Eribulin induces micronuclei and enhances the nuclear localization of cGAS in triple-negative breast cancer cells

Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI’s effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI’s impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNβ as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.


Introduction
Eribulin (ERI) is one of the microtubule-targeting agents (MTAs) and is known as a microtubule polymerization inhibitor. 1Our study assumes pivotal signi cance in unraveling the distinct anti-tumoral e cacy of ERI, a drug predominantly employed in the context of metastatic recurrent breast cancer and for tumors that have developed resistance to other MTAs, exempli ed by Paclitaxel (PTX).The inherent challenge in clinical settings lies in ascertaining the sensitivity of ERI when administered in preoperative chemotherapy.In this context, our analysis, drawing from samples collected in the preoperative chemotherapy study of ERI (JONIE-3 study), stands as a unique opportunity. 2This invaluable dataset allows us to dissect and comprehend the unadulterated anti-tumoral impact of ERI in breast cancer-a crucial endeavor that can signi cantly advance our understanding and inform clinical strategies.The outcomes of this analysis hold the potential to reshape therapeutic paradigms and re ne the clinical application of ERI in breast cancer management.
Chromosomal instability (CIN) is a phenomenon in which chromosome missegregation persists overconsecutive cell division. 3Triple negative breast cancers (TNBCs) often exhibit abnormal cell division and increased CIN with PTX treatment. 4However, the relationship between ERI and CIN has rarely been reported.Cells with mitotic defects during chromosome segregation often have micronuclei.Recent studies indicate that micronuclei, particularly nuclear membrane raptured micronuclei, can activate the cyclic GMP-AMP synthase (cGAS) pathway. 5The multifaceted roles of the cGAS pathway are recognized innate immune sensor.1][12][13][14] The ensuing binding of cGAMP to its adaptor STING triggers downstream innate immune responses. 15Dysregulation of the cGAS-STING pathway is implicated in various disorders, spanning infections, in ammatory diseases, neurodegeneration, and cancer. 16While cGAS is traditionally considered a cytosolic protein, it undergoes dynamic changes in subcellular localization. 17It transiently accumulates in the nucleus during mitotic nuclear membrane dissolution and actively translocates into the nucleus in response to DNA damage. 18triguingly, cGAS has also been reported at the plasma membrane in certain cell types. 19Despite these insights, ongoing debates persist regarding the subcellular localization and functions of cGAS across diverse biological conditions. 20This paper aims to contribute to this discourse, shedding light on the nuanced roles of cGAS in different cellular contexts.

Results
ERI but not PTX inhibited microtubule polymerization.
In our investigation of ERI's impact on cell division, we employed a tubulin polymerization assay to assess its effects, given its role as a microtubule polymerization inhibitor, similar to PTX.Con rming earlier reports, ERI exhibited a dose-dependent suppression of microtubule polymerization, effectively inhibiting this process at a concentration as low as 10 nM.Conversely, PTX, acting as a microtubule stabilizer, did not inhibit microtubule polymerization.(Supplementary Fig. 1).ERI induced distinct mitotic abnormalities compared to those caused by PTX.
Recent studies have shown that the concentration of PTX in breast cancer tumors tends to be lower than previously expected. 21In line with these ndings, an in vitro PTX concentration of ~ 10 nM has been estimated as the clinically relevant concentration.Consequently, we utilized a 10 nM concentration for both ERI and PTX in our live cell imaging experiments.
Initially, we conducted high-temporal live cell imaging to observe the differential effects of ERI and PTX during mitosis.Our ndings revealed that both ERI and PTX at their clinical concentrations caused prolonged mitosis in MDA-MB-231 (MM231) cell lines.Notably, 10 nM of ERI led to a signi cantly longer duration of mitotic arrest, lasting about 696 minutes, compared to 419 minutes with 10 nM of PTX (Fig. 1a, 1b).Moreover, we found that ERI uniquely triggered the formation of micronuclei, a phenomenon not seen with PTX.Both agents increased the presence of multinucleated cells, with PTX showing a higher propensity to induce this effect than ERI.
