Toll-like receptors (TLRs) are innate immune receptors for sensing microbial molecules and damage-associated molecular patterns released from host cells. Double-stranded RNA and the synthetic analog polyinosinic:polycytidylic acid (poly(I:C)) bind and activate TLR3. This stimulation leads to recruitment of the adaptor molecule TRIF (Toll/IL-1 resistance (TIR) domain–containing adapter-inducing interferon β) and activation of the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), classically inducing IFNβ production. Here we report that, unlike non-metastatic intestinal epithelial cells (IECs), metastatic IECs express TLR3 and that TLR3 promotes invasiveness of these cells. In response to poly(I:C) addition, the metastatic IECs also induced the chemokine CXCL10 in a TLR3-, TRIF-, and IRF3-dependent manner but failed to produce IFNβ. This was in contrast to healthy and non-metastatic IECs, which did not respond to poly(I:C) stimulation. Endolysosomal acidification and the endosomal transporter protein UNC93B1 was required for poly(I:C)-induced CXCL10 production. However, TLR3-induced CXCL10 was triggered by immobilized poly(I:C), was only modestly affected by inhibition of endocytosis, and could be blocked with an anti-TLR3 antibody, indicating that TLR3 can still signal from the cell surface of these cells. Furthermore, plasma membrane fractions from metastatic IECs contained both full-length and cleaved TLR3, demonstrating surface expression of both forms of TLR3. Our results imply that metastatic IECs express surface TLR3, allowing it to sense extracellular stimuli that trigger chemokine responses and promote invasiveness in these cells. We conclude that altered TLR3 expression and localization may have implications for cancer progression. Toll-like receptors (TLRs),5–PRR activation in cancer cells may contribute to the progression of cancer. Certain cancer cells up-regulate PRR expression and produce damage-associated molecular patterns that activate other cells in the tumor microenvironment, leading to further cytokine release and tumor-promoting inflammation . TLR3 ac9–TLR3 is widely expressed in immune cells such as macrophages and myeloid dendritic cells (mDCs) but is also expressed in fibroblasts, neurons, and epithelial cells –17. The –20–24–In this study we report that normal and cancerous intestinal epithelial cells (IECs) respond differently to TLR ligands. Cancerous IECs with metastatic potential expressed and up-regulated TLR3 and induced CXCL10 release in response to poly(I:C) addition in a TLR3-TRIF-IRF3–dependent manner. In contrast, healthy and non-metastatic IECs failed to respond to poly(I:C). Notably, metastatic IECs did not induce IFNβ in response to poly(I:C) addition and generally tolerated poly(I:C) exposure well. These cells expressed TLR3 in the plasma membrane, and CXCL10 release was observed in the absence of ligand internalization in response to low concentrations of poly(I:C), implying that TLR3 can signal from the cell surface of these cells. TLR3 activation also improved the invasive capacity of metastatic IECs. Our results imply that surface expression of TLR3 in metastatic IECs can promote the invasive capability of metastatic IECs and can induce chemokine responses to extracellular stimuli independent of ligand internalization.in vivo , the TLR4 ligand LPS, and the NLR NOD2 ligand muramyl dipeptide (MDP) for 20 h.PRR stimulation may have different effects on the survival of tumor cells, depending on which PRRs they express and the signals they mediate. To investigate differences in TLR- and NLR-mediated signaling between cancerous and healthy IECs, we initially assayed TLR- and NLR-triggered cytokine responses in five generally available and well-characterized cancer IEC cell lines  and the normal fetal IEC line FHC. We sought to compare TLR/NLR responses in cancer cell lines reported to display high metastatic ability d HCT116 ) with thd HCT116 , 32) andd HCT116 , 34. The3C and FSL-1, the TLR3 ligand poly(I:C), and the TLR4 ligand LPS following 20 h of stimulation (F). These cells still produced CXCL8 in response to TNF (F), demonstrating that these cells do induce CXCL8 production in response to inflammatory cytokines. Interestingly, only the highly metastatic cell lines SW620, HT29, and HCT116 induced CXCL8 in response to the TLR3 ligand poly(I:C) (A–C), whereas the poorly metastatic SW480 and Caco-2 cells failed to do so (D and E). In contrast to SW620 cells, which responded potently to poly(I:C) (B), SW480 cells failed to induce CXCL8 in response to poly(I:C), even at high concentrations (50 μg/ml) of ligand (D). We found this particularly interesting because the cell lines SW480 and SW620 were originally isolated from the same patient (A–C), whereas SW480 and Caco-2 cells failed to release detectable levels of CXCL10 (D and E). No detectable TNF, IL-6, MIP-1β, MIP-1α, IL-1β, IL-12p70, or VEGF-A was observed in supernatants from IECs stimulated with any of the tested TLR or NLR ligands (data not shown). Combined, these results indicate that healthy primary IECs respond poorly to TLR stimulation compared with cancerous IECs and that metastatic IECs are particularly responsive to poly(I:C) stimulation.We observed CXCL8 release in several of the cell lines in response to the TLR2 ligands Pmulation . No CXCLmulation F. These e to TNF F, demonsoly(I:C) , A–C, whto do so , D and Eoly(I:C) B, SW480 f ligand D. We fou patient . The thr patient , A–C, whf CXCL10 , D and Ee.g. by means of transfection. Because we observed that the IECs SW620, HCT116, and HT29 induced CXCL10 release upon addition of poly(I:C) in the absence of transfection reagent, we hypothesized that this response was mediated by TLR3. We initially quantified TLR3 mRNA in IECs in the absence and presence of poly(I:C) stimulation to determine whether TLR3 expression is regulated in response to stimuli in these cells. The metastatic IECs HCT116, HT29, and SW620 up-regulated TLR3 mRNA in response to poly(I:C) (A) in comparison with SW480 and Caco-2 cells (A). Notably, TLR3 expression was not up-regulated in SW480 cells in response to poly(I:C) stimulation but was strongly up-regulated in their metastatic derivatives, SW620 cells, following poly(I:C) treatment (A). We verified TLR3 expression in IECs at the protein level by staining Western blots of IEC lysates for TLR3. TLR3 protein expression was markedly up-regulated in SW620 cells in response to poly(I:C), whereas TLR3 expression was undetectable in SW480 or Caco-2 cells (B). HCT116 and HT29 cells expressed TLR3 protein and further up-regulated expression following poly(I:C) stimulation (B). These results were consistent with the results obtained at the mRNA level (A). TLR3 expression in the metastatic IECs SW620, HCT116, and HT29 parallels their ability to induce CXCL8 and CXCL10 in response to poly(I:C) addition, suggesting that this response may be mediated by TLR3.Poly(I:C) is sensed by TLR3 as well as by the cytosolic RNA helicases RIG-I and Mda-5 when it is localized to the cytosol, oly(I:C) A in comp-2 cells A. Notablreatment A. We ver-2 cells B. HCT116mulation B. These NA level A. TLR3 eC) and expressed significantly less TLR3 mRNA  compared with cells treated with NS RNA (C and D). This demonstrates that CXCL10 induction in response to poly(I:C) treatment is mediated by TLR3 in these cells. The efficiency of silencing TLR3 expression in SW620 cells with TLR3 siRNA was further verified at the protein level in poly(I:C)-stimulated SW620 cells . Cell viability was unaffected by siRNA treatment or poly(I:C) stimulation, and similar levels of silencing of TLR3 mRNA were observed with two siRNAs, TLR3.5 and TLR3.8 (data not shown). These results verify that SW620 cells express TLR3 and that CXCL10 production induced by poly(I:C) addition is TLR3-dependent in these cells.We proceeded to confirm the role of TLR3 in mediating poly(I:C)-induced CXCL10 by silencing TLR3 with siRNA. We have shown previously that CXCL10 production is impaired in HT29 cells in response to poly(I:C) addition upon silencing of TLR3 with siRNA . To deteFig. 