Fig 1: Short hairpin RNAs (shRNAs) for Ku70/80 inhibit the proliferation of ALT cells(A) shRNAs for Ku70/80 reduce the levels of Ku70/80 in CCL75.1 cells. CCL75.1 cells were infected with lentiviruses for the conditional expression of shRNAs targeting Ku70 and Ku80 or GFP and cultured for 3, 7 and10 days in the presence of 1.0 mg/ml Doxycycline (DOX). The levels of Ku70 and Ku80 were analyzed by immunoblotting with antibodies against Ku70 and Ku80. Antibodies against tubulin were used as a loading control. We estimated that 7 to 10 days after induction, Ku70/80 shRNAs-expressing cells display an 80 to 85% decrease in Ku70/80 expression compared to GFP shRNAs control. (B) Growth curves of CCL75.1 expressing shRNAs for Ku70/80 or GFP. After 5 days in Geneticin and hygromycin selection, 1.0ug/ml DOX was added to the media and the growth rate of each CCL75.1 cell line was measured by counting viable cells every 2 days. Cells were seeded at a low density, and the medium was changed every 2 days. Values represent the mean ± the standard deviation of three experiments (n = 3). (C) Detection of SA-β-gal activity. CCL75.1 cells transduced with lentiviruses for the conditional expression of shRNAs targeting K70/80 or GFP were grown for 3, 6, 9, or 12 days after addition of DOX and were stained for SA-βgal activity as previously described [14]. Values are the mean ± the standard deviation of three independent experiments (n = 3) carried out in duplicates in which 500 cells were scored for SA-β galactosidase. Student's t test was used to evaluate differences in means between two groups, and P < 0.05 was considered statistically significant. ALT cells (CCL75.1 and Saos2) were purchased from ATCC.