TINCR transcription. TINCR could bind to STAU1 (staufen1) protein, and influence CDKN2B mRNA stability and expression, thereby affecting the proliferation of GC cells. Together, our findings suggest that E2F1/TINCR/STAU1/CDKN2B signaling axis contributes to the oncogenic potential of GC and may constitute a potential therapeutic target in this disease.Recent evidence indicates that E2F1 transcription factor have pivotal roles in the regulation of cellular processes, and is found to be dysregulated in a variety of cancers. Long non-coding RNAs (lncRNAs) are also reported to exert important effect on tumorigenesis. E2F1 is aberrantly expressed in gastric cancer (GC), and biology functions of E2F1 in GC are controversial. The biological characteristics of E2F1 and correlation between E2F1 and lncRNAs in GC remain to be found. In this study, integrated analysis revealed that E2F1 expression was significantly increased in GC cases and its expression was positively correlated with the poor pathologic stage, large tumor size and poor prognosis. Forced E2F1 expression promotes proliferation, whereas loss of E2F1 function decreased cell proliferation by blocking of cell cycle in GC cells. Mechanistic analyses indicated that E2F1 accelerates GC growth partly through induces  Thus, novel prognostic and diagnostic factors that are associated with GC progression would be of great clinical relevance.Gastric cancer (GC) is still one of the most significant health problems in the world with particularly high frequencies in East Asia.6 and the activating E2F transcription factors E2F1, E2F2 and E2F3 are central to regulation of the cell cycle genes.7 E2F1 is the most thoroughly investigated member of the E2F family in human malignancies. E2F1 has pivotal roles in tumor progression by modulation of both coding and non-coding transcripts,9 and was reported to act as oncogenes or tumor suppressors to modulate tumorigenesis depending on different cell context.11 Accumulating evidence revealed E2F1 exert important effect on GC progression; however, the biology functions remain argued.14The E2F transcription factors are key participants in a number of cellular events such as cell cycle, DNA synthesis or nuclear transcription. The E2F family of transcription factors is composed of activator (E2f1-3a) and repressor  factors and is predominantly regulated by the Rb family of proteins ,TINCR, a long non-coding RNA (lncRNA) producing a 3.7-kb transcript, was first reported to bind to staufen1 (STAU1) protein and mediate differentiated mRNA stabilization.15 STAU1 protein is a double-stranded RNA-binding protein, and has various roles in gene expression. STAU1 binds to an STAU1-binding site in the 3′-untranslated region (3′-UTR) of its target mRNAs inducing mRNA degradation, which is termed STAU1-mediated mRNA decay (SMD).16 SMD is a translation-dependent mechanism that occurs when STAU1, together with the nonsense-mediated mRNA decay factor UPF1, is bound sufficiently downstream of a termination codon.16 Recently, we found that the expression of TINCR was elevated at the mRNA levels in GC cells and tissues and the upregulation of TINCR is induced by the transcription factor SP1.17TINCR regulates cell growth, cell cycle progression by affecting KLF2 mRNA stability via SMD.17TINCR in tumor development and GC cell growth. In this study, we found that: (a) E2F1 could promote GC proliferation and cell cycle progression; (b) patients with high E2F1 expression in their GC cells have a poor prognosis; (c) E2F1 could induce TINCR transcription activation; and (d) TINCR forces cell growth, cell cycle progression by affecting CDKN2B mRNA stability via SMD.Here we report a novel pathway involved in E2F1 and P<0.0001), with the sensitivity and specificity were 0.923 and 0.846, respectively  and advanced TNM stages  and overall survival (OS)  in GC, which was supported by Kaplan–Meier plotter analysis (www.kmplot.com), using microarray data from 876 GC patients18  (26 paired cancer and noncancer tissues) was obtained to analyze E2F1 mRNA expressed between GC and paired non-tumor tissues. The result showed that E2F1 mRNA was 3.34-fold higher in gastric tumor tissues (T) compared with paired adjacent normal tissues (ANTs) . We plotectively . We furtectively . E2F1 exP<0.001, . We alsoP=0.037, . We furtP=0.037, . In additients18 .in vitro (in vivo athymic (nu/nu) mouse models, the results showed that E2F1-transfected cells developed significantly larger tumors than empty vector-transfected cells  to knockdown endogenous E2F1 in BGC823, which have relative high E2F1 expression. In addition, E2F1 was overexpressed by transfecting the pmaxGFP-E2F1 vector into MGC803 cell lines, which have relative low E2F1 expression. The depletion and ectopic expression of E2F1 in cells was confirmed by western blot . MTT , −257 to −239 bp (E2), −136 to −124 bp (E3), −41 to −30 bp (E4), −16 to 0 (E5) and +56 to +73 bp (E6) in the TINCR promoter (TINCR promoter fragment (nucleotides −1000 to +163) into pGL3 vector for a luciferase activity assay. TINCR transcriptional activity was induced by E2F1 overexpression  assays to address whether E2F1 bind to the TINCR promoter region. The ChIP assay revealed that endogenous E2F1 bound to the TINCR promoter   . Hence, TINCR could promote GC cell line BGC823 and SGC7901 proliferation.17 Here, we further confirm the result in MGC803 and AGS cell lines. We used chemically synthesized siRNAs to knockdown endogenous TINCR in MGC803 and AGS cell lines, which both were considered appropriate for TINCR depletion  provided by online GEO data sets (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE40121), and found that CDKN2B are also bound to TINCR mRNA  mRNA. The two latter of these served as a positive and a negative control, respectively, for STAU1-FLAG binding.16 Anti-FLAG could immunopurifiy Rluc-CDKN2B 3′-UTR, endogenous TINCR and Rluc-ARF1 SBS, but not MUP mRNA , and their inhibitors, and deregulation of CDK inhibitors is a common feature in tumor cells.21CDKN2B serve as potent growth inhibitors of cell cycle checkpoints.21 Notably, consistent with our recent report, CDKN2B was found to be remarkably upregulated upon TINCR or E2F1 knockdown in MGC803 and AGS cells. Taken together, CDKN2B could be crucial TINCR and E2F1 target.Our data revealed that silencing E2F1 expression led to significant inhibition of cell proliferation, whereas E2F1 overexpression contributed to cell growth and tumorigenicity. Knockdown of E2F1 expression contributed to G1 phase arrest and an S phase reduction, whereas ectopic overexpression of E2F1 promoted cell cycle progression. Accumulation data revealed that E2F1 exert cell cycle modulation function by regulation of both coding and non-coding transcripts. A novel lncRNA, named 23 and TINCR is a predominantly cytoplasmic lncRNA in GC cells, indicating its action in post-transcriptional gene regulation. The results of RNA IP and RNA pull-down assays show that TINCR could bind STAU1, which is consistent with our previous data.17 STAU1 is a cytoplasmic protein and exerts multiple effects as a post-transcriptional regulator. Our teams have identified that TINCR targets KLF2 transcript through TINCR–STAU1 complex formation. Here, this study found that CDKN2B is also a target of STAU1. In addition, CDKN2B mRNA stability and the effects of binding to STAU1 are influenced by TINCR depletion. As evidenced above, TINCR may affect CDKN2B expression through SMD by TINCR-STAU1 complex formation. The pathway via which E2F1 and TINCR regulate cell cycle and cells proliferation has been depicted in TINCR overexpression. TINCR recruits STAU1 to the 3′-UTR of CDKN2B mRNA, degrading CDKN2B through the UPF1-dependent mRNA decay mechanism. Subsequently, CDKN2B depletion promotes cell cycle progression and tumorigenicity. Here, we explored a novel pathway involved in E2F1, TINCR and CDKN2B in GC development.LncRNAs can act together with specific proteins to perform various functions depending on their subcellular location,, TINCR, STAU1 and CDKN2B. Further insights into the functional and clinical implications of the pathway may contribute to early GC diagnosis and help with GC treatment.We describe here a novel mechanism underlying GC cell proliferation through a molecular cross talk between E2F1CDKN2B 3′-UTR and Rluc-ARF1 SBS, pLuc luciferase vector  carrying the Renilla luciferase (Rluc) reporter gene was digested with an endonuclease and ligated to the corresponding fragment that encodes human CDKN2B 3′-UTR and ARF1 SBS mRNA. The CDKN2B 3′-UTR and ARF1 SBS mRNA fragments were amplified by PCR using a cDNA library from MGC803 cells as a template and the primers listed below: for CDKN2B 3′-UTR: 5′-cacaactcgagCACCCCCACCCACCTAATTC-3′ (sense) and 5′-tgaagatctTGCCAGGTGGCTTCGAAAAT-3′ (antisense), where the bold nucleotides specify the XhoI site, and the italic underlined nucleotides specify the BglII site.pmaxGFP-E2F1 expression vector was purchased from Addgene (plasmid #16007) . To construct Rluc-gtcgacGTGAACGCGACCCCCCTCCCTCTCACTC-3′ (sense) and 5′-aaggatccCCAGGTGCCCATGGGCCTACATCCCC-3′ (antisense), where the bold nucleotides specify the SalI site, and the italic nucleotides specify the BamHI site. To construct the luciferase reporter vectors, the core promoter of the TINCR gene  and the relative deletion of binding sites were respectively subcloned into the pGL3 basic firefly luciferase reporter. siRNAs for specifically knockdown E2F1, TINCR and STAU1 were chemically synthesized , and the sequences of the oligonucleotides synthesized for RNAi have been listed in For ARF1 SBS mRNA: 5′-cacaa24 Antibodies used in the study were: E2F1 , CDKN2B , STAU1 , FLAG-tagged antibodies , and GAPDH antibody was used as control.The human gastric adenocarcinoma cancer cell lines MGC803, BGC823, MKN45, AGS and SGC7901 and the normal gastric epithelium cell line (GES-1) were obtained from the Chinese Academy of Sciences Committee on Type Culture Collection Cell Bank . Western blot analysis was conducted according to our previous protocol.In this study, 80 patients underwent primary GC resection at the First Affiliated Hospital of Nanjing Medical University and the Affiliated