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After observing the ATM-dependent interaction between IFI16 and p53, we then used WT and STING-/- HaCaTs treated with Etoposide for 0, 2, or 4 hours, to test if this interaction also
depended on STING. Upon IFI16 immunoprecipitation we found that IFI16 and STING interact at steady state and this interaction increases slightly at 2 hours’ post-treatment, and
148 Figure 35: Involvement of DDR components in the innate immune response to DNA damage in fibroblasts.
a-c. MRC-5 fibroblasts were pre-treated with KU55933 or DMSO control for 1 hour prior to a 6-hour treatment with 50uM Etoposide or DMSO. Cells were then lysed for measurement of IFN-β (a), IL-6 (b) and CCL20 (c) mRNA by qRT-PCR.
d-f. MRC-5 fibroblasts were treated with non-targeting (NT) or p53-targeting siRNA over 48 hours before treatment with DMSO or 50μM Etoposide for 6 hours. Cells were then
lysed for measurement of IFN-β (d), IL-6 (e) and CCL20 (f) mRNA by qRT-PCR.
Data are presented as mean values of biological triplicates. Error bars indicate standard deviations. N.s. = non-significant; * p≤0.05; ** p≤0.01 as determined by Student’s t-test. Data are representative of at least two experiments.
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immunoprecipitate with p53, however this interaction was not ablated in STING-deficient
cells, indicating that the IFI16-p53 interaction is not STING-dependent. We then reversed the
order of the immunoprecipitation, using WT and IFI16-/- HaCaT cells and
immunoprecipitating STING. In this way, we observed that STING also immunoprecipitates with p53 at 2 hours and at 4 hours, and that this interaction was dependent on IFI16 function,
as the interaction is ablated in IFI16-/- cells (Figure 38b).
To investigate the cellular mechanics of this response we looked to see the cellular
localisation of these components by fractionation (Figure 38c). IFI16 is mainly nuclear, but is
present in the cytoplasm in WT cells. P53 is also nuclear and it can be seen to increase in
expression after Etoposide treatment (Figure 38c). This increase also leads to small yet
detectable levels of p53 in etoposide treated cytoplasmic fractions (Figure 38c). Lamin A/C
and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were used as fractionation
loading controls (Figure 38c).
STING overexpression alone has been previously shown to drive Type-I IFN expression in HEK293T cells (Ishikawa, 2008). We confirmed this in our cells using an IFN-β luciferase
construct and titration of STING-HA plasmid (Figure 39a). We then selected a lower
concentration of STING, which did not drive IFN-β promoter activity on its own, and
transfected this alongside increasing concentrations of IFI16. We found that IFI16 could drive
IFN-β promoter activity in conjunction with as little as 5ng of STING, whereas Empty Vector
(EV), STING alone or IFI16 did not drive this activity (Figure 39b). With the same low
concentration of STING plasmid (5ng), increasing concentrations of p53 plasmid did not
drive IFN-β promoter activity unless cells were also treated with 50µM Etoposide for 16
hours prior to cell lysis (Figure 39c). P53’s synergistic activity with STING could be seen in
the absence of Etoposide treatment when the STING concentration was raised to 20ng,
which was still low enough to prevent STING alone driving the IFN-β promoter (Figure 39d).
When IFI16 was added to STING and p53 together, this led to a significant increase in IFN-
β-luciferase expression (Figure 39e). These data indicate that STING, IFI16, and p53 can
150 Figure 36: PARP-1 is Involved in the innate immune response to DNA damage in keratinocytes.
a-c. HaCaT cells were treated with PARP inhibitor PJ-34 or vehicle only control for 1 hour
before stimulation with DMSO control, 50μM Etoposide, Lipofectamine control, or 1μg/ml
HT-DNA for 6 hours before lysing cells for qRT-PCR analysis of IFN-β (a), IL-6 (b), and
CCL20 (c) mRNA.
Data are presented as mean values of biological triplicates. Error bars indicate standard deviations. N.s. = non-significant, p>0.05; ** p≤0.01, *** p≤0.001 as determined by Student’s t-test. Data are representative of at least two experiments.