2C and Ref. Fig. 2F). Efficient silencing of TRIF was confirmed by assessing the mRNA expression of TRIF in samples by qPCR . Similar results were obtained in the poly(I:C)-responsive IEC cell line SW620 (data not shown). These results imply that TRIF is required for CXCL10 induction in response to poly(I:C) in metastatic IECs and support a role for TLR3 in mediating this response.TLR3 signaling requires the signaling adaptor protein TRIF/TICAM-1 . BecauseFig. 2A). Initial CXCL10 protein production was detected after 6 h of stimulation with poly(I:C) (2.5 μg/ml) and plateaued after 12 h of stimulation . It has been shown previously that IFNβ is induced in HT29 and SW480 cells in response to transfection with poly(I:C) by a mechanism dependent on the cytosolic RNA helicase RIG-I . IFNβ protein was also detected in the supernatants of HT29, SW620, and Caco-2 cells in response to transfected poly(I:C) but not in response to poly(I:C) addition . IFNα protein was not detected (<12.5 pg/ml) in the same supernatants when measured by ELISA (data not shown), signifying that IFNα is not produced in any of these IECs in response to poly(I:C) addition or transfection. HT29 cells were further stimulated with poly(I:C) at intervals between 3–24 h and assayed for IFNβ and CXCL10 mRNA to determine whether IFNβ is induced at earlier time points in these cells in response to poly(I:C) addition. Although CXCL10 induction was observed in these cells after 3–6 h of poly(I:C) addition, only a slight and transient induction of IFNβ mRNA was observed at 3 h of stimulation in HT29 cells . Poly(I:C) also failed to activate the IFNβ promoter in SW620 cells transfected with an IFNβ reporter luciferase, although marked IFNβ activation was observed in response to poly(I:C) transfection in these cells following 21 h of treatment (data not shown). These results show that metastatic IECs induce CXCL10 release but do not elicit IFNβ in response to poly(I:C) addition. This does not appear to be due to a defect in signaling leading to IFNβ because IFNβ induction was observed in these cells in response to poly(I:C) transfection. Thus, the mode of poly(I:C) delivery dictates the response induced in these IECs.CXCL10 is an interferon-inducible cytokine that is efficiently induced by IFNβ. Poly(I:C) is an effective inducer of IFNβ in many cell types, and type I interferons are known to inhibit proliferation, impair angiogenesis, and have apoptotic effects in several cancers . We therFig. 3se RIG-I . In lineHT29 cells were pretreated with the protein synthesis inhibitor cycloheximide to verify that CXCL10 is induced directly in response to poly(I:C), independent of IFN signaling. The stimulation time (8 h) was chosen to be short enough to avoid to the toxic effects of cycloheximide but sufficient to observe CXCL10 mRNA expression and protein production in mock-treated cells stimulated with poly(I:C). We observed potent induction of CXCL10 mRNA, even in the presence of cycloheximide  F, whereG), indicating that these cells are highly resistant to the apoptotic effects of poly(I:C). The viability of IECs following poly(I:C) addition or poly(I:C) transfection was also compared. The viability of HT29, SW620, SW480, and Caco-2 cells was unaffected by addition of poly(I:C) but was markedly impaired in these IECs in response to transfected poly(I:C) (2 μg/ml) (H). HCT116 cells displayed some reduction in viability following exposure to poly(I:C) for 43 h but were considerably more sensitive to poly(I:C) transfection (H). Poly(I:C) transfection is therefore much more effective in inducing apoptosis in cancerous IECs than addition of poly(I:C) alone. This is in line with reports documenting the apoptotic effects of poly(I:C) upon transfection (Poly(I:C) induces apoptosis in some cancer cells in a TLR3-dependent manner. High TLR3 expression in tumor cells has therefore been appreciated as a way to target these cells using poly(I:C). To test whether cancerous IECs were sensitive to poly(I:C) treatment, we initially assayed the viability of HT29 cells following exposure to poly(I:C) for 20 h using the MTT assay. The viability of these cells was unimpaired even after exposure to high concentrations of ligand (50 μg/ml) G, indica2 μg/ml) H. HCT116sfection H. Poly(Isfection . CombineA). Persistent IRF3 phosphorylation was, however, observed in HT29 cells in response to transfected poly(I:C) after 1200 min but not in response to poly(I:C) addition . Similar results were obtained in SW620 cells (data not shown), which also induce CXCL10 but not IFNβ, in response to poly(I:C) addition . Western blots of stimulated HT29 cells stained with anti-p65Ser-536 showed a similar degree of phosphorylation in response to addition or transfection of poly(I:C), indicating that p65 is efficiently phosphorylated at this site in response to both treatments . Thus, the absence of IFNβ response in HT29 in response to poly(I:C) addition does not appear to be due to failure to activate IRF3 or NF-κB, although more sustained phosphorylation of IRF3 was observed in response to transfected poly(I:C).IRF3 is a central transcription factor in driving the gene expression of IFNβ in response to poly(I:C) . We therFig. 4B and C), indicating that both transcription factors are activated in response to both addition and transfection of poly(I:C). Persistent nuclear localization of IRF3 was, however, observed after overnight stimulation in response to transfected poly(I:C) but not in response to added poly(I:C) (B). IRF1 expression in the nucleus persisted after overnight stimulation in response to both poly(I:C) addition and poly(I:C) transfection (C), indicating that the duration of IRF1 activation is similar in response to the two treatments. These results imply that IRF3 is activated in HT29 cells both in response to poly(I:C) addition and poly(I:C) transfection, although poly(I:C) transfection induces more persistent IRF3 activation, which, in turn, may be required for IFNβ production in these cells.We further assessed nuclear localization of IRF3 in HT29 in response to poly(I:C) addition or poly(I:C) transfection. The transcription factor IRF1, which has also been implicated in IFNβ induction, was assayed in parallel. HT29 cells stimulated with poly(I:C) alone or transfected with poly(I:C) were stained with antibodies against IRF3 or IRF1 and visualized by high-throughput immunofluorescence microscopy. Nuclear localization of both IRF3 and IRF1 was observed in response to both poly(I:C) addition and poly(I:C) transfection following 3 h of treatment , B and Coly(I:C) B. IRF1 esfection C, indicaF), we proceeded to determine whether IRF3 binds the CXCL10 promoter directly in HT29 cells in response to poly(I:C) addition. We did this by performing ChIP of HT29 cells stimulated with poly(I:C) for 3 h, using antibodies against IRF3 or RNA polymerase II. We then assessed CXCL10 promoter binding by qPCR. The ChIP analysis revealed that both IRF3 and RNA polymerase II are recruited to the CXCL10 promoter in HT29 cells in response to poly(I:C) addition (D), implying that IRF3 is directly involved in driving CXCL10 induction. The role for IRF3 in driving CXCL10 expression was verified by treating HT29 cells with siRNA against IRF3 prior to poly(I:C) stimulation. Silencing IRF3 in HT29 cells diminished CXCL10 production following poly(I:C) addition (E), demonstrating the importance of IRF3 in this response. IRF7 has also been implicated in driving CXCL10 production and type I IFNs in response to TLR activation (F). In summary, these results demonstrate that poly(I:C) addition induces activation and binding of IRF3 to the CXCL10 promoter in HT29 cells. Thus, the inability of these cells to express IFNβ expression in response to poly(I:C) addition is not due to impaired IRF3 activation.CXCL10 is an IFN-inducible protein but has also been shown to be a primary response  and can Fig. 3addition D, implyiaddition E, demonstivation . Normal tivation F. In sumA). This is in line with previous observations in HEK293 cells (B and C), indicating that internalization of ligand is not necessary for TLR3 activation and suggesting that TLR3 may be expressed on the surface of these cells. Using the same assay, we compared the high-molecular-weight (HMW) poly(I:C) (1.5–8 kb) with low-molecular-weight (LMW) poly(I:C) (0.2–1 kb) and found that coated HMW poly(I:C) induces robust CXCL10 release in HT29 cells, whereas coated LMW poly(I:C) failed to induce CXCL10 release in these cells . LMW poly(I:C) added in solution induced similar levels of CXCL10 release as HMW poly(I:C) . These results suggest that TLR3 is expressed in the plasma membrane and can be activated at the surface by long strands of poly(I:C), presumably by effectively multimerizing TLR3 as described previously , further proposing that these cells express surface TLR3 and that signaling can be inhibited by blocking TLR3.Poly(I:C) is endocytosed by a clathrin-dependent pathway in mDC and HEK293 cells , 42. TLRFig. 