Levels of CTCF are increased in undifferentiated and differentiated HPV positive cells.

A). Whole cell extracts were isolated from undifferentiated normal human foreskin keratinocytes (HFK) and CIN 612 cells grown in monolayer culture and examined by Western blot analysis with antibodies to CTCF. Cytokeratin 10 served as a differentiation control. GAPDH served as a loading control. CTCF knockdown results in a statistically significant reduction in protein levels, p≤.01. B). Knockdown of CTCF with shRNA blocks viral genome amplification. CIN612 were infected with lentiviruses encoding shRNA to CTCF and after 48 hours total cell extracts were isolated and analyzed by Western blot for levels of CTCF. shRNA knockdown of CTCF blocks HPV-31 differentiation-dependent viral amplification as shown by Southern blot. Southern blot analysis of CIN 612 cells infected with lentiviruses encoding shRNAs to CTCF and induced to differentiate in methylcellulose for 48 hours. Total DNA was isolated and examined by Southern analysis for HPV amplification. Similar results were observed in three independent experiments using two different shRNAs against CTCF. Quantification of band intensities represents averages of three independent experiments and were determined by densitometry using FIJI software. CTCF knockdown results in a reduction in amplification, p≤.05.