While it is unclear why ERI tends to induce micronuclei and PTX leads to multinuclear formation, these effects are likely due to a higher frequency of unaligned chromosomes and mitotic slippage, which are both major mitotic defects. 22Consistent with the MM231 results, both PTX and ERI induced prolonged mitosis in Retinal Pigment Epithelium cell lines (RPE1) cells; in particular, ERI signi cantly prolonged mitosis more than PTX (Fig. 1c, 1d).In summary, while both ERI and PTX at clinical concentrations lead to extended mitosis and chromosome misalignment, ERI notably causes more severe mitotic arrest and a higher incidence of micronuclei.
Recent studies have demonstrated that micronuclei, rather than multinuclei, are capable of activating the cGAS-STING pathway. 23We posited that treatment with ERI might induce a more pronounced activation of the cGAS-STING pathway compared to PTX, given ERI's propensity to preferentially induce micronuclei.To evaluate this hypothesis, we conducted quantitative immuno uorescence analyses of cGAS and IFNβ in the above established cell lines.Our ndings revealed that ERI treatment resulted in an upregulation of cGAS and IFNβ expression, with a notable increase in cGAS expression in cells treated with ERI over a longer duration compared to a shorter one.Remarkably, ERI treatment led to heightened cGAS expression and its nuclear accumulation (Fig. 2a, 2b) Consistent with the results observed in MM231 cells, ERI also increased the expression of both cGAS and IFNβ in RPE1 cells, suggesting a conserved effect.(Fig. 2c,  2d).
We quanti ed the frequency of cells positive for cGAS, IFNβ, multinucleation, and micronuclei in MM231 and RPE1 cells treated with short and long durations of PTX or ERI (Fig. 2e).In both cell types, PTX treatment resulted in a signi cantly higher frequency of multinucleated cells compared to ERI treatment.Furthermore, ERI treatment uniquely increased the presence of micronuclei in MM231 and RPE1 cells relative to PTX treatment.These observations align with those from high-temporal live cell imaging studies (Fig. 1a, 1c).Notably, ERI treatment signi cantly enhanced the frequency of cGAS-, and IFNβpositive cells compared to PTX in both MM231 and RPE1 cells.In MM231 cells, prolonged ERI treatment further augmented the incidence of micronuclei, cGAS-and IFNβ-positive cells compared to shorter treatment durations, suggesting that longer exposures to lower concentrations of ERI, more akin to clinical conditions, may further activate the cGAS pathway.These results highlight the distinct mitotic defects induced by ERI and PTX, with ERI uniquely stimulating the cGAS pathway.

ERI upregulates cGAS expression and accumulates in the nucleus
To determine if ERI treatment upregulates overall cGAS expression, we performed western blotting (WB) to measure cGAS protein levels in PTX-or ERI-treated cells (Fig. 2f).In line with quantitative immuno uorescence (IF) results, both MM231 and RPE1 cells exposed to ERI showed increased expression of cGAS and STING, a crucial cGAS binding partner, compared to control and PTX-treated cells.Additionally, phospho-IRF3 and IFNβ, activators downstream of the cGAS-STING pathway, were elevated in ERI-treated cells.Extended exposure to ERI (ERI-long) further enhanced cGAS-STING and its downstream activators, suggesting that prolonged ERI exposure intensi es the activation of the cGAS-STING pathway.Observing enhanced nuclear accumulation of cGAS in ERI-treated cells (Fig. 2a), we assessed the cGAS protein levels in nuclear versus cytoplasmic fractions.To do this, we extracted proteins from the MM231 cell line, dividing them into cytoplasmic and nuclear fractions (Fig. 2g).We found that ERI treatment not only increased cGAS levels in the cytoplasmic fraction but also in the nuclear fraction, aligning with the quantitative IF results, compared to control or PTX-treated cells.Overall, these ndings suggest that cells treated with ERI exhibit heightened activation of the cGAS-STING pathway relative to control or PTX-treated cells.
Knockdown-cGAS accelerates proliferation of triple negative breast cancer cells.