593 cells . Becauseeviously . We furtF), suggesting that receptor-mediated endocytosis is not crucial for stimulation of these cells. To further confirm that TLR3 signaling can occur independently of clathrin-mediated endocytosis, we transfected HT29 cells with two siRNAs (siC2 and siC3) against clathrin heavy chain 1 (CHC) prior to stimulation with poly(I:C) for 8 h. CXCL10 content in the cell supernatant was determined by ELISA. Knockdown efficiency of CHC following siRNA treatment was confirmed in HT29 lysate by staining Western blots with antibody against CHC. Although we achieved very efficient silencing of CHC , we only observed a partial impairment in CXCL10 production in poly(I:C)-stimulated HT29 cells upon silencing of CHC (G), indicating that ligand internalization is not crucial for signaling in these cells. Combined, these results suggest that TLR3 signaling in response to poly(I:C) can occur independent of ligand endocytosis in these cells, although poly(I:C) internalization may be required for full CXCL10 induction.To verify that TLR3 signals from the cell surface, we pretreated HT29 cells with Dynasore, an inhibitor of receptor-mediated endocytosis, prior to poly(I:C) stimulation for 8 h. CXCL10 expression was subsequently determined by qPCR. We found that CXCL10 induction was not significantly impaired in poly(I:C)-stimulated HT29 cells upon inhibition of endocytosis with Dynasore F, suggesg of CHC G, inset,g of CHC G, indica+-ATPase (V-ATPase), inhibits endosomal and lysosomal acidification and has been shown previously to inhibit TLR3 signaling in several types of cells (H). This indicates that acidification of these compartments is important for enhancing TLR3 signaling in these cells, although endocytosis does not appear to be an absolute requirement for signaling.We also assessed the role of endosomal and lysosomal maturation on TLR3 signaling in HT29 cells. Bafilomycin A1, an inhibitor of vacuolar-type Hof cells , 44, 45.of cells H. This iA). Only full-length TLR3 appeared to be up-regulated in the plasma membrane fraction upon poly(I:C) stimulation, although we cannot exclude contamination of endosomal membranes in this fraction . Cleaved 70-kDa TLR3 was, however, clearly present in the plasma membrane of HT29 cells, both in the absence and presence of stimuli . Immunoblots of SW620 cells showed weak bands of both full-length and cleaved 70-kDa TLR3 in the plasma membrane fraction of SW620 cells , indicating that TLR3 is already present at the cell surface in unstimulated cells, although it is difficult to detect in whole-cell lysate . Strong up-regulation of both full-length and cleaved TLR3 was observed in the plasma membrane fraction of SW620 cells following poly(I:C) stimulation for 24 h . Eea-1 staining was minimal in the plasma membrane fractions from SW620 cells, demonstrating the purity of these plasma membrane fractions . These results show that these cells express both full-length and cleaved TLR3 in the plasma membrane, allowing TLR3 to sense extracellular dsRNA.The finding that poly(I:C) internalization does not seem to be essential for TLR3 signaling in SW620 and HT29 cells implied that TLR3 is expressed at the surface of these cells. Still, we also observed that CXCL10 production in response to poly(I:C) addition was strongly impaired following treatment with bafilomycin A, indicating that acidification of these compartments is required for TLR3 signaling in these IECs. TLR3 resides in the ER following synthesis and is transported via the Golgi to endosomes, where it is subject to proteolytic cleavage in a process dependent on acidification and cathepsins , 25, 27.Fig. 6C), whereas Caco-2 and SW480 cells failed to do so . This correlated with the ability of these IECs to induce CXLC10 in response to poly(I:C) , suggesting that UNC93B1 is involved in TLR3 signaling in these cells  but not critical for CXCL10 production. In contrast, poly(I:C)-induced CXCL10 production in SW620 cells was greatly impaired following UNC93B1 silencing (F and G), showing that TLR3 signaling in these cells is highly dependent on UNC93B1. Thus, UNC93B1 appears to play a role in TLR3 signaling in poly(I:C)-responsive IECs, but these cells display differences with regard to the extent to which they rely on UNC93B1. Although HT29 appear to be less dependent on UNC93B1, this transporter protein seems to be crucial for TLR3 signaling in SW620 cells. This, in turn, may be linked to differences in the abundance of cleaved TLR3 at the plasma membrane of these cells.The chaperone protein UNC93B1 is required for transporting TLR3 from the ER to endosomes and is crucial for TLR3 signaling , 22. UNCFig. 6oly(I:C) . We furtA). We further performed the same invasion assay using SW620 cells treated with siRNA targeting TLR3 to determine whether this effect was TLR3-dependent. Indeed, we found that the increased invasive ability of SW620 cells following poly(I:C) stimulation was highly dependent on TLR3, as the effect was abolished when this receptor was silenced (B). The efficiency of silencing TLR3 was verified by qPCR (C). These results indicate that triggering TLR3 can promote the invasive capability of metastatic IECs.Because we observed that metastatic IECs up-regulated TLR3 and responded to TLR3 stimulation, we proceeded to determine how TLR3 activation affects the invasive properties of metastatic IECs. The invasive capability of metastatic SW620 and non-metastatic SW480 cells following poly(I:C) treatment was determined using a transwell cell invasion assay. Cells were seeded into an upper chamber in serum-free medium and were either left untreated or stimulated with poly(I:C) for 20–24 h before assessment of the number of cells able to traverse an extracellular matrix–coated membrane. Interestingly, we found that the invasive ability of the metastatic cell line SW620 was up-regulated 3-fold following poly(I:C) stimulation, whereas SW480 cells were unaffected by poly(I:C) treatment in this assay A. We fursilenced B. The ef by qPCR C. These In this study, we report differences in the responsiveness of healthy and cancerous IECs to TLR ligands. In particular, we report key differences in TLR3 expression and responsiveness between non-metastatic and metastatic cancerous IECs and that TLR3 activation can enhance the invasive ability of these cells. Only highly metastatic IECs expressed detectable levels of TLR3 and induced the chemokine CXCL10 in response to poly(I:C) addition in a TLR3- and TRIF-dependent manner. Although the observed distinctions between IECs could be explained in terms of genetic variation, we observed interesting differences between SW480 and SW620 cells, which were originally isolated from the same donor, before and after tumor metastasis, respectively. Although SW480 cells did not express detectable levels of TLR3 and failed to respond to poly(I:C) addition, its metastatic counterpart, SW620 cells, up-regulated TLR3 and produced CXCL10 in a TLR3-dependent manner following poly(I:C) stimulation, suggesting that increased TLR3 expression could be a characteristic of progressed cancer. SW480 and SW620 cells share the same genetic background but display epigenetic differences  that appThe majority of studies of the role of TLR3 in cancer have focused on applying dsRNA analogues in cancer therapy because of their ability to induce growth arrest and apoptosis in some TLR3-expressing tumors , 11, 49.52–56–61–et al.  by a mechanism dependent on the cytosolic RNA helicase RIG-I but not in response to added poly(I:C) . This iset al. ; they ob , we speculate that additional posttranslational modifications of IRF3 may be induced by the RIG-I pathway that are required for IFNβ induction. Additional co-activators may also be recruited to the IFNβ enhanceosome in response to