Hospital of Yangzhou University. The study was approved by the ethics committee on Human Research of the First Affiliated Hospital of Nanjing Medical University and the Affiliated Hospital of Yangzhou University. Written informed consent was obtained from all patients. The clinicopathological characteristics of the GC patients have been summarized in Total RNAs were extracted with TRIzol reagent , and quantitative real-time PCR (qRT-PCR) analyses were conducted according to the manufacturer’s instructions . The primers sequences have been listed in Cytoplasmic and nuclear RNA were isolated and purified using the Cytoplasmic & Nuclear RNA Purification Kit , according to the manufacturer’s instructions.To quantify protein expression, both the intensity and extent of immunoreactivity were evaluated and scored. In the present study, staining intensity was scored as follows: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The scores of the extent of immunoreactivity ranged from 0 to 3, and were determined according to the percentage of cells that showed positive staining in each microscopic field of view . A final score ranging from 0 to 9 was achieved by multiplying the scores for intensity and extent. Using this method, the expression of proteins was scored as 0, 1, 2, 3, 4, 6 or 9. In case of disagreement (score discrepancy 0.1), slides were reexamined and a consensus was reached by the experts.Renilla luciferase activity.Cells were first transfected with appropriate plasmids in 24-well plates. Next, the cells were collected and lysed for luciferase assay 48 h after transfection. The relative luciferase activity was normalized with 24Cell proliferation assays and colony formation assays were performed as previously reported.24Cell cycle and cell apoptosis were analyzed by flow cytometry and detected as previously reported.μM EdU labeling media were added to the 96-well plates and they were incubated for 2 h at 37 °C under 5% CO2. After treatment with 4% paraformaldehyde and 0.5% Triton X-100, cells were stained with anti-EdU working solution. DAPI was used to label cell nuclei. The percentage of EdU-positive cells was calculated after analyses of fluorescent microscopy. Five fields of view were randomly assessed for each treatment group.5-Ethynyl-2-deoxyuridine (EDU) labeling/detection kit  was used to assess the cell proliferation. Cells were grown in 96-well plates at 5 × 103 cells per well. Forty-eight hours after transfection, 50 ChIP assays were performed using the EZ ChIPTM Chromatin Immunoprecipitation Kit (Millipore), according to the manual. The primer sequences were listed in We performed RIP experiments using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. The STAU1 and FLAG-tagged antibodies used for IP were from Millipore  and Cell Signaling Technology (8146S), respectively. The details of the primers for RT-PCR and qPCR have been provided in in vitro with the Biotin RNA Labeling Mix  and T7 RNA polymerase (Roche Diagnostics), treated with RNase-free DNase I (Roche Diagnostics) and purified with an RNeasy Mini Kit . Next, 1 mg whole-cell lysates from MGC803 cells was incubated with 3 μg of purified biotinylated transcripts for 1 h at 25 °C. Complexes were isolated with streptavidin agarose beads . The beads were washed briefly three times and boiled in sodium dodecyl sulfate buffer, and the retrieved protein was detected using the standard western blot technique. The RNA present in the pull-down material was detected using reverse transcription polymerase chain reaction (RT-PCR) and qPCR analysis. The RT-PCR and qPCR primer pairs were provided in Biotin-labeled RNAs were transcribed μg/ml). Cells were collected at different time points, and RNA was extracted using Trizol reagent . Reverse transcription was performed using oligo (dT) primers and mRNA levels were measured using qRT-PCR.To analyze RNA stability, GC cells were treated with actinomycin D (1 http://www.ncbi.nlm.nih.gov/geo/). One data set GSE51575 consisted of 26 paired primary gastric adenocarcinoma tissues and surrounding normal fresh frozen tissues was included. All the tissues were obtained after curative resection and pathologic confirmation at Samsung Medical Center (Korea cohort). The raw CEL files from the Agilent arrays  for GSE51575 were processed and normalized using the Robust Multichip Average as previously described.25 All statistical analyses were performed using SPSS 20.0 software . The significance of differences between groups was estimated using the Student’s t-test, χ2 test, Fisher’s exact test, Mann-Whitney test, Kruskal–Wallis test or Wilcoxon test, as appropriate. A ROC curve was established to evaluate the diagnostic value for differentiating between GC and benign diseases. FP survival (FPS) and OS rates were calculated by the Kaplan–Meier method with the log-rank test applied for comparison. Pearson correlation analysis was performed to investigate the correlation between TINCR and E2F1 mRNA expression. Two-sided P-values were calculated, and a probability level of 0.05 was chosen for statistical significance.GC gene expression data was obtained from the NCBI GEO, (


FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that  RPMI 1640 culture medium  and chicken serum (CS) were acquired from Life Technologies . FBS and CS were heat inactivated at 56°C for 30 min. DT40 cells were maintained as described in our previous report . The DT4FANCD2 gene, the guide RNA sequence (5′-TTTGGAAAGTGGTTGCTTCC-3′) was designed against exon 2 and cloned into the pX330 (Addgene) plasmid after digestion with BbsI. To generate the targeting vectors, the left and right arms were amplified by polymerase chain reaction (PCR) using 5′-GCGAATTGGGTACCGGGCCCATTGAGTCTCAAATTTGGT-3′/5′-CTGGGCTCGAGGGGGGGCCAATATGGCAAATAGTAAAGG-3′ (for the left arm), and 5′-TGGGAAGCTTGTCGACTTAACAGAATCAACTAGGTAATAT-3′/5′-ACTAGTAGGCGCGCCTTAAGCTTCTTTTGGAAAGCTATT-3′ (for the right arm), respectively. The amplified fragments of the left and right arms were assembled by Seamless Cloning  into the DT-ApA/RNEO  and DT-ApA/RPURO cassettes /log 2.6-benzylguanine (OBG) and used for both the DDR and MN assays to see if MGMT inhibition changes the cellular response. Prior to treatment, cells were pre-incubated with 10 μM of OBG for 2 hours and exposed to chemicals. No additional OBG was supplemented during the exposure.Cells were pretreated with O6 cells were exposed to chemicals and harvested after a 1, 2, 4, 8 or 16 hour treatment period. Cells were washed once with PBS and resuspended in 200 μL of 1X Fixation solution and stored on ice for 20 minutes. After fixation, cells were washed with PBS and resuspended in 100 μL of 1X Permeabilization solution, in which 3.5 μL of anti-phospho-Histone H2A.X (Ser139) was added. After incubation on ice for 20 minutes, 100 μL of 1X Wash solution was added. The cells were harvested by centrifugation and resuspended in 200 μL of PBS. The samples were analyzed by flow cytometry , and the data was analyzed by FlowJo .To determine the percentage of γH2AX-positive cells, IPMS-treated cells were harvested and stained with a FITC conjugated γH2AX antibody using an H2A.X phosphorylation Assay Kit for Flow Cytometry  according to the manufacturer's instructions. Briefly, 1 × 10oC. Cells were then washed in PBS, incubated in 1:250 anti-mouse Alexa Fluor 555 (Life Technologies) and Alexa Fluor 488 antibodies  for 1h. After washing with PBS, slides were mounted with a cover slip using Vectashield with DAPI (Vector Laboratories) and sealed using nail polish. Slides were analyzed using confocal microscopy (Zeiss CLSM 700). Optical sections through the nuclei were captured at 0.5 μm intervals, and the images were obtained by maximum projection of the individual sections.TK6 cells (0.5 million/ml) were exposed to IPMS (750μM) for 24 hours. Cells were then fixed using 2% formaldehyde / PBS for 20 min, washed in PBS, and spotted on to double frosted microscopic glass slides (Fisher Scientific) at a concentration of 2 million/ml. Blocking was achieved using 5% (w/v) bovine serum albumin (BSA) in PBS-TT for 30 min. Cells were then incubated with 1:500 rabbit polyclonal anti-53BP1 antibody  and mouse γH2AX antibody  in 1% BSA/PBS-TT for overnight at 450) values for each cell line using Graphpad Prism 5 . The obtained LC50 in each mutant was compared against the LC50 in wild-type cells.For DDR assay data sets, we calculated the lethal concentration 50  values (U.S.EPA/100/R-12/001 2012) were obtained using the EPA BMD software v 2.5. These values represent a one standard deviation departure from control values, and the BMDL refers to the corresponding lower limit of a one-sided 95% confidence interval on the BMD. A log10 (concentration+0.5) was used to avoid taking logarithms of zero control values. In each instance, the best fitting result from Hill, exponential, and polynominal response non-constant variance models was used.In order to represent the point of departure values for concentration-response in the MN assay in wild-type and P-value was less than 0.05.Fisher's exact test was used to examine whether the micronucleus frequency differed between the OBG pretreated cells and untreated cells following IPMS or nPMS treatment. The results were determined to be statistically significant when the


