Knockdown of SMC1 with shRNA blocks viral genome amplification.
A). CIN612 were infected with lentiviruses encoding shRNAs to SMC1 and after 48 hours total cell extracts were isolated and analyzed by Western blot for levels of SMC1. Quantitation of band intensity revealed an approximate 50% knockdown of SMC1 protein levels on average across three experiments. The knockdown is statistically significant between mock and shSMC and shCTRL and shSMC, p≤.0001, and p≤.001 respectively. B). shRNA knockdown of SMC1 blocks HPV-31 differentiation-dependent viral amplification. Southern blot analysis of CIN 612 cells infected with lentiviruses encoding shRNAs to SMC1 and induced to differentiate in methylcellulose for 48 hours. UD stands for undifferentiated while D stands for differentiated. Total DNA was isolated and examined by Southern analysis for HPV amplification. Similar results were seen in four independent experiments using two different shRNAs as well as the combination of the two. Quantification of band intensity was determined by densitometry using Image J software and represents an average of three independent experiments. SMC knockdown results in a reduction in amplification that is statistically significant, p≤.05