poly(I:C) transfection. Abnormal expression of negative regulators that specifically target TLR3-mediated IFNβ induction in metastatic IECs could also explain the absence of IFNβ induction in IECs in response to added poly(I:C). A few negative regulators of TLR3-induced IFNβ have been described that permit TLR3-IRF3–mediated CXCL10 induction as well as RIG-I–induced IFNβ. One of these regulators is the bromodomain and extraterminal family member BRD4, which is essential for poly(I:C)-induced recruitment of IRF3 and c-Jun to the IFNβ promoter. Inhibitors of BRD4 impair IFNβ induction without affecting IRF3 phosphorylation or IRF3 nuclear localization . Represset al. -containing endosomes upon stimulation . Endosomet al. , 44 demoet al. . FHC cells were cultured in DMEM:F12 supplemented with 10% FCS, cholera toxin (10 ng/ml), transferrin (5 μg/ml), hydrocortisone (100 ng/ml), and 10 mm HEPES in a humidified atmosphere of 37 °C and 5% CO2.All cell lines were of human origin. The intestinal epithelial cancer cell lines Caco-2 , SW480 , HCT116 , SW620 , and HT29  and HEK293 cells were obtained from the ATCC. All IEC lines were cultivated in RPMI 1640 supplemented with 10% FCS, 2 m3Cys-Ser-Lys4 (P3C) and fibroblast-stimulating lipopeptide 1 (FSL-1) were from EMC . LPS from Escherichia coli O111:B4, MDP, and poly(I:C) were purchased from Invivogen . Vaccigrade poly(I:C)  was used in most experiments. HMW  and LMW poly(I:C)  were used for comparison of high- and low-molecular-weight poly(I:C). Recombinant TNF was purchased from Peprotech .The synthetic lipopeptides Pam3C (100 ng/ml), poly(I:C) (50 μg/ml), LPS (100 ng/ml), FSL-1 (100 ng/ml), MDP (1 μg/ml), or TNF (100 ng/ml) for 20 h.HT29, SW620, HCT116, SW480, Caco-2, and FHC cells were treated with medium, PIECs were stimulated with poly(I:C) by adding ligand (0.15–50 μg/ml) to cells or transfecting ligand into cells by complexing poly(I:C) (1–2 μg/ml) with Lipofectamine RNAiMAX (1 μg:1 μl ratio) in RPMI for 15 min prior to addition to cells. Cells were treated for 8–24 h.HT29 cells were pretreated with polyclonal anti-TLR3 antibody  or control goat IgG  for 1 h before stimulation with poly(I:C) (2 or 5 μg/ml) for 10 h.m), cycloheximide , bafilomycin A , or DMSO control for 30 min prior to addition of poly(I:C) stimuli.Cells were pretreated with the dynamin inhibitor Dynasore , CXCL10 , or IFNβ  by ELISA (R&D Systems) according to the protocol of the manufacturer.m siRNA against TLR3  or non-silencing control siRNA  using Lipofectamine RNAiMAX (Invitrogen) for TLR3 knockdown or with siRNA against TICAM-1/TRIF  for TICAM/TRIF knockdown using Lipofectamine RNAiMAX. siRNA and transfection reagent  were preincubated for 15 min in RPMI medium before being added to newly seeded cells. Knockdown of targets were confirmed in cell lysates by qPCR.SW620 or HT29 cells were transfected for 24–48 h with 10–20 nm siRNA against IRF3  or IRF7.1 (SI00448672) or non-silencing control siRNA  using Lipofectamine RNAiMAX (Invitrogen). siRNA and transfection reagent (ratio 1:2) were preincubated for 15 min in RPMI before being added to newly seeded cells. Knockdown of targets was confirmed by qPCR.HT29 cells were transfected for 24 h with 10 nm) with siLentFect (Bio-Rad)  in Opti-MEM (Invitrogen) for 30 min before cells in solution were added to the transfection mixture and plated in 24-well plates. After 26 h of transfection, cells were treated with a new transfection mixture and incubated for an additional 20 h before stimulation experiments. To silence CHC, HT29 cells were transfected with non-silencing RNA  or siRNA against CHC 2 (GCAATGAGCTGTTTGAAGA) or CHC 3 (TGACAAAGGTGGATAAATT) . To determine CHC protein knockdown, HT29 cells were lysed and assayed by Western blotting using an antibody against clathrin heavy chain 1  or α-tubulin  as an endogenous control. To silence UNC93B1, cells were transfected with negative control siRNA  or a pool of UNC93B1_4 and UNC93B_6 FlexiTube siRNA  (10 + 10 nm). Knockdown of UNC93B1 was confirmed in cell lysates by qPCR.CHC and UNC93B1 were silenced in HT29 or SW620 cells by complexing siRNAs (20 nTM. Perfecta qPCR FastMixTM from Quanta and TaqMan probes (Life Technologies) for TLR3 (Hs01551078_m1), IFNβ (Hs01077958_s1), CXCL10 (Hs01124251_g1), UNC93B1 (Hs00276771_m1), IRF3 (Hs01547283_m1), IRF7 (Hs01014809_g1), TBP (TATA-box-binding protein) (Hs 00427620_m1), and GAPDH (Hs99999905_m1) were used. Gene expression data were analyzed using a generalized version of the comparative cycle threshold (CT) method for relative quantification with normalization to expression of the reference genes GAPDH or TBP.Total RNA was extracted using the NucleoSpin 96 RNA kit (Macherey-Nagel), and reverse transcription of RNA to cDNA was performed using the Applied Biosystems high-capacity RNA-to-cDNA kit following protocols of the manufacturer. The purity and concentration of RNA were determined using NanoDrop (Thermo Fischer Scientific). Real-time thermal cycling was performed with Applied Biosystems StepOnePlusFLAG  as a control for TLR3 expression using GeneJuice (Novagen) according to the instructions of the manufacturer. Cells were lysed, and gel electrophoresis was performed with the NuPAGE system using 10% BisTris gels (Invitrogen) following the protocol of the manufacturer. Proteins were transferred from gels to nitrocellulose membranes using the iBlot blotting system (Invitrogen). Membranes were blocked in 5% BSA in 0.01 %Tween 20/Tris-buffered saline (TBS-T) for 1 h at room temperature and stained with primary antibodies at 4 °C for 24–72 h in 5% BSA in TBS-T. Anti-TLR3  was used to determine TLR3 expression, and anti-GAPDH  was used as a loading control. Antibodies against IRF3  and phospho-IRF3Ser-396  were used to determine IRF3 activation in IECs upon poly(I:C) treatment, and antibodies against NF-κB p65  and phospho-NF-κB p65Ser-536  were used to determine activation of NF-κB in response to poly(I:C) stimulation. Isolation of plasma membrane proteins was performed using the Minute Plasma Membrane Protein Isolation Kit (Invent Biotechnologies) according to the manufacturer's protocol The resultant protein pellets were dissolved in lysis buffer. Anti-Na,K-ATPase  was used as a positive control for plasma membrane protein fractions, whereas an antibody against early endosome antigen 1 (EEA1)  was used as a marker for the presence of endosomal membranes. Anti-clathrin heavy chain 1  was used to determine expression and confirm knockdown of clathrin heavy chain 1 after siRNA treatment, and α-tubulin  was used as a loading control in these experiments. Blots were stained with secondary HRP-conjugated anti-rabbit or anti-mouse antibody  for 1 h, developed with Super Signal West Femto substrate (Pierce), and imaged using the Odyssey Fc system (Li-Cor).HT29 or SW620 cells were cultured with or without poly(I:C) alone or poly(I:C) complexed with Lipofectamine RNAiMAX (Invitrogen). HEK293 cells were transfected for 24–48 h with pcDNA3 (Invitrogen) or TLR3m NH3OH on a shaker for 30 min. The optical density at 570 nm (MTT assay) or luminescence (Cell Titer Glo assay) was measured using the Walla VictorTM3 1420 multilabel counter (PerkinElmer Life Sciences).Cell viability was determined by MTT assay or using the Cell Titer Glo assay (Promega) according to the instructions of the manufacturer. For the MTT assay, cells were cultured in medium with 3--2,5-diphenyltetrazolium bromide  for 3 h before converted dye was solubilized in alkaline isopropanol with 0.25% 1 HT29 cells were added to Rhodamine-labeled poly(I:C) (Invivogen) for 2 h before cells were washed and fixed with ice-cold fixation buffer (R&D Systems) for 10 min at room temperature. Cells were subsequently stained with antibody against Na,K-ATPase  for 1 h at 4 °C before staining with A488-conjugated anti-mouse secondary antibody (Invitrogen) for 30 min at 4 °C. Cells were observed with an Axiovert 100-M inverted confocal microscope equipped with an LSM 510 laser-scanning unit and a ×63 1.4 Plan Apochromat oil immersion objective .m K-Pipes (pH 6.8), 5 mm EGTA, 2 mm MgCl2, and 0.05% saponin), quenched of free aldehyde groups with 50 mm NH4Cl in PBS with 0.05% saponin (PBS-S), and blocked with 20% human serum (HS) in PBS-S. Cells were stained with antibodies against IRF3  or IRF1  (1:200 dilution) in 1% HS/PBS-S overnight at 4 °C, stained with A647-labeled secondary antibody  for 15 min, and post-fixed with 4% paraformaldehyde/PBS. Nuclei were stained with Hoechst 3342 (200 ng/ml) in PBS-S. Automated imaging was done with the ScanR system (Olympus) using a ×20 objective, 1-s exposure time. 30 frames were captured for each well (∼1000–2000 cells) performed in triplicate. Automated image analysis was done with the ScanR software v1.3.0.3.In nuclear translocation studies, HT29 cells were seeded in 96-well glass-bottom plates  and treated with poly(I:C) alone or complexed with Lipofectamine RNAiMAX for 3 h or overnight (15–24 h) before intracellular staining for IRF1 or IRF3 . In brie−ΔΔCT method (CXCL10 promoter occupancy by IRF3 was investigated in HT29 cells stimulated with poly(I:C) (2 μg/ml) for 3 h by ChIP as described previously . The antT method  with PowCoating experiments were performed to determine whether SW620 and HT29 cells elicit CXCL10 independent of poly(I:C) internalization. 