Reovirus is under development as a therapeutic for numerous types of cancer. In contrast to other oncolytic viruses, the safety and efficacy of reovirus have not been improved through genetic manipulation. Here, we tested the oncolytic capacity of recombinant strains (rs) of prototype reovirus laboratory strains T1L and T3D  in a panel of non-small cell lung cancer (NSCLC) cell lines. We found that rsT1L was markedly more cytolytic than rsT3D in the large cell carcinoma cell lines tested, whereas killing of adenocarcinoma cell lines was comparable between rsT1L and rsT3D. Importantly, non-recombinant T1L and T3D phenocopied the kinetics and magnitude of cell death induced by recombinant strains. We identified gene segments L2, L3, and M1 as viral determinants of strain-specific differences cell killing of the large cell carcinoma cell lines. Together, these results indicate that recombinant reoviruses recapitulate the cell killing properties of non-recombinant, tissue culture-passaged strains. These studies provide a baseline for the use of reverse genetics with the specific objective of engineering more effective reovirus oncolytics. This work raises the possibility that type 1 reoviruses may have the capacity to serve as more effective oncolytics than type 3 reoviruses in some tumor types. Oncolytic viruses are an emerging class of cancer therapeutics that selectively replicate in and kill transformed cells while sparing non-transformed cells ,2. MammaICP34.5 gene, which enhances tumor cell lysis. ICP34.5 also is required for replication in the central nervous system (CNS) and its deletion further improves the safety profile of the virus [ICP34.5 locus was replaced with the gene encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that functions to recruit and activate antigen presenting cells that facilitate T-cell recognition and killing of cancer cells [Phase I and II trials indicate that the majority of oncolytic viruses under development, including reovirus, are safe for use in humans . Howeverhe virus . The ICPer cells . Other per cells , adenovier cells , coxsacker cells , measleser cells , and veser cells  also haver cells .In contrast, reovirus has not undergone genetic manipulation to increase its therapeutic potency ,20. The Reovirus can be genetically modified using plasmid-based reverse genetics ,29,30,31To assess the oncolytic capacity of recombinant reoviruses, we infected a panel of NSCLC cell lines  with recWe performed a fluorescent focus assay to assess reovirus infectivity in the NSCLC cell panel . Each ceAlthough rsT1L and rsT3D infectivity did not differ in H661 cells, rsT1L infected H1299 cells more efficiently than rsT3D. A greater level of infection by rsT1L could result in more killing of H1299 cells than rsT3D. To test whether the disparity in infectivity caused strain-specific differences in killing of H1299 cells, we performed cell viability assays in which the infectious dose of rsT1L and rsT3D was normalized based on fluorescent focus unit (FFU) titers that were determined on H1299 cells. We infected H1299 cells with 10,000, 1000, or 100 FFU/cell and measured ATP at 72 h post-infection as an indicator of cell viability . For reoTo assess viral protein production in the NSCLC cell panel, each cell line was mock infected or infected with rsT1L or rsT3D at an MOI of 10 PFU/cell . Whole cTo assess viral replication of the NSCLC cells, we infected each cell line with rsT1L or rsT3D at an MOI of 1 PFU/cell and quantified viral titers at 0, 24, and 48 h post-infection . We usedIn many cell types, including L929 and HeLa cells, T3 reoviruses induce higher levels of apoptosis than T1 reoviruses . To deteTo directly test whether caspases are required for reovirus killing of NSCLC cells, we assessed the effect of the pan-caspase inhibitor Z-VAD-FMK (Z-VAD) on reovirus cell killing . Each luTo identify reovirus gene segments responsible for differential killing of the two large cell carcinoma lines by rsT1L and rsT3D, we used reverse genetics to engineer reciprocal panels of T1L × T3D single-gene reassortant viruses. We generated a complete set of 20 single-gene reassortant viruses in which all ten gene segments were individually replaced in rsT1L or rsT3D genetic backgrounds. We infected H661 and H1299 cells with rsT1L, rsT3D, or the reassortant viruses at an MOI of 100 PFU/cell and measured ATP content at 72 h post-infection as an indicator of cell viability . In bothWe next assessed replication of L2, L3, and M1 single-gene reassortment viruses in H661 and H1299 cells . We focuOur results indicate that recombinant reoviruses effectively kill NSCLC cell lines . ImportaIt remains to be determined why the large cell carcinoma lines (H661 and H1299) differed in susceptibility to reovirus killing . One posThe difference in cell killing between rsT1L and rsT3D in H661 and H1299 cells also could result from distinct cell death mechanisms induced by T1 and T3 reoviruses. In cultured cells and in vivo, T3 reoviruses induce apoptosis, whereas T1 reoviruses induce minimal apoptosis and kills cells by an undefined mechanism ,47,48,49We found that the L2, L3, and M1 genes associate with differences in killing between rsT1L and rsT3D in H661 and H1299 cells. The L2, L3, and M1 genes encode viral proteins λ2, λ1, and μ2, respectively . ProteinIt is important to note that although rsT3D and Reolysin originally derive from laboratory strain T3D, each has a unique passage history and genetic differences between the two strains that could impact oncolytic efficacy. It remains to be determined whether H661 and H1299 cells are more resistant to Reolysin than rsT1L. Next generation sequencing of Reolysin stocks revealed 32 nucleotide changes between Reolysin and T3D reference sequences in Genbank, 18 of which resulted in protein coding changes . A subseIn this study, we assessed the oncolytic potential of recombinant reoviruses generated by plasmid-based reverse genetics. To our knowledge, this is the first characterization of cancer cell killing by recombinant reoviruses generated using plasmid-based reverse genetics . Our finl-glutamine. L929 cells were maintained in spinner culture at 37 °C in Joklik’s MEM  supplemented to contain 5% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 25 ng/mL amphotericin B (Sigma-Aldrich).Lung cancer cell lines H661, H1299, H1437, H1563, and H1975 were obtained from the American Type Culture Collection (ATCC). All lung cancer cells were maintained at 37 °C in 5% CO2 in RPMI Medium 1640  supplemented to contain 10% fetal bovine serum (FBS) and 2mM 3 CsCl gradients, and centrifuged at 29,000 rpm for 4 h. Bands corresponding to virions (1.36 g/cm3) [2, 10 mM Tris-HCl (pH 7.4)). Plaque assay using L929 cells was used to determine viral titer [Non-recombinant reovirus strains T1L and T3D are laboratory stocks. Recombinant reoviruses were generated using plasmid-based reverse genetics as described ,29. Puri6 g/cm3)  were colal titer .4 cells/well) were mock infected or adsorbed in triplicate with reovirus at room temperature (RT) for 1 h. The MOIs were calculated on PFU titers determined on L929 cells or FFU titers determined on H1299 or 1563 cells, as indicated. Monolayers were washed twice with phosphate-buffered saline (PBS) and incubated at 37 °C in completed media for various intervals. CellTiter-Glo  was used to assess cell viability [Monolayers of cells in 96-well plates  were adsorbed with reovirus at a range of multiplicities at RT for 1 h. The monolayers were washed twice with PBS and incubated in completed media at 37 °C. At the indicated time points, cells were frozen and thawed twice, and viral titers in lysates were determined using plaque assay on L929 cells [tx is the time post-infection.Monolayers of cells in 24-well plates  in 96-well plates were mock infected or adsorbed in triplicate with reovirus at the indicated multiplicities at RT for 1 h. The monolayers were washed twice with PBS and incubated at 37 °C in completed media for various intervals. The cells were cooled to RT, 100 μL of Caspase-Glo 3/7 (Promega) was added to each well and the plates were incubated in the dark at RT for 1 h. Luminescence was measured using a FluoStar Omega plate reader (BMG LabTech).Monolayers of cells (1 × 105 cells/well) in 12-well plates were mock infected or adsorbed with reovirus at an MOI of 10 PFU/cell for 1h at RT. The monolayers were washed twice with PBS and incubated at 37 °C in completed media for various intervals. Cells were lysed with RIPA buffer  with 1% protease inhibitor cocktail (Sigma-Aldrich) and 1% phosphatase inhibitor cocktail (Sigma-Aldrich). Proteins were separated by SDS-PAGE and immunoblotted [Monolayers of cells (5 × 10oblotted  using re4 cells/well) were adsorbed with reovirus at an MOI of 100 PFU/cell for 1h at RT. The monolayers were washed twice with PBS and incubated at 37 °C in completed media for 24 h. The media was removed and cells were fixed in methanol for 30 min at −20 °C. Fixed cells were incubated with PBS + 5% bovine serum albumin (BSA) for 15 min at RT followed by staining with reovirus-specific polyclonal antiserum (1:500 dilution in PBS + 0.5% Triton X-100) for 30 min at 37 °C. The cells were washed three times with PBS and incubated with Alexa-488-conjugated goat-anti-rabbit IgG  and DAPI (1:1000 dilution) at 30 min at 37 °C. Cells were washed three times with PBS and visualized with an EVOS-FL Auto imaging system . Infected cells were quantified by counting the number of reovirus antigen-positive cells along with the number of DAPI-stained nuclei  in three separate fields of view in two independent experiments. The percentage of infected cells was calculated by dividing the number of reovirus antigen-positive cells by the number of DAPI-positive nuclei.Monolayers of cells in 96-well dishes . p values <0.05 were considered to be statistically significant.Means for cell viability experiments were determined for three independent experiments, each of which contained three replicates. Differences were determined using random effects analysis of variance. Differences in cell killing between recombinant and non-recombinant viruses, cell infectivity, viral replication, and caspase-3/7 activity were determined using an unpaired Student’s