151 7.8 Conclusions
In this chapter, we have shown that DNA damage repair factors ATM (Figure 31), p53,
(Figure 34), and PARP-1 (Figure 36) are required for the innate immune response to DNA damage. Both ATM and PARP-1 have been reported to be essential for NFκB activation after DNA damage (Hinz, 2010). We confirmed this involvement for ATM by western blot (Figure 31a), real-time PCR analysis of the NFκB-induced gene IL-6 (Figure 31c), and
confocal microscopy observing p65 translocation (Figure 32). p53, which is activated by
ATM, was found to associate with IFI16 and STING inducibly upon DNA damage dependent
on ATM activity and p53 Ser15 phosphorylation (Figure 37). The interaction between STING
and p53 required IFI16 but STING was not required for the IFI16 and p53 interaction,
indicating that IFI16 and p53 interact first and then move to STING (Figure 38a, b). Using
cellular fractionation, movement of small amounts of both IFI16 and p53 can be detected in
the cytoplasm (Figure 38c). By luciferase assay, we then showed that the co-expression of
STING, IFI16, and p53 led to a cumulative effect on the IFN-β response (Figure 39).
Together, this data indicates that DDR factors ATM, PARP-1, and p53 signal to the innate immune response through IFI16 and STING, to activate the innate immune response we observe to Etoposide.
152 Figure 37: IFI16 and STING interact with p53 after ATM-dependent p53 phosphorylation
a. WT HaCaT cells pre-treated for 1 hour with ATM inhibitor KU55933, or mock control, were
further treated with 50μM Etoposide for 0, 2, or 4 hours before lysis for protein analysis by Western blot. Lysates were immunoprecipitated with anti-IFI16 antibody, anti-STING
antibody, or anti-IgG antibody as a control, as indicated, and blotted for interaction partners.
b. HEK293T cells were transfected with the indicated combination of IFI16, WT p53, S15A
p53, or S15D p53 plasmids for 24 hours before lysis for protein analysis by Western blot. Lysates were immunoprecipitated with anti-IFI16 antibody, or IgG as a negative control, and blotted for interaction partners.
c. HEK293T cells were transfected with the indicated combination of STING-Flag, WT p53,
S15A p53, or S15D p53 plasmids for 24 hours before lysis for protein analysis by Western blot. Lysates were immunoprecipitated with anti-Flag antibody, or IgG as a negative control, and blotted for interaction partners. Data are representative of at least two experiments.
153 Figure 38: IFI16, p53, and STING form a complex dependent on ATM activation after DNA damage.
a. WT and STING-deficient HaCaT cells were treated with 50μM Etoposide for 0, 2, or 4
hours before lysis for protein analysis by Western blot. Lysates were then immunoprecipitated with anti-IFI16 antibody and blotted for interaction partners.
b. WT and IFI16-deficient HaCaT cells were treated with 50μM Etoposide for 0, 2, or 4 hours
before lysis for protein analysis by Western blot. Lysates were then immunoprecipitated with anti-STING antibody and blotted for interaction partners.
c. WT and IFI16-deficient HaCaT cells were treated with DMSO (D) or 50μM Etoposide (E)
for 4 hours before non-stringent cell lysis to isolate the Cytoplasmic cell fraction (Cyto). Remaining cell pellets were then lysed in a more stringent buffer to release the contents of the Nuclear cell fraction (Nuc). Both Cytoplasmic and Nuclear fractions were compared by Western blot to determine protein localisation. Data are representative of at least two experiments.
154 Figure 39: IFI16, p53, and STING work together to promote IFN-β promoter activity.
a. HEK293T cells were transfected with IFN-β-luciferase and Renilla plasmids and stimulated
with increasing concentrations of STING plasmid (0, 2, 5, 10, 20, 50, 100, and 200ng). Cells were then grown for a further 24 hours before lysis in assay lysis buffer and lysates analysed with either luciferase substrate, to measure IFN-β activity, or coelenterazine, to measure Renilla levels in cells. Luciferase levels were normalised to Renilla levels and presented as a fold change of untreated cells.
b. Cells treated as in (a) were stimulated with 5ng of STING as indicated and EV, IFI16 alone,
or increasing concentrations of IFI16 plasmid (0, 50, 100ng) before lysis and analysis of luciferase activity.
c. Cells treated as in (a) were stimulated with 5ng of STING as indicated and EV or 50ng, or
100ng of p53 as indicated for 24 hours. Cells were then treated with DMSO or 50μM
Etoposide 16 hours prior to lysis and analysis of luciferase activity.
d. Cells treated as in (a) were stimulated with 20ng of STING as indicated and EV, p53 alone,
or increasing concentrations of p53 plasmid (0, 25, 50, 100ng) before lysis and analysis of luciferase activity.
e. Cells treated as in (a) and stimulated with indicated plasmids for 24 hours before lysis and
analysis of luciferase activity. Data are presented as mean values of biological triplicates. Error bars indicate standard deviations. * p≤0.05, ** p≤0.01 as determined by Student’s t-test.
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