with siUNG (200 nM) or siMM (200 nM) for 24 h and then analyzed for fragmented DNA by comet assay under alkaline conditions. Representative pictures are shown.

(C) Quantitation of damaged DNA in siMM- and siUNG-transfected LNCaP, DU145 and PC3 cells as measured by the comet assay image analysis. The extent of the DNA damage induced by UNG knockdown is quantified by determining the tail moment. The tail moment of cells transfected with siMM is expressed as 100%. Results represent the average of three independent experiments with 100 cells (nuclei) analyzed per experiment; bars, ±SD. Significant difference from control (siMM) is represented by asterisks (*, P < 0.01).