96-well plates  were coated overnight at 4 °C with PBS or titrations of poly(I:C) or double-stranded DNA poly(dA:dT) and then washed. SW620 or HT29 cells were plated  in coated or uncoated wells. Cells in uncoated wells were stimulated by adding titrations of poly(I:C) or poly(dA:dT) in solution. Coating experiments comparing HMW poly(I:C) with LMW poly(I:C) were conducted in the same way.TM 96-well cell invasion assay (Cell Biolabs), using polycarbonate membrane transwell inserts (8-μm pore size); the upper surface of the insert membranes was coated with a layer of dried basement membrane matrix solution to discriminate between invasive and non-invasive cells. The assay was performed according to the instructions of the manufacturer. Briefly, siRNA or untreated cells were serum-starved overnight before cells were seeded into the upper chamber in serum-free medium . Bottom wells were filled with 150 μl of RPMI medium supplemented with 10% FCS. The cells were either left untreated or treated with poly(I:C) (10 μg/ml) and incubated for 20–24 h at 37 °C and 5% CO2. The inserts were then placed in detachment buffer for 30 min to dissociate the invasive cells from the membrane. Upon detachment, cells were lysed and quantified using CyQuant GR fluorescent dye. The fluorescence of each well was determined at 480 nm/520 nm using the fluorescent plate reader function of the Walla Victor3TM 1420 multilabel counter (PerkinElmer Life Sciences).Cell invasion was determined with the CytoSelectt test. Two or more groups were compared with one-way ANOVA with Holm-Sidak post-test or two-way ANOVA with Bonferroni post-test for multiple testing, as indicated in the figure legends.Statistical analysis was performed with GraphPad Prism version 5 or 6. The differences between two groups were determined by two-tailed Student's M. B. designed and conducted the majority of the experiments, analyzed and interpreted the results, and participated in writing the manuscript. B. B. designed and conducted the ChIP experiment shown in Figs. 4


M2-cyclin D3 interaction was validated through GST pull-down and recapitulated in influenza A/WSN/33-infected cells. Knockdown of Ccnd3 by small interfering RNA significantly enhanced virus progeny titers in cell culture supernatants. Interestingly, the increase in virus production was due to cyclin D3 deficiency per se and not merely a consequence of cell cycle deregulation. A combined knockdown of Ccnd3 and Rb1, which rescued cell cycle progression into S phase, failed to normalize virus production. Infection by influenza A virus triggered redistribution of cyclin D3 from the nucleus to the cytoplasm, followed by its proteasomal degradation. When overexpressed in HEK 293T cells, cyclin D3 impaired binding of M2 with M1, which is essential for proper assembly of progeny virions, lending further support to its role as a putative restriction factor. Our study describes the identification and characterization of cyclin D3 as a novel interactor of influenza A virus M2 protein. We hypothesize that competitive inhibition of M1-M2 interaction by cyclin D3 impairs infectious virion formation and results in attenuated virus production. In addition, we provide mechanistic insights into the dynamic interplay of influenza virus with the host cell cycle machinery during infection.To identify new host factors that modulate the replication of influenza A virus, we performed a yeast two-hybrid screen using the cytoplasmic tail of matrix protein 2 from the highly pathogenic H5N1 strain. The screen revealed a high-score interaction with cyclin D3, a key regulator of cell cycle early G The threat of influenza infection is felt globally, and the disease leads to an estimated 3–5 million cases of severe illness and about 250,000–500,000 deaths each year . Althoug2–Drosophila -based genome-wide screening strategies in osophila  and mammosophila –9 to othsophila –, 11, yeasophila –, 12, andsophila –, 13–15, hila –13–, 16 or shila –13–. Althoughila –13–. Host inet al.  infection resulted in host cell cycle arrest in G0/G1 phase, which was accompanied by cyclin D3 relocalization and degradation. Using a combination of small interfering RNA (siRNA)-mediated genetic analyses we further showed that cyclin D3 restricts IAV production, independent of its role in the cell cycle. The restriction of cyclin D3 on IAV life cycle did not impair viral protein synthesis but interfered with M1-M2 binding, which may result in defective assembly and release of progeny virions. The role of cyclin D3 in the context of influenza infection has not been described previously. More interestingly, our results suggest a novel function of cyclin D3 that is beyond its classical function in cell cycle regulation.Altogether, these studies provide evidence that influenza M2, especially the CT domain, plays a critical role in multiple steps of the virus life cycle. Hence, the identification of cellular interactors of M2 would provide mechanistic insights into influenza pathogenesis and possibilities for development of novel strategies to interfere with multiple steps of the infection process. By using M2-CT as bait, we screened a human placenta complementary DNA (cDNA) library to identify host proteins that either facilitate or restrict viral infection. Cyclin D3, a key regulator of cell cycle GA). The screening revealed human cyclin D3 (GenBankTM accession number NM_001760.2) as one of the most prominent interactors of M2-CT, as summarized in Fig. 1B). The M2-binding domain on cyclin D3 is largely outside of its cyclin box, which is responsible for binding to and activation of cyclin-dependent kinases , which are functionally related with tissue-specific expression profiles (–C). Altogether, these results confirm that cyclin D3 binds to the IAV M2-CT.Cyclin D forms a complex with and functions as a regulatory subunit of cyclin-dependent kinases 4 and 6 (CDK2 and -4), whose activity is required in the cell cycle early G1 phase . There aprofiles –43. Our rofiles –C. AltogeD (lane 1), M2 was found to co-purify with cyclin D3, demonstrating their physical interaction during virus infection, either directly or through a common interactor. No interaction was observed in control samples, where cells were either mock-transfected and immunoprecipitated with anti-FLAG antibody  or transfected with cyclin D3-FLAG and immunoprecipitated with isotype control antibody . These data validate cyclin D3 as a bona fide interactor of influenza M2, corroborating our results from the Y2H screening.To examine the intracellular association of M2-CT with cyclin D3 during the IAV life cycle, co-immunoprecipitation experiments were performed from virus-infected cells. HEK 293T cells were transiently transfected with a plasmid encoding FLAG-tagged cyclin D3 (cyclin D3-FLAG) and infected with influenza A/WSN/33 virus. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-M2 antibody. As shown in Fig. 1E).The specificity of interaction between cyclin D3 and M2 was tested using a panel of exogenously expressed cyclins . Although M2 appears to interact with other cyclins to some degree when they are overexpressed, we consistently found that it has the strongest interaction with cyclin D3 E.A, top panels). The effect of M2 protein on cyclin D3 relocalization was recapitulated in analogous experiments using A549 cells expressing M2 protein alone , which resulted in the apparent reduction of cyclin D3 from the nucleus, similar to the phenotype induced by viral infection.To understand the functional relevance of cyclin D3-M2 interaction in virus-infected cells, we tested their subcellular distribution through immunofluorescence imaging. To achieve unbiased analysis, A549 cells were infected with influenza A/WSN/33 virus at an MOI of 0.5 to have a mixed population of infected and uninfected cells in the same sample. Cells were fixed at 24 h postinfection (p.i.) and stained for cyclin D3 and M2 proteins. In uninfected cells, cyclin D3 resides both in the nucleus and in the cytoplasm with only the nuclear CDK4/6-cyclin D3 complex displaying retinoblastoma (Rb)-directed kinase activity . InteresFig. 2B and C, top). Densitometric analysis indicates that 10% of total cyclin D3 was found in the nucleus of uninfected cells. This is very likely an underestimation because cyclin D3, being soluble, would leak from the nuclear into the cytoplasmic fraction during the experimental procedures. The percentage of nuclear cyclin D3 dropped to <4% at 8 h p.i. , indicating translocation of cyclin D3 from the nucleus to the cytoplasm in virus-infected cells. This significant (>60%) reduction of nuclear cyclin D3 detected through biochemical measurement confirmed our immunofluorescence data on its subcellular relocalization.To address the reduction of cyclin D3 from the nucleus upon virus infection, we fractionated both control and infected cells into cytosol and nuclear material to measure cyclin D3 levels. Cytoplasm- and nucleus-enriched extracts were prepared from A/WSN/33-infected (MOI of 2 to achieve a more homogeneous infection of the cell culture) and uninfected A549 cells. Lamin B1 and GAPDH were used as markers to verify clear separation of nuclear and cytoplasmic fractions. Total intracellular level of cyclin D3 in virus-infected cells decreased to ∼60% of that observed in mock-infected cells . Most of cyclin D3 appeared in the cytosolic fraction under these conditions. Interaction of cyclin D3 with M2, combined with its enrichment in the cytosol upon M2 expression strongly suggests that this association must occur in the cytosol, either directly or as a complex presumably en route to assembly and release of intact progeny virions.We further verified colocalization of cyclin D3 with that of M2 through infection of A/WSN/33 in cyclin D3-expressing cells. Because at 24 h p.i. most of cyclin D3 is degraded, we selected an earlier time point and treated mock- and virus-infected cells with MG-132 to increase the signal strength of cyclin D3. Indeed, at 15 h p.i., we could capture colocalization of influenza M2 and cyclin D3 along the secretory pathway D. Most o1 phase regulator, cyclin E, were measured through immunoblotting. Cyclin D3 was dramatically down-regulated by IAV infection to almost undetectable levels compared with uninfected cells at 24 h p.i. . A time course analysis revealed that the steady-state levels of cyclin D3 started declining at 6 h p.i. and decreased by ∼50% at 9 h p.i. . Cyclin E, which is activated at the late stage of cell cycle G1 phase to facilitate G1/S transition, displayed only a moderate reduction under identical conditions (A), contrary to the finding by He et al.  (1 phase (G0/G1 phase) of the cell cycle machinery.The induction of D-type cyclins is growth factor-dependent, and both mRNAs and proteins exhibit moderate oscillations in continuously proliferating cell populations , 45–48. 45–4 h p.i. A, top. A9 h p.i. A, bottomnditions A, contrae et al. , where csee also A . Thus, an obvious rescue of cyclin D3 was observed after one round of infection (9 h p.i.) through the use of proteasome inhibitors. The regulation of other cell cycle moderators was also investigated. Although viral infection also resulted in cyclin D1, CDK4, and CDK6 down-regulation on protein level (A), the level of down-regulation is not as robust as that of cyclin D3, and their protein levels could not be rescued by protease and calpain inhibitor when compared with cyclin D3 (B). These findings suggest that IAV posts a cyclin D3-specific effect during infection.To further investigate the mechanism of virus-induced cyclin D3 down-regulation, we determined the involvement of the proteasomal degradation pathway in this process. Cells were infected with A/WSN/33 virus in the absence or presence of proteasome inhibitors, MG-101 or MG-132, and tested for cyclin D3 levels. MG-101, also known as calpain inhibitor I, inhibits both proteasome and calpain activities . CalpainFig. 3in level A, the leyclin D3 B. These Our data indicate that IAV infection results in reduced cyclin D3 levels in the nucleus of virus-infected cells. This phenotype is triggered by translocation of nuclear cyclin D3 into the cytoplasm, where IAV facilitates proteasomal degradation of cytoplasmic cyclin D3.1 phase when compared with the uninfected group (data not shown). To reduce variability due to a mixed population of cells in differential phases of the cell cycle, we first synchronized them before infection in G1 using lovastatin, a fungal metabolite shown to affect cell cycle distribution in a reversible manner (A). The overlay of cell cycle patterns of virus-infected and uninfected cells indicates that infection decreased the peaks representing cells in S and G2/M phases (B). The percentages of cells in G1 phase exhibited a progressive, time-dependent drop from 83.0 ± 2.1 to 80.1 ± 0.9% and subsequently 45.7 ± 2.8%  in the mock-infected samples. This reduction reflects progression of cells through G1 phase, followed by entry into the later stages of the cell cycle. In contrast, cells infected by A/WSN/33 virus remained in G1 with the percentage of cells in G1 phase hovering above 80% at all time points (C). Our results demonstrate that IAV infection impairs progression of cell cycle through the G1/S intersection.The principal role of the D-type cyclins is to act as growth factor sensors, converting environmental signals into fuel for the cell cycle engine. The interaction between cyclin D3 and M2-CT during the viral life cycle implies a potential regulation of host cell cycle by IAV. This is supported by our observation that cyclin D3 is actively removed from the nucleus and degraded by the proteasome in virus-infected cells. To address whether the cell cycle is compromised, a quantitative profiling of cell cycle kinetics was performed on virus-infected A549 cells. Using an asynchronously growing cell population, we found that virus-infected cells accumulated at Ge manner . Cells we manner A. The ovM phases B. The pee points C. Our re1 phase, we used a separate approach. Cells were preaccumulated in G2/M phase by treatment with RO-3306, a CDK1 inhibitor (D). Similar to the result obtained with lovastatin, the cell cycle pattern of virus-infected cells showed lower peaks for both S and G2/M phases when compared with uninfected cells (E). Upon recovery of cells from drug restriction, both uninfected and infected cells resumed division and initiated a new round of cell cycle, displaying an increased percentage of cells representing G0/G1 phase at 9 h p.i.; only uninfected cells, however, progressed further into S phase. The percentage of cells in G0/G1 phase dropped from around 85 to 65% in the control sample, whereas it remained constant in IAV-infected cells (F). Collectively, these results indicate that IAV infection perturbs normal cell cycle progression in human cell lines and installs a block at G0/G1 phase.To further prove that IAV infection induced cell cycle arrest in Gnhibitor . At 0 h nhibitor D. Similaed cells E. Upon red cells F. CollecA). Furthermore, serine 807/811 phosphorylation on Rb (ppRb Ser-807/811), which affects the S phase of the cell cycle displayed a similar trend , an observation consistent with the virus-induced block at G0/G1 phase. In line with the down-regulation of ppRb proteins, accumulation of hypophosphorylated Rb (pRb) was observed .D-type cyclins activate CDK4/6, which initiate phosphorylation of Rb at multiple sites. Hyperphosphorylated Rb (ppRb) releases transcription factor E2F and leads to the activation and transcription of cell cycle progression-responsive