Figure 3: Blocking EGFR signalling decreases IFRD1 levels and rescues cytokine production by hrHPV+KCs.

(a) Microarray intensities for EGFR in KCs (n=4) and hrHPV+KCs (n=4) represented in a box plot. (b) Histogram of EGFR surface protein expression on KCs and HPV16+KCs, as determined by flow cytometry. (c) RT–qPCR of EGFR expression in KCs transfected with complementary DNA for E2, E5, E1+E2+E6+E7 or empty control. (d) RT–qPCR of IFRD1 expression in KCs and HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml⁻¹ anti-EGFR or anti-CD20. (e) IFRD1, RelA K310 acetylation and total RelA levels in KCs and HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml⁻¹ anti-EGFR or anti-CD20. (f) Quantified protein levels of IFRD1, RelA K310 acetylation and RelA over β-actin in HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml⁻¹ anti-EGFR (two-dimensional western blot). The expression levels of the 0 μg ml⁻¹-treated HPV+KCs were set as 100%. (g) RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated, anti-CD20- or anti-EGFR-treated HPV16+KCs (left) and KCs (right). (h) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h non- or IFN-γ- and TNF-α-stimulated, anti-CD20- or anti-EGFR-treated HPV16+KCs (left) and KCs (right). (i) RT–qPCR of IFRD1 expression in HPV16+KCs treated with inhibitors of PI3K (LY94002, 25 μM), mTOR (rapamycin, 50 nM), MEK1 (PD98059, 50 μM), RAF (GW5074, 20 μM) and JNK (SP60025, 20 μM). Gene expression was normalized using GAPDH as the calibrator gene. Fold changes over control were calculated and depicted. These data are representative for at least three independent experiments, except for h that was performed once. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t-tests. *P<0.05, **P<0.01 and ***P<0.001.