We examined the effects of knockdown-cGAS (KD-cGAS) on cell growth with the combination of PTX or ERI treatments.To this, we rst evaluated the knockdown e ciency of four different siRNAs (siRNA5, siRNA6, siRNA7 and siRNA8) of cGAS (Supplementary Fig. 2a), con rming that cGAS was knocked down in all four siRNAs by WB and RT-PCR, with a knockdown e ciency of approximately 70% (Supplementary Fig. 2b).While cGAS knockdown did not alter the cell proliferation as compared to control, KD-cGAS or control cells treated with ERI or PTX declined cell growth.KD-cGAS or control cells treated with ERI or PTX declined cell growth (Fig. 3a).To further determine cell survival, we counted live and dead cells at 24 hours after PTX or ERI treatment in control and KD-cGAS cells.We found that KD-cGAS increased the number of viable cells.Furthermore, KD-cGAS treated with ERI slightly increased the ratio of viable cells compared to ERI-short, while PTX did not alter the ratio of viable cells to dead cells much.(Fig. 3b).In IF of KD-cGAS cells, cGAS staining was reduced compared to non-KD-cGAS, but no difference in mitotic morphology was observed between KD-cGAS and non-KD-cGAS cells (Supplementary Fig. 2c).
It has been reported that both cGAS-STING pathway and the DNA damage response are tightly linked. 23- 26We then assessed RAD51 protein expression to examine the nuclear effects of cGAS.KD-cGAS upregulated RAD51 expression compared to non-KD-cGAS (Fig. 3c).DNA damages are known to induce cGAS translocation into the nucleus. 5To test whether ERI-treated cells induce DNA damages, we stained γH2AX and RAD51, which are speci cally accumulated at the DNA damage cites.Interestingly, compared to DMSO we found that KD-cGAS-DMSO seemed to increase the accumulation of RAD51 and reduced γH2AX in the nucleus.Compared to ERI short, KD-cGAS-ERI also seemed to increase the accumulation of RAD51 and decreased γH2AX in the nucleus (Fig. 3d).Then we focused on the foci formation of RAD51 and γH2Ax in the nucleus.KD-cGAS-DMSO signi cantly increased RAD51 foci and decreased γH2Ax foci predominantly compared to non-KD-cGAS-DMSO.Moreover, KD-cGAS-ERI increased RAD51 foci and decreased γH2Ax foci compared to ERI-short (Fig. 3e).These results indicate that foci formation of RAD51 may be partially involved in the cGAS pathway in breast cancer cells.

Patients with low cGAS and high RAD51 associated with non pCR after chemotherapy
In order to assess how cGAS expression levels in uences the effects of ERI in clinical outcomes, we rst performed immunohistochemistry (IHC) of 56 biopsy specimens obtained by breast cancer patients who were enrolled the JONIE-3 clinical trial for neoadjuvant setting. 2 This study compared ERI treatment group with PTX treatment group for 12 weeks, followed by 4 cycles of uorouracil, epirubicin and cyclophosphamide (FEC).The pathological complete response (pCR) rates were 20.7% in the ERI group and 29.8% in the PTX group, and there was no signi cant difference between them.The method of the patients-extraction is shown in Fig. 4a.
We examined if cGAS, STING and IFNβ expression were associated with pCR in the PTX and ERI groups.Representative IHC images with each expression score of cGAS, STING and IFNβ are shown in Fig. 4b (ERI) and 4c (PTX).The intensity of the staining was evaluated by Histoscore (H-score) at hot spot. 27GAS (H-score: 0-175) and IFNβ (H-score: 0-274) were predominantly localized in both the cytoplasm and nucleus; STING (H-score: 0-220) was stained in the cytoplasm and RAD51 (H-score: 0-270) was stained in the nucleus.
We quanti ed and compared cGAS and IFNβ levels between pCR cases and non pCR cases (Fig. 4d).We found that the expression of low cGAS was signi cantly correlated with non pCR (p = 0.0375) in the ERI group, while the correlation between cGAS and pCR was not observed in the PTX group (p = 0.2983) (Fig. 4e).Similar to cGAS pro les, low IFNβ also correlated with non pCR (p < 0.0001) in the ERI group, while there was no signi cant correlation between IFNβ and pCR (p = 0.2983) in the PTX group.Furthermore, cGAS and IFNβ showed a moderate positive correlation in the ERI group (R = 0.4690) but not in the PTX group (R = 0.0140) (Supplementary Fig. 3, Supplementary Table 1).Notably, in the ERI group, ~ 50% of the patients with high cGAS and IFNβ achieved pCR.Interestingly, in both ERI and PTX groups, STING was not signi cantly correlated with pCR (Table.1a).These results suggest that a sensitivity to ERI was decreased in the cells with low cGAS or IFNβ in a treatment-naïve breast cancer.On the other hand, in the ERI group, high RAD51 tended to be related to non pCR, but the difference was not statistically signi cant (p = 0.1233).Especially, high cGAS and low RAD51 cases are more likely to achieve pCR compared to the others (Table.1b).