genes , 57, 58.Fig. 6ar trend  . Expression levels of p16 from the INK4 family of CKI, which specifically inhibits the catalytic subunits of CDK4 and CDK6, were also measured. No increase in p16INK4A was observed even after prolonged infection time (data not shown).Inhibition of cell cycle can also occur through induction of cyclin-dependent kinase inhibitors (CKIs). P21e arrest . Interes pathway , A and BFrom the results described above, we conclude that IAV infection down-regulates cyclin D3 levels, which in turn inhibits CDK4/6 activation and ultimately results in reduced Rb phosphorylation. Consequently, pRb sequesters the transcription factor E2F, which prevents cell cycle entry into S phase .Ccnd3 knockdown on virus replication. To avoid off-target effects imposed by the use of siRNA pools, we used an individual siRNA duplex from the published literature (CCND3 #1) for generating a knockdown (A). Control (NT siRNA-treated) and cyclin D3-deficient cells were infected with A/WSN/33 at an MOI of 0.01 to allow multiple rounds of infection. Viral titers obtained from cyclin D3-deficient cells were at least 10-fold greater than those from control cells . Because cyclin D3-M2 interaction was identified using avian M2-CT, we measured the effect of Ccnd3 knockdown on the replication of highly pathogenic H5N1 avian influenza virus and observed a more >10-fold higher progeny virus production in cyclin D3-deficient cells when compared with control group at 24 h p.i. . The difference in production of H5N1 virus was less significant at 48 h p.i. (∼2.5-fold), most likely due to the cytopathic effect of H5N1 virus in a limited cell culture system.To establish a functional relevance for cyclin D3 in IAV pathogenesis, we studied the effect of nockdown . siRNA Cnockdown A. Controol cells B, left. 4 h p.i. B, right.Ccnd3 and Rb1 rescued cells from the G0/G1 phase block imposed by cyclin D3 deficiency, allowing us to measure the effect of cyclin D3 alone on virus replication. In addition, we included a Ccne1 knockdown, which induced cell cycle arrest at the G1/S interface without affecting cyclin D3 levels. To gain further confidence in the specificity of the cyclin D3 siRNA, we included a second siRNA duplex specific for cyclin D3 (CCND3 #2). Both individual siRNAs and siRNA combinations resulted in significant reduction of the desired gene expressions (C). In the control siRNA-treated cell population, around 55% of cells were found in G0/G1 phase of the cell cycle. As expected, knockdown of Ccnd3 alone arrested cell cycle progression in A549 cells with ∼65% (CCND3 #1) or ∼70% (CCDN3 #2) of cells in G0/G1 phase when compared with cells treated with NT siRNA. A concomitant Ccnd3/Rb1 knockdown rescued cell cycle progression into S phase. Knockdown of Ccne1 expression in A549 cells induced a more robust cell cycle G0/G1 phase accumulation than Ccnd3 knockdown with ∼80% cells in G0/G1 phase (D). For all siRNA-mediated knockdowns, cells were subjected to infection with A/WSN/33 virus at an MOI of 0.01 at 72 h post-transfection for progeny virus production. Ccnd3 knockdown with the two individual siRNA duplexes increased progeny virus titers by approximately 1 log. Despite the rescue in cell cycle progression, a double knockdown of Ccnd3/Rb1 did not normalize virus production when compared with NT siRNA-treated cells. A 5-fold difference in viral titers persisted between control cells and those that harbored a double knockdown in Ccnd3/Rb1. Virus titers from cells with Ccne1 knockdown were comparable with those from Ccnd3/Rb1 double knockdown .To differentiate whether the increased progeny virus titer was intrinsic to the absence of cyclin D3 protein or a consequence of cell cycle arrest induced by cyclin D3 silencing, we next used combinations of siRNAs targeting various steps in the cell cycle to modulate its progression. Concomitant knockdown of ressions C. In theG1 phase D. For alnockdown E, left.E, middle and right panels).To study whether the inhibitory role of cyclin D3 on viral replication is also applicable to the other IAV strains, two clinical isolates were tested. At both 24 and 48 h p.i., equivalent or higher virus titers for influenza A/HK/1/68 (H3N2) and A/HK/54/98 (H1N1) viruses confirmed our results with A/WSN/33 , comparable protein amounts of cyclin D3 were detected in A549, HBEC, and pneumocytes (B). Cyclin D2 expression could be detected in the two primary cells but not in A549 (B). Collectively, cyclin D3 is expressed in tissues along the natural infection route of IAV. To validate its functional relevance in these cells, we performed a knockdown of cyclin D3 in HBECs to study its effect on influenza virus production. As observed with A549 cells, cyclin D3 deficiency in HBEC resulted in ∼10-fold increase in viral titers, supporting our conclusions from experiments performed in actively dividing cells (C and D).To examine the distribution of D-type cyclins in the context of natural infection, we determined their expression in the human airway, in particular human bronchial epithelial cells (HBECs) and pneumocytes. Although cyclin D3 mRNA was expressed at lower levels compared with the other two D-type cyclins in both cell types A, comparumocytes B. Cyclin in A549 B. Collecng cells , C and DCcnd3/Rb1 double knockdown. Multiple time points were selected to measure viral protein synthesis within a single cycle of infection. Knockdown cells did not display enhanced viral protein synthesis compared with control cells . Upon overexpression of cyclin D3, the level of M1 that co-precipitated with M2 was significantly lower than the amount obtained in the absence of cyclin D3 . Densitometric analysis revealed that a reduction of ∼70% in binding of M1 to M2 was observed when cyclin D3 was overexpressed . An isotype control antibody used in parallel did not bind to M1 . Our results demonstrate that (i) cyclin D3 does not affect viral protein synthesis during IAV life cycle and (ii) interaction between M1 and M2 is significantly attenuated in the presence of cyclin D3. We propose that through competition with M1 for M2 binding, cyclin D3 restricts IAV production either through reduced or defective progeny virion formation.The biogenesis of viral particles involves proper packaging and assembly of viral proteins and RNA to form intact virions at discrete budding sites. M2 is a multifunctional protein; its cytosolic tail interacts with M1, promoting the recruitment of viral internal proteins and vRNA to the plasma membrane for efficient virus assembly . We postFig. 10C, more M2 was detected at the plasma membrane in cyclin D3-deficient cells. This suggests that without the blockage imposed by cyclin D3, a greater amount of M2 could reach the cell surface and/or at a faster pace, thus facilitating the recruitment of viral internal components to the plasma membrane for assembly and budding. This trend could be observed at both time intervals, as summarized in Fig. 10B. In addition, we could validate these results using confocal imaging. Overexpression of cyclin D3 resulted in both M1 and M2 being predominantly confined to the early secretory network compared with control cells with endogenous cyclin D3 levels, where a significant fraction of M2 appeared at the plasma membrane . Interestingly, although the absence of cyclin E1 (a weak interactor of M2), facilitates IAV progeny virus production, this effect occurs through a different mechanism because M2 surface expression remained unaffected in cells depleted of cyclin E1 (data not shown).To test this hypothesis, cyclin D3-deficient cells and control cells were infected with A/WSN/33 to measure plasma membrane expression of viral M2 protein within one cycle of infection. As illustrated in Fig. 6+/K+-ATPase, heat shock protein 40 (Hsp40), and p58IPK (inhibitor of protein kinase) (–The search for the interactome of M2 protein of IAV in host cells has been limited so far. Host proteins that M2 has been shown to exploit include Na kinase) –67. In akinase) –. Withdrakinase) –. We prevkinase) –. More rekinase) –, 71. A hCcnd3 knockdown by default leads to cell cycle G0/G1 phase arrest, providing a favorable environment for IAV replication. Our results indicate that cyclin D3 functions beyond its role in cell cycle in the context of influenza