Fig 2: Cas down-regulates NF-?B activity by interfering with RelA-p300/CBP interaction in the nucleus.(A) Absence of suppressive effect of nuclear export signal (NES)–attached Cas on RelA acetylation. HEK293 cells transfected with Cas, NES-Cas, or their control vector were analyzed by immunoblotting. (B) GFP-Cas–dependent decrease in acetylation, but not phosphorylation, of RelA in MEFs. Cas-deficient MEFs infected with either control (Cas-/-) or GFP-tagged Cas (Cas-/- + GFP-Cas)–expressing retroviruses were analyzed by immunoblotting. (C) Alleviation of RelA-induced NF-?B activation by Cas. HEK293 cells cotransfected with the luciferase reporter vectors together with combinations of FLAG-Cas, HA (hemagglutinin)–RelA, and their respective controls were subjected to a luciferase assay. Values were normalized against the control data, which was set at 1 (left, n = 3). The expression levels of transfected genes were evaluated by immunoblotting (right). (D) Decreases in RelA-induced expression of NF-?B target genes by exogenous Cas expression. HEK293 cells, transfected with combinations of FLAG-Cas and HA-RelA as in (C) except for the luciferase reporter vectors, were analyzed for IL-8 and TNF-a mRNA expression. Values were normalized against the controls, which were transfected with parent vectors and set at 1 (n = 3). (E and F) Down-regulation of TNF-a–induced NF-?B activation by Cas. HEK293 cells cotransfected with pNF-?B Luc and phRL-TK together with FLAG-Cas (E) or Cas-targeted small interfering RNA (siRNA) (si-Cas) (F) or their respective controls, either left untreated or treated with TNF-a (10 ng/ml, 8 hours), were subjected to a luciferase assay as in (C) (n = 3). The expression level of Cas was evaluated by immunoblotting (E and F, right). (G) Down-regulation of TNF-a–induced NF-?B target gene expression by Cas. HEK293 cells, transfected with either control (si-Cas -) or Cas-targeted siRNA (si-Cas +) as in (F) except for the luciferase reporter vectors, were either left untreated or treated with TNF-a (10 ng/ml, 2 hours) and analyzed as in (D) (n = 3). (H) Cas-mediated down-regulation of NF-?B activation and acetylation by constitutively active IKKß. HEK293 cells transfected with the luciferase reporter vectors together with combinations of HA-IKKßSS/EE, FLAG-CasWT, and FLAG-Cas15YF were subjected to a luciferase assay as in (C) (n = 3; top). HEK293 cells transfected likewise except for the luciferase reporter vectors were analyzed by immunoblotting (bottom). (I) Absence of suppressive effect of NES-Cas on RelA-enhanced NF-?B transcriptional activity. HEK293 cells, cotransfected with combinations of RelA, Cas, NES-Cas, and their respective controls as in (A), together with the luciferase reporter vectors, were subjected to a luciferase assay as in (C) (n = 3). (J) Absence of suppressive effect of exogenous Cas expression on the transcriptional activity of NF-?B in which Lys310 of RelA is mutated. HEK293 cells transfected with various RelA mutants and the luciferase reporter vectors, together with and without exogenous Cas, were subjected to a luciferase assay as in (C) (n = 3). (K) Competition of Cas with RelA for p300 binding. HEK293 cells, cotransfected with p300-HA and FLAG-RelA together with incremental FLAG-Cas, were analyzed by anti-HA immunoprecipitation (IP) and subsequent anti-FLAG and anti-HA immunoblotting. The levels of RelA and Cas recovered from coimmunoprecipitates with p300 were inversely correlated (compare the top and second rows). (L) Inverse correlation between endogenous RelA-p300 and Cas-p300 interactions in MLO-Y4 osteocytes. Note that the RelA-p300 association is enhanced in Cas-knockdown cells (compare lanes 2 and 3).

Fig 3: (a) Primary cortical neurons were exposed to 1 h OGD and then 24 h later to 3 h OGD. The next day, cell viability was measured by LDH assay. Sub-lethal ischemic injury totally prevented the 3 h OGD-mediated neurotoxicity. Bars are means±S.E.M. of three separate experiments run in triplicate; *P<0.01 versus the corresponding control value. (b) Activation of p50 and RelA was evaluated by ELISA analysis in nuclear extracts from cortical cells exposed to 1 or 3 h OGD. Bars are means±S.E.M. of three separate experiments; *P<0.05 versus the corresponding control value. (c) Representative picture of co-immunoprecipitation analysis of p50, RelA and c-Rel dimers in nuclear extracts from primary cortical neurons showed a high level of p50/RelA complex activation after 1 or 3 h OGD, whereas the activation of c-Rel dimers decreased slightly

Fig 4: 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 ß-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-?- and TNF-a-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-?- and TNF-a-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.

Fig 5: Entinostat treatment reveals involvement of HDAC1/3 in RelA deacetylation in HPV16+KCs.(a) RelA K310 acetylation and total RelA levels in KCs and HPV16+KCs treated with decreasing doses of entinostat (40, 20, 10 and 2 µM), SAHA (10, 5 and 1 µM), TSA (5, 1 and 0.333 µM) or NaBu (10, 5 and 1 mM). RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 expression in steady state (b) or 24-h non- or IFN-?- and TNF-a-stimulated (c) control or entinostat (10 µM) pre-treated HPV16+KCs. (d) Total RelA levels and RelA K310 acetylation in non- or entinostat-treated control or RelA knockdown (KD) HPV16+KCs. (e) RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 expression in 24-h non- or IFN-?- and TNF-a-stimulated non- or entinostat-treated control or RelA knockdown (KD) HPV16+KCs. (f) RT–qPCR of EGFR expression in KCs and HPV16+KCs treated with increasing doses of entinostat (0, 10 or 40 µM). Gene expression was normalized using GAPDH as the calibrator gene. (g) IFRD1 in control or entinostat (10 µM) pre-treated HPV16+KCs. (h) RT–qPCR of IFRD1 and EGFR expression in control or IFRD1 KD HPV16+KCs. Gene expression was normalized using GAPDH as the calibrator gene. Histogram (i) and geomean (j) of EGFR expression on control or IFRD1 KD HPV16+KCs, as determined by flow cytometry. s.e.m. of two independent experiments. These data are representative for at least two independent experiments. 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.