Cdh23c.753A), common to many inbred mouse strains, accelerates age-related hearing loss (AHL) and can worsen auditory phenotypes of other mutations. We used homologous recombination in C57BL/6 NJ (B6N) and 129S1/SvImJ (129S1) embryonic stem cells to engineer mouse strains with reciprocal single base pair substitutions (B6-Cdh23c.753A>G and 129S1-Cdh23c.753G>A). We compared ABR thresholds and cochlear pathologies of these SNV mice with those of congenic (B6.129S1-Cdh23Ahl+ and 129S1.B6-Cdh23ahl) and parental (B6N and 129S1) strain mice. Results verified the protective effect of the Cdh23c.753G allele, which prevented high frequency hearing loss in B6 mice to at least 18 months of age, and the AHL-inducing effect of the Cdh23c.753A allele, which worsened hearing loss in 129S1 mice. ABR thresholds differed between 129S-Cdh23c.753A SNV and 129S1.B6-Cdh23ahl congenic mice, and a linkage backcross involving these strains localized a Chr 10 QTL contributing to the difference. These results illustrate the large effects that strain background and congenic regions have on the hearing loss associated with Cdh23c.753alleles. Importantly, the B6-Cdh23c.753Gstrain can be used to eliminate the confounding influence of the Cdh23c.753Avariant in hearing studies of B6 mice and mutant mice on the B6 background.A single nucleotide variant (SNV) of the cadherin 23 gene ( Age-related hearing loss (AHL) is the most prevalent sensory impairment in elderly individuals and is becoming a major health concern with world population aging256910ahl) on Chr 10ahl with cadherin 23 (Cdh23) and the correspondence of specific Cdh23 alleles with hearing loss severity in multiple inbred strains provided evidence that a single nucleotide variant (SNV) of Cdh23 underlies the ahl phenotypers257098870 and is at the 753rd nucleotide position from the ATG translation start site of Cdh23 cDNA (c.753), which corresponds to the last nucleotide of exon 7 in GenBank sequence AF308939 and to the last nucleotide of exon 9 in Ref Seqs NM_023370 and NM_001252635. A G nucleotide at this site (Cdh23c.753G) results in normal exon splicing, whereas an A nucleotide (Cdh23c.753A) disrupts the canonical donor splice site sequence and causes in-frame exon skippingCdh23c.753G allele is associated with resistance to AHL and is dominant to the recessive Cdh23c.753A allele, which is associated with AHL susceptibility.A major genetic factor contributing to AHL in B6 mice was mapped to a locus  cells. These strains were chosen because of their widespread use and the availability of strain-matched ES cells and BAC clones. We compared, over an 18-month time course, the auditory phenotypes (hearing loss and cochlear pathology) of these single nucleotide variant mice with those of mice from their parental strains and with mice from corresponding congenic strains. Our results provide insight into the genetic and pathological mechanisms underlying progressive hearing loss in these two commonly used inbred strains of mice with potential implications for human genetic studies of age-related hearing loss.Here, we describe our analyses of C57BL/6NJ mice with a Cdh23c.753A and B6-Cdh23c.753G SNV strains  and rs13480546 (24.28 Mb position) to a breakpoint between rs13480629 (67.62 Mb position) and rs13480646 (71.25 Mb position). The 7–18 Mb B6J-derived Chr 10 congenic region of 129S1.B6-Cdh23ahl extends from a breakpoint between rs3661754 (44.97 Mb position) and rs3696307 (53.74 Mb position) to a breakpoint between rs257098870 (60.53 Mb position) and rs29330969 (63.15 Mb position).For comparisons with the SNV strain mice, we generated two corresponding congenic strains, B6.129S1-Cdh23 alternative exon splicing by RT-PCR (14Cdh23c.753A variant. Our results (Cdh23ahlcongenic (lane 5), and 129S-Cdh23c.753A SNV (lane 7) strains, but not in 129S1 parental (lane 3), B6.129S1-Cdh23Ahl+ congenic (lane 4), and B6-Cdh23c.753G SNV (lane 6) strains. For each of the three strains exhibiting exon skipping, we compared the image density of the gel band corresponding to the wildtype allele  to the density of the band corresponding to the exon-skipped allele  using the ImageJ software program. From these gel band density ratios, we estimated that the wildtype allele comprises about 45% of the Cdh23 transcripts in cochleae of B6 mice, about 24% in 129S1.B6-Cdh23ahl congenic mice, and only about 18% in 129S-Cdh23c.753A SNV mice.RNA extracted from eight cochleae (four mice) from each of the SNV and congenic strains and from the B6 and 129S1 parental strains were evaluated for y RT-PCR . As show results  showed eCdh23c.753G SNV mice remained at normal levels throughout the 18 month duration of the study  compared with parental B6N mice  OHC and IHC losses in 9-month-old 129S1.B6-J strain  indicatiCdh23ahl congenic and 129S-Cdh23c.753A mice, we produced F1 hybrids between these two strains and measured their ABR thresholds at three months of age, when ABR thresholds differ significantly between the parental strains  variant. A total of 182 N2 generation progeny were produced from the backcross, and each mouse was assessed for ABR thresholds at three months of age and genotyped for 13 SNPs that differ between B6- and 129S1-strain DNA and that span the 20–70 Mb region of Chr 10. SNP marker genotypes were used to assign haplotypes to individual N2 mice to better define the 129S1.B6-Cdh23ahl congenic region and to identify informative crossover events for narrowing the Mahl candidate interval. The strain of origin for the Cdh23c.753A allele was distinguished by the presence or absence of the 104 bp PGK-Neo insertion remnant . The Cdh23c.753G allele, however, did not fully prevent progression of lower frequency (8 kHz) hearing loss  but is much reduced in more basal regions corresponding to higher frequencies. Taken together, the results of these studies suggest that genetic factors other than the Cdh23c.753A variant are responsible for the less commonly recognized low frequency hearing loss exhibited by B6 mice.Our results using homologous recombination in ES cells to produce single base pair substitutions in excision  does notexcision  and cochexcision  results ing loss  and apicing loss  in B6 miCdh23 knockout mice exhibit defects in hair bundle development and are congenitally deaf, whereas mice with a missense mutation of Cdh23 exhibit normal hair bundle development but have defective tip links and exhibit a progressive hearing lossCdh23 mutations may cause age-related hearing loss. The Cdh23c.753A variant disrupts a splice donor site and is predicted to cause in-frame skipping of an exon encoding part of the 2nd and 3rd ectodomains of CDH2314Cdh23c.753A splice site variant causes exon skipping , had the smallest estimated proportion of wild-type Cdh23 transcripts (18%) followed by 129S1.B6-Cdh23ahl congenic mice  and B6N mice . These results suggest that genetic factors that modify the fidelity of Cdh23 exon splicing may contribute to some of the hearing loss variability associated with the Cdh23c.753A variant in different inbred mouse strains.skipping . In our Cdh23c.753A variant (ahl) not only influences the progression of hearing loss in many inbred mouse strainsAtp2b2dfw Adgrv1frings Sod1tm1Leb Myo7ash1-8J Actbtm1.1Erv Ush1gjs Cdh23c.753G strain, which can be used to eliminate the potentially confounding effects of the Cdh23c.753A variant when assessing other auditory phenotypes of mutations produced on the commonly used C57BL/6 J and C57BL/6 N sub-strains. To accomplish this, B6 mice carrying the mutation to be studied would first be crossed with B6-Cdh23c.753G mice. The resulting F1 hybrid progeny (Cdh23c.753A/G) that have inherited the mutation would then be selected and backcrossed to mice of the B6-Cdh23c.753Gstrain. Finally, N2 progeny that have inherited the mutation and are homozygous for the Cdh23c.753G allele would be interbred to establish a new mutant line for phenotype evaluation. An advantage of using the B6-Cdh23c.753G strain described here is the ability to easily identify the targeted Cdh23c.753G allele by PCR genotyping of size differences due to the closely linked 104 bp remnant of the PGK-Neo selection cassette  all have the Cdh23c.753A allele, thereby excluding this variant as the underlying cause of the Phl2-related hearing loss. The 101/H strain and the 129S1 strain were derived from a recent ancestorPhl2 locus. If so, the deleterious 129S1 allele at this locus (Phl2/Mahl) would be present in 129S-Cdh23c.753A SNV strain mice (which exhibit high ABR thresholds), but replaced by the non-deleterious B6J allele in 129S1.B6-Cdh23ahl congenic strain mice (which exhibit lower ABR thresholds).On the basis of map position and inbred strain ancestry, we suspect that the Mahl to the 58–63 Mb position on Chr 10, which includes the Cdh23 gene. A comparative search of whole genome sequences of mouse inbred strains (http://www.sanger.ac.uk/science/data/mouse-genomes-project) did not reveal any sequence differences between C57BL/6 J (or C57BL/6NJ) and 129S1/SvImJ DNA that would affect splicing or cause amino acid changes in any of the 65 protein-coding genes located in the ~5 Mb Mahl candidate region. These results suggest that DNA differences in non-coding sequences or regulatory elements may be responsible for the modifier effect of the Mahl locus on hearing loss caused by the Cdh23c.753A variant. Non-coding DNA differences within or surrounding the Cdh23 gene itself cannot be ruled out, including those that may influence Cdh23 expression levels or exon splicing fidelity.We narrowed the candidate region for Cdh23c.753A variant in the context of the 129S strain background (129S-Cdh23c.753A SNV or 129S1.B6-Cdh23ahl congenic mice) accelerates the progression of hearing loss  has no effect on hearing thresholdsCdh23c.753A variant has on hearing loss thus can vary greatly depending on strain background, having little or no effect on the CBA backgroundCdh23c.753A-related hearing loss has potential implications for genetic studies of human age-related hearing loss. Specific screens of CDH23 mutations in human subjects have identified variants that are weakly associated with both age-relatedCdh23c.753A variant in mice, the phenotypic expression of hypomorphic CDH23 mutations in human populations may depend on the diverse genetic backgrounds of individuals, and the resulting decreased phenotypic penetrance would make associations with age-related hearing loss difficult to detect by GWAS.The ing loss . In conthttp://www.jax.org/), and all procedures involving their use were performed at The Jackson Laboratory and approved by the Institutional Animal Care and Use Committee. All methods used in the study were performed in accordance with the guidelines and regulations of the U.S. National Institutes of Health (NIH) Office of Laboratory Animal Welfare (OLAW) and the Public Health Service (PHS) Policy on the Humane Care and Use of Laboratory Animals. The Jackson Laboratory is accredited by the American Association for the