Discussion
TNBCs have the strongest tumor immunogenicity among all breast cancer subtypes, and immunotherapies targeting PD-1 and PD-L1 have shown e cacy, 28 but therapies targeting cGas/STING are not yet practical and the e cacy of TNBCs as therapeutic targets is still unclear.cGAS, a known sensor of foreign DNA in pathogens and tumors, and known to activate type I IFNs by STING, has recently attracted attention as a good target for cancer therapy. 7The present study reveals a new aspect of ERIs, which are microtubule polymerization inhibitors, but their mechanism for anti-tumor activity is not fully understood.
DNA damage can result from exposure to drugs or radiation, leading to various abnormal cell division processes.Conversely, recent studies have indicated that mitotic defects themselves may be a source of DNA damage.Major mitotic defects, such as misaligned chromosomes, lagging chromosomes and chromosomal bridges, can lead to the formation of micronuclei. 5,29While both ERI and PTX disrupt proper microtubule dynamic instability, 30 our ndings show that ERI treatment predominantly induces micronuclei, whereas PTX treatment results in multinuclei.This distinction is critical, as micronuclei are known to trigger cGAS-STING activation.Consequently, ERI has the potential to enhance the effectiveness of immune checkpoint inhibitors.
Although cGAS is also thought to localize to the nucleus and binds to chromatin, 31 and there are a few reports on the role of nuclear cGAS.The following roles for nuclear cGAS have been reported: regulation of innate immune responses, 32,33 suppression of homologous recombination. 24Another previous study reported that knockdown of cGAS inhibited tumor growth through stabilization of the replication forks in lung cancer cells. 25On the other hand, it has also been reported that the promotion of DNA repair might accelerate cell proliferation, and furthermore, RAD51 inhibitor enhanced sensitivity to radiation or drugs. 34,35,36Our results indicate that ERIs accumulate nuclear cGAS and increase their own sensitivity through delayed DNA damage repair.PTX and ERI are both microtubule inhibitors, but have different detailed mechanisms.It is interesting to note how these mechanistic differences can have a profound impact on the effects they have on cancer cells, such as CIN and inhibition of DNA repair.
It was highly signi cant that immunostaining was performed with a sample of a drug-naïve tumor before the effects of ERI.Because clinical indication of eribulin is later line after failure of microtubule inhibitors in metastatic breast cancer, and it is impossible to determine the effect of pure eribulin in a normal clinical specimen.We also considered evaluating if the cGAS-STING pathway is associated with overall survival and recurrence-free survival.However, due to the small number of cases (Fig. 4a), the relapses (2 in total) and the lack of prognostic follow-up period in the JONIE-3 trial, this could not be evaluated in the present study.Focusing on the pCR rate, which is known to be related to prognosis used instead of prognostic analysis, cases with low cGAS, low IFNβ and high RAD51 were less able to achieve pCR.Considering these clinical data together with Fig. 3b, nuclear cGAS which inhibits RAD51 may increase susceptibility to ERI and may be a biomarker to infer ERI e cacy.
In the present report, ERI activated the cGAS-STING pathway more than PTX, similar to previous reports. 37,38We considered that it may be clinically important to note that ERI in particular accumulates cGAS in the nucleus and subsequently delays DNA damage repair pathways.Hence, the present study has newly identi ed the possible role of ERIs in a continuous positive loop, whereby ERI increases DNA damage, leading to increased nuclear cGAS accumulation, which then inhibits homologous recombination and subsequently further increases DNA damage.Since strategies targeting DNA repair pathways, such as PARP inhibitors, are still extremely important as a treatment strategy for TNBC, and are expected to be further developed in the future, our data seems to be an important study for future breast cancer treatment.

