FIG 2.

TWIST1 RNA and protein levels are downregulated by E2 and the HPV16 genome in N/Tert-1 cells. (A) qRT-PCR of N/Tert-1 lines with E2 (lane 2), HPV16 (lane 3), E6 and E7 (lane 4), or empty pCDNA3.1 vector (lane 1). Results are expressed as fold change from that observed in the vector control N/Tert-1 cells (lane 1). (B) Western blot analysis was carried out on protein extracted from N/Tert-1 cells with empty vector (lane 1) or those with E2, HPV16, or E6 and E7 (lanes 2 to 4). GAPDH is shown as an internal control. Western blotting was visualized using a Li-Cor system. (C) Western blotting was quantitated, and TWIST1 protein expression was calculated relative to vector using ImageJ. (D) Repression of TWIST1 by E2 and HPV16 leads to reduction in EMT marker expression. N/Tert-1 cells were harvested for RNA and processed for cDNA. qRT-PCR was performed for TWIST1 target genes CDH2 and VIM, which encode the proteins N-cadherin and vimentin, respectively. Results are expressed as fold change from that observed in the vector control N/Tert-1 cells (lane 1). Data in panels A, C, and D represent the averages of at least 3 independent experiments, and error bars indicate standard error of the mean. *, P < 0.05.