Accreditation of Laboratory Animal Care.All mice examined in this study originated from The Jackson Laboratory  into the genomes of 129S1 and B6J, respectively. To accomplish this introgression, mice from the B6J and 129S1 parental strains were first intercrossed to generate F1 hybrids. The F1 hybrids were then backcrossed to B6J mice to produce N2 generation mice for development of the B6.129S1-Cdh23Ahl+ strain and to 129S1 mice to produce N2 mice for development of the 129S1.B6-Cdh23ahl strain. Repeated backcrossing of hybrid progeny (selected on the basis of Chromosome 10 marker genotypes) to the parental (B6J or 129S1) strain continued until 10 backcross generations (N10) were completed. Finally, the N10 generation mice of each line were sibling inbred to produce the two homozygous congenic strains. To define the extent of the congenic regions in each strain, single nucleotide polymorphisms (SNPs) along the length of Chromosome 10 were genotyped.The B6.129S1-Cdh23 gene of C57BL/6 (B6) and 129S1/SvImJ (129S1) strain mice were produced using a combination of recombineering31Cdh23 gene containing the targeted nucleotide (c.753) was retrieved from bacterial artificial chromosome (BAC) clones and integrated into embryonic stem (ES) cells by homologous recombination , and to produce 129S-Cdh23c.753A knock-in mice, the Cdh23 fragment from a B6 BAC clone was incorporated into the genome of 129S1-derived ES cells (ES-CJ7). The Cdh23c.753A/G single nucleotide polymorphism (SNP) is the only DNA sequence difference between B6 and 129S1 in the entire targeted region , and the natural strain variants precluded the need to create mutations in the targeting vectors.Single base pair substitutions in the bination . To prodHindIII and Xho1, spaced by Kpn1) for retrieval and Not1 for construct release was cloned into the pBlight-TK plasmidCdh23 fragments from BAC DNA by gap repair in competent bacteria, and DNA was sequenced to confirm the presence of the targeted nucleotide. A PGK-Neo cassette with flanking LoxP sites and homology arms was synthesized and inserted by recombineering into the Cdh23 fragment of the targeting vector, 178 bp downstream of the targeted c.753 nucleotide. The final plasmid vector was confirmed by DNA sequence analysis, linearized with NotI and electroporated into ES cells. ES cells under G418 positive selection were screened by a loss of allele assay against the PGK-Neo insertion site, and positive ES cell clones were confirmed by Southern blot and DNA sequence analysis.To create the targeting vector, a synthetic gene  consisting of a 300 bp homology arm  and a 320 bp homology arm  and including restriction enzyme sites (Cdh23c.753G substitution. The floxed PGK-Neo cassette then was deleted by mating with B6N.Cg-Tg(Sox2-cre) 1Amc/J mice (Stock #14094). Mice heterozygous for the Cdh23c.753G substitution, with the PGK-Neo cassette excised and lacking the Cre transgene, were mated to C57BL/6NJ mice, and progeny heterozygous for the Cdh23c.753G substitution then were intercrossed to establish a homozygous B6-Cdh23c.753G line, confirmed by DNA sequence analysis (Cdh23tm2.1Kjn/Kjn (Jackson Laboratory Stock #18399).Verified B6N JM8A3 ES cell clones were microinjected into 3.5 dpc blastocyst-stage albino C57BL/6 J host embryos. After surgical transfer of the microinjected embryos and development to term, chimeras were identified by coat color and high chimera males were mated with C57BL/6NJ females. Progeny were screened for the presence of PGK-Neo and the targeted analysis . The forCdh23c.753A substitution. The floxed PGK-Neo cassette then was deleted by mating with 129S/Sv-Tg(Prm-cre)58Og/J mice (Stock #3328). Mice heterozygous for the Cdh23c.753A substitution and lacking the PGK-Neo cassette and Cre transgene were mated to 129S1/SvImJ mice, and progeny heterozygous for the Cdh23c.753A allele then were intercrossed to establish the homozygous 129S-Cdh23c.753Astrain, confirmed by DNA sequence analysis (Cdh23tm1.1Kjn/Kjn (Stock #16208).Verified 129S1/SvImJ ES cell clones were microinjected into 3.5 dpc blastocyst-stage non-agouti B6 host embryos. After surgical transfer of the microinjected embryos and development to term, chimeras were identified by coat color, and high chimera males were mated with 129S1/SvImJ females. Progeny were screened for the presence of PGK-Neo and the closely linked targeted analysis . The forCdh23c.753 SNVs.A simple PCR-based genotyping method was developed to distinguish wild type alleles from those with genetically engineered single base pair substitutions . The forCdh23 gene-specific primer  rather than random primers.For each of the six strains analyzed in this study, total RNA from eight cochleae  was extracted and purified with the RNeasy Mini Kit  using the manufacturer’s procedures. Total RNA quantity and quality were determined using the Agilent 2100 Bioanalyzer . First strand synthesis of cDNA was performed using the High Capacity cDNA Reverse Transcription Kit  in accordance with manufacturer’s instructions, but using a Cdh23 exons that flank the variably skipped exon: forward primer  and reverse primer RT-Cdh23R (the same primer shown above for reverse transcription). The same RT-PCR primers were used in a previously reported assay of Cdh23 exon skippinghttps://imagej.nih.gov/ij/) was used to process digital images of the gels and quantify the densities of gel bands corresponding to alternatively spliced transcripts.To evaluate alternative splicing, RT-PCR primers were designed from DNA sequences located in the ABR thresholds were measured at 8, 16 and 32 kHz in a sound attenuating chamber using the SmartEP auditory evoked potential diagnostic system from Intelligent Hearing Systems  as described previously2, decapitated, and their bullae quickly removed and opened to expose the inner ear. A small hole was carefully made in the round window and 10% formalin fixative was gently perfused through the opening. Afterward, the cochlea was immersed in fixative and shipped to University of Buffalo for analysis. Cochleae were stained with Ehrlich’s hematoxylin solution. A person, blind to the ABR results, dissected out the organ of Corti into two to three half turns. The samples were then mounted as a flat surface preparation in glycerin on glass slides, coverslipped and examined with a light microscope . Inner hair cells (IHC) and outer hair cells (OHC) were counted along successive 0.12–0.24 mm intervals from the apex to the base of the cochlea by a second person blind to the experimental conditions. A hair cell was counted as present if both the cuticular plate and hair cell nucleus were clearly visible and considered missing if either were absent. Using lab norms and custom software, the percentages of missing IHC and OHC were determined for each animal and a cochleogram constructed showing the percentage of missing OHC and IHC as a function of percent distance from the apex of the cochlea. Position in the cochlea was related to frequency using a mouse tonotopic mapCdh23c.753A mice, at 9 months of age for 129S1/SvlmJ and 129S1.B6-Cdh23ahl mice, and at 18 months of age for C57BL/6NJ, B6.129S1-Cdh23Ahl+ and B6-Cdh23c.753G mice. Cochlear surface preparations were photographed with a digital camera  attached to a Zeiss Axioskop microscope, processed with imaging software , Adobe Photoshop 5.5.Our procedures for preparing cochleograms have been described in detail in earlier publications34Cdh23c.753A × 129.B6-Cdh23ahl) × 129S-Cdh23c.753A backcross, tail-tip samples from 182 individual N2 progeny mice were provided to the Jackson Laboratory’s Genotyping Service, which outsources SNP genotyping to LGC Genomics (Beverly MA). SNP genotyping was performed for 13 selected SNPs on Chr 10 that differed between the B6 and 129S1 strains. For genotyping the Cdh23c.753 locus, 129S1- and B6-derived alleles were distinguished by the presence or absence of the of the 104 bp PGK-Neo insertion remnant . Statistical significance of the differences among means was determined by one-way ANOVA with post-hoc Tukey HSD test for multiple pair-wise comparisons.ABR data analysis was performed using the JMP 7.0 interactive statistics and graphics software program .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress. Drug-resistant influenza viruses commonly arise due to frequent genetic changes and current antiviral drugs are not highly efficient. These underscore the need for new antiviral therapies effective against influenza viruses. Understanding how influenza virus uses cellular proteins for infection can potentially identify novel targets for pharmacological intervention. Influenza virus modulates cellular pathways to promote its replication and avoid immune restriction. Here we reveal the interplay between the cellular protein mTOR, which functions in two distinct protein complexes, and influenza virus infection. mTOR complex 1 (mTORC1) is activated during influenza virus infection through a cascade of specific modifications, or phosphorylation events, and by reducing the levels of another cellular protein termed REDD1, which is an mTORC1 inhibitor. Activation of mTORC1 results in additional phosphorylation events that together promote viral protein expression and replication. On the other hand, mTOR complex 2 (mTORC2) phosphorylates AKT at a specific site during infection, which is a process mediated by the viral NS1 protein that is known to regulate viral-mediated cell death. Since these effects occur midway through the virus life cycle in the infected cell, mTORC1 and mTORC2 activation are likely important to regulate the cellular environment in order to facilitate the late stages of viral infection. Influenza virus is a major human pathogen for which there are few treatment options. To search for novel potential therapeutic targets while systematically investigating viral-host interactions, comprehensive proteomics screens ,2 and va+-adenosine triphosphatase ATPase (v-ATPase) and its interaction with the Ragulator complex of scaffold proteins [mTOR is a highly conserved serine/threonine kinase that resides in two functionally distinct multi-protein complexes termed mTOR complex 1 and 2 (mTORC1 and mTORC2), which