Fig 1.
High risk HPVs upregulate cGAS levels at the level of transcription.
(A) Western blot analysis of cGAS and STING protein levels in primary keratinocytes HFKs along with HPV16 (HFK16) and HPV31 (HFK31) positive keratinocytes. (B) Quantitative PCR analysis of mRNA levels of cGAS and STING expressed in HFKs, HFK16 and HFK31 cells normalized to GAPDH as a control. (C) Western blot analysis comparing cGAS protein levels between spontaneously immortalized keratinocytes NIKS and primary HFKs from two different donors. Data shown is representative of three independent experiments.
Fig 2.
cGAS-STING canonical pathway is active and responsive in high risk HPV positive keratinocytes.
(A) Western blot analysis of the canonical cGAS-STING factors upon 2’3’-cGAMP treatment. Primary keratinocytes HFKs and HFK16 cells keratinocytes were permeabilized with digitoinin and treated with 5μG/mL cGAMP for 4hrs (B) Response of downstream factors in cGAS pathway to different concentrations of cGAMP. HFKs, HFK-31 and CIN612 cells derived from a CIN lesion were examined. Cells were digitonin permeabilized and treated with 2 or 20μG/Ll cGAMP for 4hrs followed by western analysis. (C) Quantitative PCR analysis for IFN-β mRNA following cGAMP treatment as described above. Each set includes 3 technical replicates. Levels are shown relative to untreated HFKs. To determine significance of IFN production in HFKs statistical analysis used: two-tailed unpaired T-test; p<0.05 indicates significance compared to control HFK. Where (*) <0.05 and (****) <0.0001. Unpaired T-test to determine significance in HFKs. (D) Western analysis of HFK and CIN612 cells for phosphorylated STING and IRF3 at 4hrs post transfection of 1uG/mL poly (dA:dT).
Fig 3.
E6 is responsible for increased cGAS expression.
(A) Western analysis of HFK, HFK-HPV31E6, HFK- HPV31E7, HFK- HPV31E6/E7 and HFK31 cells for factors in canonical cGAS pathway activity in response to digitonin permeabilization and treatment with 5μG/mL 2’3’cGAMP for 4hrs. (B) Time course examining cGAS protein levels in HFK-HPV31E7 cells following p53 knockdown at 1 to 3 days post p53 siRNA transfection.
Fig 4.
Lack of cGAS has minimal effects on episome maintenance and amplification.
(A) Southern blot analysis (left) of CIN612 pLenti control pooled cultures and two cGAS-KO pooled cultures. Cells were examined as undifferentiated monolayer cultures or at 72 hours post calcium induced differentiation. Western blot analysis (right) for levels of cGAS and differentiation markers cytokeratin 10 and loricrin. (B) Southern (left) and western blot (right) analyses of clonal CIN612 pLenti control and cGAS-KO cell lines. Densitometry values of episomes in both both (A) and (B) were calculated using ImageJ.
Fig 5.
DNA damage induces cGAS expression in HPV positive cells while IFN has no effect.
(A) Western blot analysis of cGAS, RIG-I, and pCHK2 of HFKs, HFK-31, CIN612 parental, CIN612 pLenti control and CIN612 cGAS-KO cells following etoposide treatment. Cells were treated with 50uM etoposide for 16hrs prior to analysis. (B) qPCR analysis of IFN-β, IFN-κ mRNA levels in same cells upon etoposide treatment. Levels of expression are relative to that in untreated HFKs. (C) Effect of IFN-β treatment on levels of cGAS along with two known ISGs, ISG15 and RIG-I. Cells were treated for 16hrs with 200 IU/mL of IFN-β prior to analysis. All data representative of 2 or more independent experiments. Statistical analysis: two-tailed unpaired T-test, where p<0.05 indicates significance (***) <0.001 and ns indicates not significant.
Fig 6.
cGAS localization in keratinocytes upon DNA damage induction.
Immunofluorescence analysis for cGAS and γH2AX proteins in HFKs, CIN612 pLenti controls, and CIN612 cGAS-KO keratinocytes following treatment with 50μM etoposide for 24hrs.
Fig 7.
cGAS localizes in DNA damage induced micronuclei.
Micronuclei formation in HPV positive cells and cGAS co-localization (left panel) post etoposide treatment for 24hrs at 50μM. Quantitation of micronuclei from three independent experiments and two different clones of CIN612 pLenti control or cGAS-KO (right panel). Analysis was performed on 100 cells for each condition. Statistical analysis: two-tailed unpaired T-test; p<0.05 indicates significance compared to the respective sample group. Where (*) <0.05 and (***) <0.001.
Fig 8.
HPV positive cells lacking cGAS display lower levels of γH2AX and cleaved caspases 3/7.
(A) Time course western blot analysis for DNA damage repair factors and cleaved caspase 3/7 in CIN612 pLenti control and CIN612 cGAS-KO cells in response to 50 μM etoposide treatment or DMSO for the indicated times. (B) Time course western analysis of same cells as in (A) in response to 5μM cisplatin treatment or PBS for 24 and 48 hrs. Similar results were seen in three or more independent experiments.
Fig 9.
pDNA-PK levels are decreased in absence of cGAS.
(A) Western blot analysis of total and phosphorylated DNA-PKcs levels in HFKs, HFK-31 and CIN612 cells. CIN612 pLenti control and cGAS-KO cells were screened for levels of cGAS, DNA-PKc, pDNA-PKc Ser2056 and γH2AX following treatment with etoposide (50μM) (B) and cisplatin (5μM) (C) for 24hrs. Densitometry values were determined using ImageJ.
Fig 10.
cGAS promotes DNA damage induced apoptosis of HPV positive cells.
(A) Levels of DNA breaks in CIN612 pLenti control and cGAS-KO cells as determined by alkaline COMET assays at 2 hrs following etoposide treatment. Representative images of comets are shown in right panel and quantitation of comet percent tail DNA from three independent experiments including 2 different clones of pLenti control and 2 clones of cGAS-KO cells are shown in left panel. Tail length was calculated using OpenComet software for FIJI. Statistical analysis: two-tailed unpaired T-test, where p<0.05 indicates significance and ns indicates not significant. (B) Flow cytometric analysis of apoptotic cells as measured by Annexin V and PI staining at 24hrs post 10μM etoposide treatment. Graphical analysis plotted and quantitated using FlowJo software. Similar results were seen in three independent experiments.