Presence/absence of viral DNA in 123 breast cancer tumors.

<p>Presence/absence of viral DNA in 123 breast cancer tumors.</p

Profile of the IBC and non-IBC patients.

<p>IBC, inflammatory breast cancer.</p><p>Profile of the IBC and non-IBC patients.</p

Presence/absence of viral DNA according to IBC status.

<p>*Fisher exact chi-square test</p><p>**Presence of DNA of any of the following viruses: BKV, KIV, JCV, MCV, WUV, TSV, HPyV6, HPyV7, HPyV9, SV40, CMV, EBV1, EBV2, HSV1, HSV2 and 46 types of HPV (21 alpha-HPV and 25 beta-HPV)</p><p>Presence/absence of viral DNA according to IBC status.</p

Presence/absence of viral DNA in IBC/non-IBC tumors, stratified by triple-negative status.

<p>*Fisher exact chi-square test</p><p>Presence/absence of viral DNA in IBC/non-IBC tumors, stratified by triple-negative status.</p

Presence/absence of viral DNA according to ER/PR/HER2 status.

<p>*Fisher exact chi-square test</p><p>**Presence of DNA of any of the following viruses: BKV, KIV, JCV, MCV, WUV, TSV, HPyV6, HPyV7, HPyV9, SV40, CMV, EBV1, EBV2, HSV1, HSV2 and 46 types of HPV (21 alpha-HPV and 25 beta-HPV)</p><p>Presence/absence of viral DNA according to ER/PR/HER2 status.</p

In situ hybridization for Epstein-Barr encoding region (EBER) in breast carcinoma shows (A) a few malignant epithelial cells and lymphocytes that express EBER (magnification x10) and (B) one malignant mammary epithelial cell with strong nuclear labeling indicating EBER expression (magnification x40).

<p>(C and D) Hematoxylin and eosin (H&E) staining performed on the same breast carcinoma (magnifications x10 and x40).</p

IRF6 and not IRF8 is recruited to the IL-1β promoter which is blocked by HPV16E6.

<p>(A) HEK293 cells were co-transfected with IL-1β promoter luciferase construct along with the empty vector pUNO, IRF8 or IRF6 plasmid at the indicated concentration. Post 48 h cells were lysed and luciferase activity measured. n = 4. (B) Oligo pulldown assay for WT or the mutated ISRE site using protein lysates from HEK293 cells transfected with IRF6 or IRF8. Bound proteins were assessed by immunoblotting for IRF8 or IRF6. Input controls (10%). n = 4. (C) Immunoblot analysis of IRF6 protein levels in in pLXSN and 16E6 transduced human primary keratinocytes. n = 4. (D) IRF6 relative levels were measured in pLXSN, 16E6 and 16E7 transduced human primary keratinocytes by RT-qPCR. n = 4. (E) Immunofluorescent staining of IRF6 in human keratinocytes transduced with pLXSN or HPV16E6. Left, semi-quantative analysis of IRF6 was examined by calculating immunofluorescent intensity. The mean and S.E.M of five fields were plotted. n = 4. (F) Immunoblot analysis of IRF6 protein levels in C33A and NIKs. n = 4. (G) IRF6 mRNA levels detected by RT-qPCR in NIKs and CaSki cells. n = 4. (H) NIKs and CaSki cells were co-transfected with IL-1β promoter luciferase construct ± siRNA for 16E6. Post 48 h cells were lysed and luciferase activity measured. (I) C33A cells were treated with control PsV or 16QsV at different v.g.e per cell for 24 h and IRF6 protein levels were examined by immunoblot. n = 3. (J) C33A cells were treated with control PsV or HPV16 at different v.g.e for 24h and IRF6 mRNA levels were examined by RT-qPCR and (below) viral DNA expression of E7 vs β2-microgloubulin. n = 3. (K), ChIP using IgG, IRF6 or IRF8 antibodies was performed for the ISRE site on C33A cells infected with HPV16 or PsV for 24 h. n = 3. Data are representative of n independent experiments performed in triplicate. Panels A, E, J and K are shown as the mean ± SEM with ***, P < 0.0001, * P, < 0,01, based on a two way ANOVA test. Panels D and G are shown as the mean ± SEM with ***, P < 0.0001 based on an unpaired T test. For immunoblotting data, 1 out of 4 experiments is shown. For immunoblotting data, 1 out of 4 experiments is shown.</p

Oligo sequences.

<p>Oligo sequences.</p

Loss of p53 inhibition by 16E6 restores IRF6 activity.

<p>(A) Table defining the 16E6 mutations that alter p53, E6AP or PDZ binding sites. (B) NIKs were co-transfected with IL-1β promoter luciferase construct along with the HPV16E6 WT and mutated constructs at the indicated concentration. n = 5. (C) Human primary keratinocytes were transfected with cas9/sgRNA for p53 or control and 36h later cells were examined for mRNA levels for p53, IL-1β or IRF6. n = 4. (D) siRNAE6AP (+) or siRNA scramble control (-) was transfected into PLXSN or 16E6 transduced human keratinocytes for 48 h, cells were harvested for protein and RNA. Western blot analysis for p53 and β-actin. Left top, RT-qPCR for IL-1β and, left below IRF6. n = 3, (E) NIKs were co-transfected with the IL-1β promoter and pLXSN, E6, p53 or E6 with p53. Luciferase activity was measured 48 h post infection. n = 4. (F) 16E6 transduced primary keratinocytes were transfected with vector control (-), p53 or IRF6 expression vectors. Twenty-four hours later cells were harvested and pro-IL-1β, p53 or IRF6 levels were examined by immunoblotting. n = 4. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, based on an one or two way (applicable to > 2 conditions) ANOVA test. For immunoblotting data, 1 out of 4 experiments is shown.</p

HPV16 induces transient IL-1β secretion by keratinocytes.

<p>(A) Human primary keratinocytes were treated as indicated with 16QsV or PsV (at 200 viral genome equivalents (v.g.e) per cell). IL-1β transcripts were determined by RT-qPCR. n = 5. (B) As in A, E1, E6 and E7 mRNA relative levels were determined by RT-qPCR. n = 4. (C) Human keratinocytes were treated at 4, 8 and 24 h with 16QsV at different v.g.e per cell. Supernatants were harvested and IL-1β or IL-18 production was measured by ELISA. PsV or L1/L2 fractions were added as controls. n = 5. (D) Human keratinocytes were treated with 16QsV or PsV at decreasing v.g.e per cell for 24 h ± recombinant IL-1β (200pg/ml). Cells were harvested and E7 mRNA levels were measured by RT-qPCR. n = 5. (E) Human keratinocytes were treated with 16QsV or PsV (200 v.g.e) for 24 h ± IL-1R inhibitor (Anakinra). Cells were harvested and E1 mRNA levels were measured by RTqPCR. qPCR. n = 5. Data are representative of n independent experiments performed. Shown are the mean ± SEM with ***, P < 0.0001, based on a two way ANOVA test.</p