are defined by association with the proteins Raptor or Rictor, respectively. mTORC1 regulates cellular protein synthesis, growth and proliferation in response to nutrients, such as amino acids and growth factors. Amino acid stimulation promotes mTORC1 recruitment to lysosomes, which results in mTORC1 activation . This prproteins . The Ragproteins . Growth proteins . Activatproteins .3) activates mTORC2 in response to growth factors or insulin [The proteins Rictor and SIN1 distinguish mTORC2 from Raptor-containing mTORC1. mTORC2 regulates the cytoskeleton, cell survival and has a more recently identified role in translation –13. The  insulin ,15. mTOR insulin .The influenza virus non-structural protein 1 (NS1) has been shown to interact with the p85β subunit of PI3K, which results in activation of AKT S473 ,17 Fig . AKT actInfluenza virus infection promotes AKT phosphorylation at amino acid S473 –18,32, bRictor-/- MEFs and in A549 cells where Rictor was knockdown, respectively Fig 1A). ectively . These rRictor-/- MEFs to a similar degree as in Rictor+/+ cells when normalized to total AKT levels  phosphorylation was largely reduced in Pdpk1-/- cells whereas p-AKT(S473) was slightly decreased after normalization to total AKT  but in the absence of p-AKT(S473), these findings point to the AKT T308 phosphorylation site as important to stimulate mTORC1 activity during infection  and in the absence or barely detectable levels of p-AKT(S473)  . TherefoTo clarify our understanding of viral factors involved in mTORC1 activation, we first set out to better characterize mTORC1 signal induction during influenza virus infection. To understand the kinetics of mTORC1 activation during infection, S6K phosphorylation was examined at multiple times post-infection. mTORC1 activation was evident at 4 hours post-infection and increased over time . To ensuWe and others have previously shown that mTORC1 is activated by WSN ,6 and A/Atg5-/- cells  . Influen/- cells . Thus, iMavs-/- and Ifitm3-/- MEFs compared to their wild-type counterparts  approach was taken. Small-interfering RNAs (siRNAs) against individual viral mRNAs were employed to reduce specific viral protein levels followed by assessment of mTORC1 activation. Viral siRNAs were specific to their targets with the exception of M2, which reduced both M1 and M2  since thWe then investigated how the AKT-TSC1/2-Rheb axis might be regulated during infection to prompt mTORC1 signaling by assessing REDD1, which converges on and inhibits the pathway. Under stress conditions, TSC2 activity is induced by REDD1, and therefore, REDD1 is inhibitory to mTORC1 . REDD1 wTo investigate what triggers down-regulation of REDD1 at the mRNA and protein levels during infection, we knocked down influenza virus proteins and assessed REDD1 protein levels. As expected, REDD1 protein levels decreased upon infection but dramatically increased upon knockdown of the viral NP protein, which prevents virus replication and viral protein expression, as NP functions with the virus polymerase . These rTo determine whether increased levels of REDD1 can reduce AKT T308 phosphorylation during infection as was previously observed under certain basal conditions , we emplmTORC1 and mTORC2 signaling are enhanced during influenza virus infection , and botRictor-/- MEFs would not reduce viral protein production as compared to Rictor+/+ MEFs. However, we observed reduced viral protein levels in both Rictor+/+ and Rictor-/- MEFs treated with Torin1  to distinguish peptides from the differing conditions and their method of phosphopeptide enrichment was different than the one we used [The impact of mTORC1 on influenza virus infected cells was also demonstrated with the identification of mTORC1-dependent substrates that were regulated during infection using a systematic phosphoproteomics approach. In addition to known mTORC1 substrates, we have also identified multiple mTORC1 substrates that were not previously revealed in an mTORC1 substrate screen using insulin stimulus , which c we used . Rps6ka3 we used , which i we used . Here, i we used . We also we used ,71 and p we used . In addimTORC1 regulates protein translation and other cellular processes, such as autophagy and lipid biosynthesis . Since tPdpk+/+ and -/- and Akt+/+ and -/-, obtained from Bert Vogelstein, Johns Hopkins University) [Rictor+/+ and -/- were kindly provided by Mark Magnuson [Pdpk1+/+ and -/- and Akt+/+ and -/- were graciously shared by Bert Vogelstein [Tbk1+/+ and -/- MEFs were kindly provided by Tak Mak [Mavs+/+ and -/- MEFs were generated as previously described [Ifitm3+/+ and -/- MEFs were generated from day 15 embryos according to published protocols [Atg5+/+ and -/- MEFs were kindly provided by Beth Levine . Cells were maintained at 37°C with 5% CO2. Cells tested negative for mycoplasma. For tetracycline induction of REDD1 in U2OS-REDD1 cells, cells were pre-treated for 2 h with 1 μg/mL of tetracycline and throughout the infection.Human lung adenocarcinoma epithelial cells A549, ATCC), Madin-Darby canine kidney cells , African green monkey kidney epithelial cells , mouse embryonic fibroblasts  , and MDCMagnuson . HCT116 gelstein . Tbk1+/+ Tak Mak . Mavs+/+escribed . Immortarotocols . Atg5+/+Chemical reagents include: Torin1 , HEPES (Gibco), urea (Sigma), PhoSTOP phosphatase inhibitor cocktail tablets (Roche), Complete EDTA-free protease inhibitor tablets (Roche), Trichloroacetic acid solution (Sigma), DMSO (Sigma), BX795  and Tetracycline (USB Corporation). Antibodies used in western blots to detect viral proteins were generated against Influenza A virions (Meridian Life Science B65141G) , HA (Genetex GTX127357), NP (Abcam ab20343), NA (GeneTex GTX125974), PA (GeneTex GTX 118991), PB1 (Santa Cruz sc-17601), PB2 (Santa Cruz sc-17603), M2 , NS1 (generated by García-Sastre laboratory) and VSV M protein generated by our laboratory in collaboration with Cocalico Biologicals against full-length VSV M. Antibodies from Cell Signaling Technologies for western blot analysis were against phospho-S6K(T389) (#9234), S6K (#9202), phospho-4E-BP1(T37/46) (#2855), 4E-BP1 (#9644), phospho-Akt  , Akt1 (#2967), TBK1 (#3504), PDK1 (#5662) and p-eIF4B(S422) (#3591). Additional antibodies used for western blot analysis were against Rictor (Millipore 05–1471), IFITM3 (R&D Systems AF3377), MAVS (generated by Z. Chen laboratory), β-actin (Sigma A5441), REDD1 , ATG5 , ATG7 (Sigma A2856), and LC3 . Horseradish peroxidase (Hrp)-conjugated secondary antibodies used were donkey anti-rabbit and sheep anti-mouse , as well as donkey anti-goat (Jackson Immunoresearch 705–035003). NA was expressed from a pCAGGS-NA plasmid.All virus work was performed in strict accordance with CDC guidelines for biosafety level 2 and 3 agents: BSL2  and BSL3 (A/Shanghai/1/2013). All Sh/1  virus work was performed in the BSL3 laboratory of the Icahn School of Medicine at Mount Sinai, NY.x g for 10 minutes, aliquoted and stored at -80°C.Virus strains used in these studies were VSV-GFP and the following influenza viruses A/WSN/1933 (WSN), A/PR/8/1934 (PR8), A/Shanghai/1/2013 (Sh/1), recombinant A/Shanghai/1/2013 ": recombinant virus containing A/Shanghai/1/2013 segments 1, 2, 3, 5, 7 and 8 and A/PR/8/1934 segments 4 and 6) , PR8:WSNAtg5+/+/-/- and Ifitm3+/+/-/- MEFs. DMEM infection media (DMEM substituted for EMEM in standard infection media) was used for Rictor+/+/-/-, Mavs+/+/-/- as well as Tbk1+/+/-/- MEFs and HCT116 Pdpk+/+/-/- and Akt+/+/-/- to maintain cell viability. Rictor-/- MEFs were plated at a density of 25% more than Rictor+/+ MEFs for each experiment to standardize the number of cells so that the same amount were being infected, as determined by cell counting prior to the infection. Nearly confluent cells in 12- or 24-well plates were mock infected with media only or infected with 150–200 μL virus in infection media for 1 hour at 37°C. After 1 hour, 350–800 μL of infection media was added and cells were incubated at 37°C prior to harvest. For Torin1 treated cells at a high MOI, cells were infected for 1 h at 37°C, after which the infection media was removed and media containing 250 nM Torin1 or 0.1% DMSO was added for the additional time indicated in the legend. Viral titer analyses by plaque assays were performed as described previously [For high MOI infection experiments (MOI of 2), adherent cells were serum starved overnight in infection media. EMEM infection media (see above) was used for A549, HBEC, U2OS-REDD1, Vero cells and primary, eviously  and as iFor western blot analysis, cells were washed with PBS and harvested in 2X sample buffer , boiled 10 minutes and subjected to western blot analysis. Sample loading was standardized by Bradford assay (Biorad DC Protein Assay Kit). Western blot quantification was performed using ImageJ64. Images were converted to grayscale, rectangles were drawn around the lane of bands of interest and a profile plot was generated for each lane of bands. Each band was separated using the line tool, and the density of each band was determined by the area under the peak using the wand tool. Phosphorylated protein bands were compared to the total non-phosphorylated protein bands. Values were normalized to uninfected controls as indicated.t-test was performed. A normal distribution can be assumed for all populations (p>0.05).Cell viability assays  were performed to assess cell health. In To UV inactivate WSN, virus was exposed 10 cm from the UV lamp in the laminar flow hood for 7 minutes. The amount of virus inactivated was the same amount as non-inactivated virus used in each experiment for an MOI of 2 PFU/cell. Inactivation was confirmed by plaque assay, and a hemagglutination (HA) assay was performed following the VIRAPUR HA Assay Protocol to ensure UV-inactivated virus was present and still able to fuse with turkey red blood cells .U2OS or A549 cells were forward transfected in duplicates in 12 well plates with 450 ng pcDNA3-NP, 283.3 ng pcDNA3-PA, 283.3 ng pcDNA3-PB1 and 283.3 ng pcDNA3-PB2 +/- 200 ng spLuc using 3.2 μL/well Mirus TransIT-X2 transfection reagent (MIR6004) following manufacturer's protocol. For the experiment in For influenza virus mRNA-specific siRNAs, A549 cells