Specificity of test shRNA towards HPV-16 enhancer. a Time dependent fall in the expression of HPV-16 E6 and E7 by NFκBEn–Pr+2-HPV-16–E6/E7, in SiHa cells, showed maximum suppression after 5 days (p < 0.05 at all-time points). The apparent increase in E6 and E7 mRNA on the 6th day compared with 5th day was statistically insignificant (p = 0.22). b, c Decrease in E6 and E7 mRNA levels is seen in both HPV-16 positive cell lines SiHa and CaSki and the fall in E6/E7 expression is in concordance with strength of the construct driving shRNA expression. NFκBEn–Pr+2-HPV-16–E6/E7 significantly decreased HPV-16 E6/E7 mRNA levels over PLAPPr+2-HPV-16–E6/E7 in SiHa cell line (p = 0.022 and p = 0.030 for E6 and E7, respectively) and CaSki (p = 0.041 and p = 0.017 for E6 and E7, respectively). d, e Post HPV-16 E6/E7 suppression by shRNA, significant increase in the expression of p53 target genes was observed in SiHa and CaSki cells at the mRNA level. f and g Restoration of p53 protein, post HPV-16 E6/E7 suppression corroborated with the mRNA levels of PUMA and NOXA. h and i Decrease in the HPV-16 E7 expression, post shRNA treatment, significantly reduced levels of E2FI candidate genes like cyclin A2 and E (p < 0.05).