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. 2014 Mar 6;10(3):e1003957. doi: 10.1371/journal.ppat.1003957

Figure 2. Activation of LEDGF expression by the HPV E6/E7 oncogenes.

Figure 2

(A) LEDGF transcript measurements by qRT-PCR. Primary human keratinocytes were transduced with retroviral vectors coding for HPV16 E6, -E7, or -E6/E7 and relative LEDGF transcript levels were determined (the value for keratinocytes transduced with the empty retroviral vector (-) was arbitrarily set at 1.0). Standard deviations are indicated. Asterisks above columns indicate statistically significant differences from control cells transduced with the empty retroviral vector, with p-values of ≤0.01 (**) or ≤0.001 (***). (B) Analysis of LEDGF promoter activities by luciferase reporter assay. Left panel: Primary keratinocytes were transfected with expression vectors coding for HPV16 E6, -E7, or -E6 and E7, together with a luciferase reporter plasmid (pGL4.10LEDGFp75 −723/+59) under transcriptional control of the LEDGF promoter [60]. Relative luciferase activities (RLA) are indicated above those of cells transfected with the empty expression vector (-), arbitrarily set at 1.0. Central panel: HepG2, A549 and HaCaT cells were transfected with expression vectors coding for HPV16 E6 and E7, together with pGL4.10LEDGFp75 −723/+59. RLA are indicated above those of respective control cells transfected with the empty expression vector (arbitrarily set at 1.0). Right panel: HepG2 cells were transfected with expression vectors coding for E6 and E7 from HPV6, HPV11, HPV16 and HPV18, together with pGL4.10LEDGFp75 −723/+59. Relative luciferase activities (RLA) are indicated above those of cells transfected with the empty expression vector (-), arbitrarily set at 1.0. Standard deviations are indicated. Asterisks above columns indicate statistically significant differences from cells transfected with the empty expression vector, with p-values of ≤0.05 (*),≤0.01 (**), or ≤0.001 (***). (C) Analysis of LEDGF promoter activities by luciferase reporter assay. HeLa cells were transfected with shRNA-expressing vectors blocking E6 (sh18E6-1, and -3) or E6/E7 (sh18E6/E7-1, -2, and -3) together with reporter plasmid pGL4.10LEDGFp75 −723/+59. shContr-1 and shNeg: negative controls. Luciferase activities are indicated relative to those for shContr-1-transfected cells (arbitrarily set at 1.0). Standard deviations are indicated. Asterisks above columns indicate statistically significant differences from shContr-1-transfected cells, with p-values of ≤0.05 (*),≤0.01 (**), or ≤0.001 (***). (D) Basal LEDGF promoter activities. Primary cervical keratinocytes (CxK3), HPV18-positive HeLa and HPV16-positive SiHa cervical carcinoma cells were transfected with reporter plasmid pGL4.10LEDGFp75 −723/+59. Values are indicated relative to corresponding control cells transfected with basic luciferase vector pGL4.10 (devoid of the LEDGF promoter fragment), arbitrarily set at 1.0. Standard deviations are indicated. Asterisks above columns indicate statistically significant differences from CxK3 cells, with p-values of ≤0.05 (*) or ≤0.01 (**).