Materials and Methods
All methods were performed in accordance with the relevant guidelines and regulations.
Live cell imaging and image analysis.RPE1 cells stably expressing Histone H2B-RFP or MDA-MB-231 cells stably expressing Histone H2B-mCherry were plated on 4-chaamber 35 mm glass bottom dish at least one day prior to do imaging (#1.5 glass, Cellvis).Cells were treated with DMSO (control), PTX (10 nM), or ERI (10nM) 1 hour prior to imaging.High-temporal live-cell imaging was performed using a Nikon Ti2 inverted microscope equipped with a Hamamatsu Fusion camera, spectra-X LED light source (Lumencor), Shiraito PureBox (TokaiHit) and a Plan Apo 20x objective (NA = 0.75) controlled by Nikon Element software and Metamorph (Molecular Devices).Cells were recorded at 37°C with 5% CO2 in a stage-top incubator using the feedback control to maintain the growing media's temperature (Tokai Hit, STX model).Image analysis was performed using Nikon Element software.Mitotic stages were determined by nuclear staining.The mitotic duration was measured from nuclear envelope breakdown (NEBD) to anaphase onset.Incidences of multi-nuclei, mitotic slippages and unaligned chromosome were analyzed.The experiments were independently repeated 2-3 times for mitotic duration measurements (total of n = 100), and P-values between variants were calculated by One-Way Anova and two-tailed t-test.P-values < 0.05 were considered signi cant.
Immuno uorescence (IF).MM231 cell lines(DMSO, PTX-short, PTX-long, ERI-short, ERI-long) and RPE cell lines (DMSO, PTX-short, ERI-short) were plated on the cover glass of 6-well plates at approximately 5×10 4 cells /well.After cells adhesion, the cells were treated with DMSO or PTX or ERI for 24 h.Then Cells were xed with 4% paraformaldehyde in PBS for 15 minutes at RT, permeabilized and blocked with 2.5%FBS, 0.2M glycine and 0.1% TritonX-100/PBS overnight at 4℃.The primary antibodies were diluted in PBS with 1% BSA, and incubated 1 hour at RT.The secondary antibodies were dissolved in PBS and incubated 30 min at RT with blocking out light.Cell nuclei were stained with DAPI followed by imaging using a Zeiss AxioCam HRC microscope camera using a 60x objective lens.A DNA Damage Detection Kit was used as the protocol.Characteristic cells were independently counted in each of the 10 elds of vision for each stain and statistically compared by unpaired t test.
Western blotting (WB).Cell lysates were extracted with RIPA bufer (#16488-34, Nacalai) containing protein inhibitor (#A32955, Thermo Fisher).Proteins (20 µg) were resolved by SDS-PAGE using a 15% XV PANTERA Gel, and transferred to Immobilon-P PVDF membranes.After blocking with 5% skim milk for 60 min, except phosphorylated protein blocked with 5% Bovine serum albumin, the membranes were immersed with diluted primary antibody and shaked for overnight at 4°C, followed by shaking with secondary antibody for 1 hour at RT. Proteins were visualized using Chemi-Lumi One Super (#02230) or Chemi-Lumi One Ultra (#11644) which are chemiluminescent substrates.LAS4000 UV mini were used for blot detection.The lysates and the antibodies were washed by using 5% TBST buffer.
Cell fractionation assay.The Thermo Scienti c™ NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (#78833) was used for separation of cytoplasmic and nuclear extracts from MM231 cell lines (DMSO, PTX-short, PTX-long, ERI-short, ERI-long).Nuclear components and cellular components were extracted from each cells following the protocol (https://www.thermosher.com/order/catalog/product/78833).Then we evaluated the differences of cGAS expression between nuclear components and cellular components by WB.