were reverse transfected using RNAiMAX  with 50 nM of a pool of 3 mRNA-specific siRNAs for approximately 36 h, during which cells were serum starved overnight prior to infection the next day as outlined above. Cell viability analysis  was performed to ensure that the siRNA concentrations were not toxic. Influenza virus mRNA-specific siRNAs were generated by Thermo Scientific to target mRNAs from the WSN strain (siGENOME modifications). siRNA sequences are as follows:siRNA Universal Negative Control #1 and #2 siGENOME Non-Targeting (pooled for use in experiments):#2 5'-UAAGGCUAUGAAGAGAUACdTdT-3' (Dharmacon D-001210-02)#3 5'-AUGUAUUGGCCUGUAUUAGdTdT-3' (Dharmacon D-001210-03)Influenza virus (WSN)-specific :t-test was performed. A normal distribution can be assumed for all populations (p>0.05).Cells were washed with PBS, harvested in TRIZOL (Invitrogen) and subjected to RNA extraction using Direct-zol RNA Miniprep Kit (Zymo Research). For each sample, 500 ng of RNA were used to generate cDNA by reverse transcription using the iScript cDNA Synthesis Kit (Bio-Rad) following manufacturer's protocol. cDNA was diluted 1:3, mixed with primers  and Roche 480 SYBR Green I Master real-time PCR reagents following manufacturer's protocol and subjected to quantitative real-time PCR using the Roche LightCycler 480. Real-time PCR was performed at 95°C for 5 min, 40 cycles of 95°C for 15 sec, 60°C for 15 sec and 72°C for 18 sec. A melting curve cycle was performed from 65°C to 95°C for quality assurance. Primers (Eurofins) used were REDD1 ; 18S rRNA ; Minigenome RNA (firefly luciferase RNA) . Primers to quantify viral mRNAs were previously described . For staRictor-/- MEFs were plated in 15 cm plates (6 per condition), serum starved overnight in DMEM infection media and, at near confluency, were mock- or WSN-infected the following day. Cells were infected at an MOI of 2 PFU/cell for 1 h at 37°C. Infection media was removed after 1 h, and media containing 250 nM Torin1 or 0.1% DMSO was added for an additional 6 h. After 7 hpi, cells were washed twice with cold PBS and lysed in 1.5 mL/plate fresh urea lysis buffer . Cell lysates were sonicated 12 times for 10 second pulses with 30 second cooling periods between each pulse. Lysates were then placed on ice for 30 min with vortexing every 10 min. Cell debris was removed by centrifugation at 4°C for 10 minutes at 13,000 x g. Supernatants were collected and subjected to TCA precipitation. One volume of TCA solution (Sigma) was added to four volumes of cell extract and incubated on ice for 10 minutes. Proteins were collected by centrifugation at 14,000 rpm for 5 minutes at 4°C. Supernatants were removed, and the protein pellet was washed twice with 500 μL of cold acetone. Pellets were vacuum-dried using a Labconco CentriVap Concentrator for 20 minutes and stored at -20°C. Samples were prepped, labeled, phosphopeptide enriched and analyzed by LC-MS/MS by PTM Biolabs  using the methods below.Proteins were dissolved in buffer  and the protein concentration was determined with 2-D Quant kit  according to the manufacturer’s instructions. For digestion, the protein solution was reduced with 10 mM DTT (Sigma) for 1 h at 37°C and alkylated with 20 mM IAA  for 45 min at room temperature in darkness. For trypsin digestion, the protein sample was diluted by adding 100 mM TEAB to urea concentration less than 2 M. Finally, trypsin  was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h digestion.After trypsin digestion, peptides were desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer's protocol for 8-plex iTRAQ kit (AB Science). Briefly, one unit of iTRAQ reagent (defined as the amount of reagent required to label 100 μg of protein) were thawed and reconstituted in ACN . The peptide mixtures were then incubated for 2 h at room temperature, pooled, desalted and dried by vacuum centrifugation. The sample was then fractionated by high pH reverse-phase HPLC using Agilent 300Extend C18 column . Briefly, peptides were first separated with a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate pH 10 over 80 minutes into 80 fractions. Then, the peptides were combined into 7 fractions and dried by vacuum centrifugation.4OH was added and the enriched phosphopeptides were eluted with vibration. The supernatant containing phosphopeptides was collected and lyophilized for LC-MS/MS analysis.Peptide mixtures were first incubated with IMAC microspheres suspension with vibration. The IMAC microspheres with enriched phosphopeptides were collected by centrifugation, and the supernatant was removed. To remove nonspecifically adsorbed peptides, the IMAC microspheres were washed with 50% ACN/6% TFA  and 30% ACN/0.1% TFA, sequentially. To elute the enriched phosphopeptides from the IMAC microspheres, elution buffer containing 10% NHTM Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (ThermoFisher Scientific). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were selected for MS/MS using NCE setting as 28; ion fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5.0E3 in the MS survey scan with 15.0s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the ion trap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z.Peptides were dissolved in solvent A  in 2% ACN) and directly loaded onto a reversed-phase pre-column . Peptide separation was performed using a reversed-phase analytical column  with a linear gradient of 4–22% solvent B (0.1% FA in 98% ACN) for 50 min, 22–35% solvent B for 12 min, 35–85% solvent B for 4 min and holding at 85% for the last 4 min at a constant flow rate of 300 nl/min on an EASY-nLC 1000 UPLC system. The resulting peptides were analyzed by Q ExactiveSwissProt_Mouse database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 2 missing cleavages, 5 modifications per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, phosphorylation on Ser, Thr, Tyr and acetylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification site were specified at 1%. Minimum peptide length was set at 7. For quantification method, iTRAQ-8 plex was selected. All the other parameters in MaxQuant were set to default values. The site localization probability was set as > 0.5. The resulting raw data can be found in The resulting MS/MS data was processed using MaxQuant with integrated Andromeda search engine (v.1.4.1.2). Tandem mass spectra were searched against Rictor-/- MEFs to WSN-infected + Torin1-treated Rictor-/- MEFs (mTORC1 substrates). The fold-change for each phosphorylation site between groups was calculated. Protein sites that showed +/- 1.5-fold change or more in phosphorylation are shown (http://software.broadinstitute.org/gsea/index.jsp) to reveal the top pathways that showed the best enrichment scores. Interferon regulated genes were selected based on the database http://www.interferome.org [Protein phosphorylation site ratios were generated from the LC-MS/MS values by comparing WSN-infected + DMSO(control)-treated re shown . The datrome.org .The study using embryonated chicken was carried out in strict accordance with recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The use of embryonated chicken eggs before hatching is not considered animal use. Embryonated eggs were purchased from Charles River Laboratories, inoculated with influenza viruses at day 10, incubated at 37°C for 2 days, and then incubated at 4°C overnight before allantoid fluid harvesting.S1 FigA-F) Protein bands from the indicated blots from G) A549 cells were transfected for 48 h with control siRNAs or siRNAs to knock down Rictor. Cells were then infected with WSN at MOI of 2 PFU/cell for 6h, 8h and 10h. Cell lysates were subjected to immunoblot analysis to detect the depicted proteins. (H-M) Protein bands from the indicated blots from ((TIF)Click here for additional data file.S2 FigA) p-AKT(T308) to total AKT levels and (B) p-AKT(S473) to total AKT levels after normalizing to the mock-infected controls.Protein bands were quantified by ImageJ64 analysis comparing ((TIF)Click here for additional data file.S3 FigA) A549 cells were infected at MOI of 2 PFU/cell with WSN for the indicated times. (B) Primary MEFs or (C) HBEC30KT cells were infected with WSN for 6 h or 8 h, respectively, at MOI of 2 PFU/cell. A549 cells were infected with (D) Sh/1 (H7N9), (E) rSh/1 (recombinant Sh/1) and WSN (H1N1), (F) VSV-GFP, WSN (or treated with 5% serum for 7 h) for 6h at MOI of 2 PFU/cell. Immunoblot analyses were performed for detection of viral proteins (influenza virus M1 or VSV M) or host proteins . Total S6K serves as the loading control. The upper band in the S6K/p-S6K blots is p85 S6K, whereas the lower band is p70 S6K. Data are representative of three independent experiments.((TIF)Click here for additional data file.S4 FigA) A549 cells were infected with wild-type PR8:WSN or PR8:WSNDeficientM2 at MOI of 2 PFU/cell for 6 h. (B) A549 cells were transfected with the indicated siRNAs for 48 h followed by infection with WSN at MOI of 2 PFU/cell for 6 h. (C) Atg5+/+ and Atg5-/- MEFs were infected with WSN at MOI of 2 PFU/cell for 6 h. Immunoblot analyses were performed with antibodies against the depicted proteins. Total S6K serves as the loading control. Data are representative of three (A) or two  independent experiments. (D) UV inactivation of WSN. WSN was UV-inactivated for 7 minutes under UV light. WSN and UV-inactivated WSN (UV WSN) were subjected to both plaque assay and HA assay to confirm UV inactivation prior to infection by assessing infectious virus (PFU/mL) and quantifying virions (HA unit/50 μl). These assays were carried out each time WSN was UV-inactivated prior to infection. (E) Poly(I:C) stimulation does not induce mTORC1 activiation. MEFs were non-treated or treated with rapamycin (250nM) or Torin (250nM) and transfected with high molecular weight (HMW) poly(I:C) at 1 μg/ml for the indicated time points. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Mito70 was used as loading control. (F) As control for E, MEFs were also mock infected or infected with influenza A virus at MOI of 2 PFU/cell. Cell extracts were obtained at 8h post-infection and subjected to immunoblot analysis with the depicted antibodies. (G) MEFs were mock transfected or transfected with HMW poly(I:C) at 0.5 μg/ml for 6 and 12h. Total RNA was extracted at the indicated time points post-transfection and the relative abundance of mouse IFN β was measured by real time PCR. Data from triplicate experiments were normalized to β-Actin.((TIF)Click here for additional data file.S5 FigWestern blots shown in (TIF)Click here for additional data file.S6 FigA) A549 cells were treated with 0.1% DMSO or 250 nM Torin1 for the indicated times. Cell viability was determined by measuring ATP levels and calculated as a percent of the DMSO control. (B) A549 cells were infected with WSN at MOI of 2 PFU/cell for 1 h and then treated with 250 nM Torin1 or DMSO for an additional 9 h. QPCR was performed to measure viral mRNA levels. Mean and SD are shown, n = 3, ***p<0.001. (C) A549 cells were infected for 24h with rSh/1 at MOI of 0.001 in the absence or presence of Torin. Viral titers were measured by plaque assay. Error bars are SEM, n = 9, **p<0.01. (D) A549 cells were transfected for 48 h with control siRNAs or siRNAs to knock down Rictor as in n = 3, *p<0.05.((TIF)Click here for additional data file.S1 TableThe table indicates protein names and IDs, position(s) of amino acid (aa) phosphorylation, and the ratio of phosphopeptide reads (> and < 1.5 fold or more) in Torin1-treated vs. DMSO-treated cells.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


