Research Paper Volume 3, Issue 4 pp 395—406

Figure 5. Depletion of WRN does not influence the levels of t-circles in CCL75.1 cells. (A) CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs for WRN or GFP and analyzed 3 days (shWRN) and 7 days (shWRN and shGFP) after the addition of 1.0 mg/ml DOX to the media. Protein levels were determined by Western blotting with WRN antibodies. We estimated that WRN shRNAs reduce its cognate gene expression ~75% compared to GFP shRNAs control after 7 days of DOX induction. Antibody against GAPDH was used as a loading control. (B) Genomic DNA isolated from CCL75.1 cells expressing shRNAs targeting WRN or GFP for 7 days was digested with the restriction endonucleases HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA)₄ probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate levels of t-circles present in each sample (expressed as a percentage of the total telomeric DNA) were estimated as in [14] and is shown in the upper right corner.