Figure 5: Role of IFRD1 in hrHPV+ cervical cancer cells.

(a) IFRD1, RelA acetylation and total RelA levels at steady state in three KC donors and three HPV16-induced CxCa lines. (b) RT–qPCR of IFRD1, CCL2, RANTES, IL-8 and CXCL9 expression, and IFRD1 protein levels in steady-state control or IFRD1 KD Caski cells. (c) RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 KD Caski cells. (d) Histogram of EGFR expression on three HPV16-induced CxCa lines. (e) Geomean of EGFR expression on KCs and CxCa, as determined by flow cytometry. s.e.m. of two independent experiments. (f) 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 Caski cells. (g) IFRD1 and RelA K310 acetylation status in Caski cells treated for 72 h with 0, 1 or 10 μg ml⁻¹ anti-EGFR (cetuximab) or anti-CD20 (rituximab). (h) RT–qPCR of IFRD1 expression in KCs and Caski cells treated for 72 h with 0, 0.1, 1 or 10 μg ml⁻¹ anti-EGFR or anti-CD20. (i) RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated control (dimethylsulphoxide (DMSO)) or entinostat-treated Caski cells. (j) Schematic representation of IFRD1-mediated RelA (de-)acetylation. (I) In KCs, RelA acetylation is positively regulated by KATs, resulting in the production of pro-inflammatory cytokines. HDACs may suppress this process. (II) In HPV+KCs, elevated EGFR levels can induce the expression of IFRD1, which can mediate RelA deacetylation by forming a bridge between RelA and HDAC1 and/or -3, hampering pro-inflammatory gene expression. (III) Interfering with EGFR signalling (1 and 2) or HDAC function (3) may lower IFRD1 levels, restoring the RelA acetylation balance, augmenting pro-inflammatory gene expression. 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.