RT-PCR.RNA was extracted from MM231 cell lines according to the manufacture's protocols of the RNeasy Mini Kit (QIAGEN).The RNA was reverse transcribed to cDNA using SuperScript VILO cDNA synthesis Kit and Master MixRNA (Thermo).PCR reaction solution was prepared using TB Green Fast qPCR Mix (Takara Bio) and human cGAS primers (5'-TGCAAAGGAAGGAAATGGT-3′ and 5′-TTTAAACAATCTTTCCTGCAACA-3′).PCR reactions were performed on an Applied Biosystems 7300.To evaluate the relative expression of proteins, the 2-ΔΔCT method was used to compare with control. 35roliferation assay.At 24h after siRNA (Hs_C6or 50_5, negative control siRNA) transfection into MM231, the cells were detached and seeded at 5×10 4 cells/ml (set as 0 hour).Then DMSO, PTX, and ERI were added to both MM231 cells and KD-cGAS MM231 cells.Cell proliferation was assessed by measuring absorbance at 450 nm using Cell count Reagent SF (Nacalai Tesque) and a plate reader (Bio-Rad Laboratories).
Tripan blue assay.The cell lysates from MM231 cell lines (DMSO, PTX-short, ERI-short, KD-cGAS-DMSO, KD-cGAS-PTX, KD-cGAS-ERI), which were 24 hours after PTX or ERI treatment, were extracted and diluted equally by tripan blue (Nacalai, #20577-34).The total number of cells and the viable cells were counted by Bio RAD TC20 Automated Cell Counter.The procedure was performed three times independently.
Patients and samples.All clinical samples were obtained from breast cancer patients who assigned to randomized Phase JONIE-3 clinical trial (UMIN000012817).In this multicentre randomised study, 121 patients were diagnosed with invasive breast cancer and performed neoadjuvant chemotherapy (NAC) between December 2013 and April 2016.The patients were randomly assigned to 2 different NAC groups: ERI group (eribulin followed by uorouracil, epirubicin, and cyclophosphamide; FEC) or PTX group (paclitaxel followed by FEC).The patients of both groups were performed biopsy before and after chemotherapy.In the trial, 115 cases were analyzed for safety.We de ned pCR as no invasive residual cancer in the breast.JONIE3 clinical trial protocol was approved by the institutional review board (IRB) of Tokyo Medical University on Dec. 25, 2013 (approval number: SH2588) and subsequently by all participating institutions.No tissues were procured from prisoners or vulnerable groups.Both JONIE3 clinical trial and this study were approved by the IRB at department of medicine, Chiba University (approval number: M10112) and all methods were performed in accordance with the Declaration of Helsinki and domestic relevant guidelines and regulations.Study participant names and other HIPAA identi ers are not included in all texts/ gures/tables/images.Immunohistochemistry (IHC).IHC was performed using tissue samples obtained by JONIE-3 clinical trial. 2 We can evaluate 56 clinical samples in out of 115 cases.Four cases were excluded because it was insu cient for evaluation of IHC, 55 cases were excluded because we cannot permit using the samples.For IHC staining, tissue samples were thin sliced at 4 µm thickness.Antigen retrieval was performed by autoclaving for 25 min, and endogenous peroxidase activity was inactivated with 3% hydrogen peroxide.
Following nonspeci c protein blocking with 5% BSA except for using STING antibody blocked with 5% skim milk, the slides were stained with a cGAS or IFNβ or RAD51 antibody.The sections were incubated overnight with primary antibodies at 4°C.The sections were incubated with primary antibodies overnight at 4°C, and stained with secondary antibodies (DAKO, #k4003112) for 30 min at room temperature, followed by staining with diaminobenzidine for 5 min (Nakalai Tesque).The IHC was evaluated by Hscore at hot spot. 24H-score was calculated by adding the percentage of positive cells multiplied by the weighted intensity of staining: H-score= (1×% weak positivity)+ (2×% medium positivity)+ (3×% strong positivity).A positive cGAS-score was de ned as beyond 80, a positive STING-score was de ned as beyond 120, a positive IFNβ-score was de ned as beyond 180 and a positive RAD51-score was de ned as beyond 100.The H-scores for each group were calculated and compared using the unpaired t-test.Simple linear regression was used to examine the correlation between the score of cGAS and IFNβ, or cGAS and RAD51.
Statistical analysis.Statistical analysis were performed using Graphpad Prism version 9 statistical software.In all analysis, P < 0.05 was judged statistically signi cant.(g) The cGAS expression of the cytoplasmic and nuclear fractions was evaluated by cell fractionation assay.Vinculin was used as cytoplasmic loading control and Histone H3 as nuclear loading control.

Figure 1 Live
Figure 1

Figure 2 The
Figure 2