Figure 2.

HPV16 oncogenes induce TREX1 expression in human keratinocytes and sensitize these cells to TREX1 silencing. TREX1 expression levels were determined by western blot in 30 micrograms of total protein extracts from monolayer (A) and organotypic (B) cultures of keratinocytes transduced with constructs expressing E6 and E7 from HPV16 and HPV11. (C) TREX1 expression was analyzed by immunohistochemistry in sections from organotypic cultures established from low passage number keratinocytes transduced with retroviral vectors expressing HPV16 E6 and/or E7 with. Besides, organotypic cultures established from keratinocytes transfected with HPV16 or HPV18 whole genomes and cultured for different passage number (p18 to p82) were used. Magnification: X400. (D) The effect of TREX1 silencing on cell viability was determined in control or HPV16 E6 and/or E7 transduced PHKs. Gene silencing was performed as described in Fig. 1. Cells were cultured in 96 wells plates (2000 cells/well) and after 72 hours 10 µL of Alamar blue per well were added. Cells were incubated at 37 °C and Alamar Blue’s reduction was monitored every hour in a spectrophotometer through absorbance measurement at 570 e 600 nm. (E) Clonogenic assays with low- (p4) and high-passage (p81) PHK expressing HPV16 E6 and E7 were performed as described in Fig. 1. The results shown are representative of at least three independent experiments performed in triplicate. *p-value ≤ 0.05.