Compelling evidence suggests that defective DNA damage response (DDR) plays a key role in the premature aging phenotypes in Hutchinson-Gilford progeria syndrome (HGPS). Studies document widespread alterations in histone modifications in HGPS cells, especially, the global loss of histone H3 trimethylated on lysine 9 (H3K9me3). In this study, we explore the potential connection(s) between H3K9me3 loss and the impaired DDR in HGPS. When cells are exposed to a DNA-damaging agent Doxorubicin (Dox), double strand breaks (DSBs) are generated that result in the phosphorylation of histone H2A variant H2AX (gammaH2AX) within an hour. We find that the intensities of gammaH2AX foci appear significantly weaker in the G0/G1 phase HGPS cells compared to control cells. This reduction is associated with a delay in the recruitment of essential DDR factors. We further demonstrate that ataxia-telangiectasia mutated (ATM) is responsible for the amplification of gammaH2AX signals at DSBs during G0/G1 phase, and its activation is inhibited in the HGPS cells that display significant loss of H3K9me3. Moreover, methylene (MB) blue treatment, which is known to save heterochromatin loss in HGPS, restores H3K9me3, stimulates ATM activity, increases gammaH2AX signals and rescues deficient DDR. In summary, this study demonstrates an early DDR defect of attenuated gammaH2AX signals in G0/G1 phase HGPS cells and provides a plausible connection between H3K9me3 loss and DDR deficiency. Since cThe phenotypes of H3K9me3 loss and ATM and gammaH2AX signal reductions were becoming more severe with prolonged passaging in HGPS cells. We speculated that this was due to the accumulation of progerin in HGPS cells over passages. This hypothesis was supported by previous reports that progerin over-expression directly induced H3K9me3 loss ,49. MoreAside from accumulating progerin, HGPS fibroblasts also became prematurely senescent over passages, raising the possibility that cellular senescence plays a secondary role on H3K9me3 loss and DDR deficiency in HGPS cells. In support of this idea, Scaffidi et al. have previously shown that fibroblasts from normal donors exhibited a gradual reduction of H3K9me3 with prolonged passages . In addiPreviously, we showed that methylene blue (MB) was able to improve mitochondrial functionality, alleviate nuclear blebbing and restore heterochromatin loss in HGPS fibroblasts . InteresS1 Fig(A) Representative fluorescence images of basal gammaH2AX foci in middle passage normal and HGPS fibroblasts. Scale Bar: 5um.(B) Quantification of (A) showing the number of gammaH2AX foci in each nucleus in middle passage normal and HGPS fibroblasts at a basal level. More than 100 cells were randomly picked for quantification. Results were presented as mean ± SEM. **P < 0.01.(TIF)Click here for additional data file.S2 FigWestern blotting analysis with anti-H2AX, anti-alpha-tubulin and anti-GAPDH antibodies on middle passage normal and HGPS fibroblasts.(TIF)Click here for additional data file.S3 FigCell cycle analysis of middle passage normal and HGPS fibroblasts with or without serum starvation synchronization.(TIF)Click here for additional data file.S4 Fig(A) Representative fluorescence images of RIF1 and BrdU in middle passage normal and HGPS cells. G0/G1 cells were indicated by BrdU negative staining. Scale Bar: 5um.(B) Quantification of (A), showing the number of RIF1 foci in each nucleus at each time point. More than 50 cells were picked for each group. Results were presented as mean ± SEM. ***P < 0.001.(C) Western blotting analysis with anti-RIF1 and anti-GAPDH antibodies in middle passage normal and HGPS fibroblasts.(D) Representative fluorescence images of RIF1 and BrdU in middle passage normal and HGPS cells. S phase cells were indicated by BrdU positive staining. Scale Bar: 5um.(E) Quantification of (D), showing the number of RIF1 foci in each nucleus at each time point. More than 100 cells were picked. Results were presented as mean ± SEM.(F) Western blotting analysis with anti-53BP1 and anti-GAPDH antibodies in middle passage normal and HGPS fibroblasts.(TIF)Click here for additional data file.S5 FigWestern blotting analysis with anti-pATM(S1981), anti-ATM, anti- gammaH2AX and anti-GAPDH antibodies in middle passage normal and HGPS fibroblasts treated with indicated concentrations of Dox.(TIF)Click here for additional data file.S6 Fig(A) Western blotting analysis with anti-pCHK1(S345), anti-CHK1 and anti-GAPDH antibodies on middle passage normal and HGPS fibroblasts after Dox treatment. ATR activation was indicated by phosphorylation of CHK1(S345).(B) Western blotting analysis with anti-pDNAPK(T2609), anti-DNAPK and anti-GAPDH antibodies on middle passage normal and HGPS fibroblasts after Dox treatment. DNAPK activation was indicated by phosphorylation of DNAPK(T2609).(C) Western blotting analysis with anti-pCHK1(S345), anti-CHK1, anti- gammaH2AX and anti-GAPDH antibodies on middle passage normal and HGPS fibroblasts pre-incubated with indicated concentrations of ATR inhibitor for 24h prior to Dox treatment.(D) Western blotting with anti-pDNAPK (T2609), anti-DNAPK, anti- gammaH2AX and anti-GAPDH antibodies on middle passage normal and HGPS fibroblasts pre-incubated with indicated concentrations of DNAPKcs inhibitor for 24h prior to Dox treatment.(TIF)Click here for additional data file.S7 Fig(A) Representative fluorescence images of gammaH2AX and 53BP1 in middle passage normal fibroblasts pre-incubated with or without 10uM ATM specific inhibitor prior to Dox treatment. Scale Bar: 5um.(B) Line profile analysis of (A), showing the reduced recruitment of 53BP1 to gammaH2AX foci in ATM inhibitor treated fibroblasts. Green (gammaH2AX) and red (53BP1) fluorescence intensities (y axis) were plotted against distance (x axis) along the yellow line in (A). Stars indicated strong co-localization of 53BP1 and gammaH2AX.(C) Quantification of the percentage of a population that displayed more than five 53BP1 foci in control or ATM inhibitor treated fibroblasts. Results were presented as mean ± SEM. ***P < 0.001.(TIF)Click here for additional data file.S8 Fig(A) Western blotting analysis with anti-Lamin A/C and anti-GAPDH antibodies on normal and HGPS fibroblasts at early, middle and late passages, showing the passage dependent accumulation of progerin in HGPS.(B) Quantification of (A), showing the relative band intensity of progerin  in early, middle and late passage HGPS fibroblasts.(TIF)Click here for additional data file.S9 Fig(A) Representative fluorescence images of Dox induced pATM(S1981) in late passage normal fibroblasts ectopically expressing DsRed, DsRed-lamin A (DsRed-LA) and DsRed-progerin (DsRed-PG) respectively. Scale Bar: 5um.2 were calculated using linear regression function.(B) Quantification of (A), showing the fluorescence intensities of DsRed (x axis) and pATM(S1981) (y axis) in either DsRed-LA or DsRed-PG over expressing cells, after Dox treatment. Trend line and R(C) Western blotting analysis with anti-pATM(S1981), anti-ATM, anti-lamin A/C and anti-GAPHD antibodies on late passage normal fibroblasts ectopically expressing DsRed, DsRed-lamin A (DsRed-LA) or DsRed-progerin (DsRed-PG). Cells were treated with or without Dox to induce DSBs before analysis.(D) Quantification of (C), showing the relative band intensity of pATM over total ATM before or after Dox treatment. Three independent experiments were performed. Results were presented as mean ± SEM. *P < 0.05.(TIF)Click here for additional data file.S10 Fig(A) Representative fluorescence images of Dox induced SUV39H1 foci formation in middle passage normal and HGPS fibroblasts. Scale Bar: 5um.(B) Quantification of (A), showing the number of SUV39H1 foci in each cell after Dox treatment. More than 100cells were randomly picked for each group. Results were presented as mean ± SEM. ***P < 0.001.(TIF)Click here for additional data file.S11 Fig(A) Western blotting analysis with anti-pATM(S1981), anti-ATM and anti-GAPDH antibodies on late passage normal and HGPS fibroblasts with indicated treatments.(B) Quantification of (A), showing that the relative band intensity of pATM(S1981) over total ATM was unchanged at basal level (without Dox induction) in HGPS cells with/out methylene blue treatment.(TIF)Click here for additional data file.S12 Fig(A) Representative fluorescence images of H3K9me3 and gammaH2AX responses in late passage normal and HGPS fibroblast with or without methylene blue treatment. Scale Bar: 5um.(B) Quantification of (A), showing the Spearman correlations between H3K9me3 green fluorescence intensity and gammaH2AX red fluorescence intensity in HGPS fibroblasts with or without methylene blue treatment. More than 100 cells were randomly picked for quantification in each group.(TIF)Click here for additional data file.