infection. The absence of cyclin D3 in combination with accumulation of cell cycle in G1 phase appears to have an additive effect on IAV progeny virus production, indicating that cyclin D3 deficiency specifically facilitates influenza virus production independently of the cell cycle. Instead, co-immunoprecipitation with M2 suggests that cyclin D3 impairs nascent virus production at a late stage of the virus life cycle by interfering with M1-M2 binding  is the best characterized. In addition, interferon-stimulated gene (ISG) products are a group of newly emerging host restriction factors that can control viral, bacterial, and parasite infection, as exemplified by viperin and ISG15, to name a few (–Our study provides important insights into the mechanism through which cyclin D3 restricts IAV infection. We demonstrated that the increase in progeny virus production upon me a few –81. The TM accession number 81975894) was used as bait to screen for interactors from a random-primed human placenta cDNA library, which contains 10 million independent fragments. The cloning of bait and Y2H screening were performed by Hybrigenics . Briefly, M2-CT was fused with the LexA binding domain by subcloning it into the pB27 vector. The L40ΔGAL4 yeast strain was then transformed with pM2-CT bait plasmid and mated with Y187 yeast strain transformed by the prey library , human embryonic kidney (HEK 293T), and Madin-Darby canine kidney (MDCK) cells were purchased from ATCC. Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) was used to culture and maintain all cell types used.Influenza A virus strains A/WSN/33 (H1N1), A/HK/1/68 (H3N2), A/HK/54/98 (H1N1), and A/Vietnam/1203/2003 (H5N1) were propagated in MDCK cells supplemented with 0.3% bovine serum albumin (BSA) (Sigma-Aldrich) in the presence of 1 μg/ml tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Thermo Scientific) and stored as virus stocks at −80 °C until further use. Viral titers were obtained by a plaque assay on MDCK cells. Briefly, serial 10-fold dilutions of virus stocks were adsorbed onto a confluent monolayer of MDCK cells for 1 h at 37 °C. Next, viral inoculums were removed, and cells were washed twice with phosphate-buffered saline (PBS), pH 7.4. Cells were then covered with 1% semisolid agar in DMEM complemented with 0.3% BSA and 1 μg/ml TPCK-treated trypsin. Three days after incubating the plates in an upside down manner, plaques were visualized by staining with crystal violet .Homo sapiens, GenBankTM accession number NM_001760.4) from plasmid pCMV6-CCND3-GFP (Origene Technologies) into the pCMV6-Entry vector at the MluI and SgfI restriction sites. Plasmid pCAGGS-M2-FLAG, pCAGGS-M1-HA, and rabbit anti-M1 polyclonal antibody (pAb) were kindly donated by Prof. Adolfo García-Sastre (Icahn School of Medicine at Mount Sinai).Plasmid pCMV6-FLAG-CCND3 was generated by subcloning the CCND3 open reading frame (ORF)  (clone DCS22) was purchased from Cell Signaling Technology; mouse anti-cyclin E mAb (clone HE12), mouse anti-influenza A M2 mAb (clone 14C2), mouse anti-influenza A M1 mAb (clone GA2B), and rabbit anti-p21 pAb were purchased from Santa Cruz Biotechnology, Inc.; mouse anti-influenza A M2 mAb (clone 14C2) was purchased from Thermo Scientific; mouse anti-FLAG mAb (clone M2) and mouse anti-c-Myc mAb (clone 9E10) were purchased from Sigma-Aldrich; mouse anti-human pRb mAb (clone G99-549), mouse anti-Rb (ppRb Ser-780) mAb (clone J146-35), and mouse anti-Rb (ppRb Ser-807/Ser-811) mRb (clone J112-906) were purchased from BD Biosciences; rabbit anti-cyclin D3 mAb (clone EP463E), mouse anti-influenza A nucleoprotein mAb (clone AA5H), mouse anti-lamin B1 mAb (clone ZL-5), and mouse anti-GAPDH mAb (clone 6C5) were purchased from Abcam; and Texas Red-conjugated goat anti-rabbit IgG (H+L) (pAb) and FITC-conjugated goat anti-mouse IgG (H+L) (pAb) were purchased from Life Technologies, Inc.Escherichia coli strain BL21. The resulting GST fusion protein was purified with glutathione-Sepharose beads  and added to A549 cell lysates. The empty vector expressing GST alone was used as negative control. After overnight incubation, beads were collected by centrifugation and washed five times to remove nonspecific binding. Bound proteins were resolved by SDS-PAGE and immunoblotting with the indicated antibodies.A DNA fragment encoding the M2 gene of influenza virus A/WSN/33 strain was cloned into the pGEX4T-1 vector and expressed in m Tris (pH 7.4), 150 mm sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm EDTA, and protease inhibitors (Roche Applied Science). Cell extracts were cleared by centrifugation, and supernatant was incubated with anti-FLAG or isotype control antibody overnight at 4 °C. Protein G-Sepharose beads  were then used to recover the immune complexes from whole cell lysates. Immunoprecipitated samples were washed five times with lysis buffer before being resolved by SDS-PAGE and detection by Western blotting. An interaction specificity study was performed in HEK 293T cells transfected with plasmid encoding any one of the c-Myc-tagged cyclins, D1, D2, D3, or E1, and targeted to similar treatment as described above for co-immunoprecipitation experiments.HEK 293T cells were transfected with pCMV6-FLAG-c-Myc-CCND3 plasmid in a 10-cm dish. At 48 h post-transfection, cells were infected with influenza A/WSN/33 strain at an MOI of 2 for 6 h before being lysed in buffer containing 50 mFor competitive binding assays, HEK 293T cells were co-transfected with pCAGGS-M1-HA and pCAGGS-M2-FLAG plasmids or with pCAGGS-M1-HA, pCAGGS-M2-FLAG, and pCMV6-FLAG-cMyc-CCND3 plasmids in 10 cm dishes. Bulk cell lysates were prepared as described above at 48 h post transfection. Cleared cell extracts were incubated with anti-M2 (Santa Cruz) or isotype control antibody overnight at 4 °C, followed by recovery of the immune complexes from whole cell lysates using protein G-Sepharose beads. Immunoprecipitated samples were washed six times with lysis buffer before being resolved by SDS-PAGE and detection by Western blotting.1 phase. Briefly, culture medium was removed 24 h after the initial plating of A549 cells and replaced with fresh complete medium containing 40 μm lovastatin (Sigma-Aldrich). Cells were incubated in lovastatin for 22 h to achieve cell cycle G0/G1 phase synchronization before other experimental procedures. At time 0, cells were allowed to reenter cell cycle progression by replacing the medium with fresh complete medium supplemented with 4 mm mevalonolactone (Sigma-Aldrich). The synchronization of cells at the G2/M transition by RO-3306 was as follows. At 24 h following plating A549 cells, medium was replaced with fresh complete medium containing 4 μm RO-3306  and incubated for 18 h. Cells were then washed three times with PBS and cultured in fresh complete medium to promote cell cycle progression.Lovastatin was used to synchronize cells in Gm BrdU (Invitrogen) for 1 h and fixed overnight in 70% ethanol at −20 °C. BrdU-pulsed cells were rescued from ethanol and denatured by 2 m hydrochloric acid with 0.5% Triton X-100 for 30 min at room temperature, followed by neutralization with 100 mm borate buffer (pH 8.5). BrdU labeling was revealed with Alexa Fluor 647-conjugated BrdU mouse mAb . Total DNA content was measured by 7-aminoactinomycin D (BD PharMingen) staining. Cells were then immediately analyzed on a BD LSR II flow cytometer (BD Immunocytometry Systems). Approximately 30,000 cells were acquired for each sample, and data were analyzed using FlowJo software (Treestar).Cell cycle profiles were analyzed by BrdU incorporation and DNA content staining. Briefly, presynchronized A549 cells were infected at an MOI of 2 with influenza A/WSN/33 strain or mock-infected. Immediately before collection, cells were incubated in 10 μm Tris (pH 7.4), 150 mm sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mm EDTA, and protease inhibitors. After end-to-end rotation at 4 °C for 1 h to facilitate lysis, cell lysates were cleared by centrifugation at 20,817 × g for 30 min at 4 °C. Whole cell lysates were further boiled at 95 °C for 10 min in 1× sample loading dye and 50 mm DTT before being subjected to SDS-PAGE.A549 cells were infected with A/WSN/33 virus at an MOI of 2 or mock-infected. At various time points after infection, cells were washed twice with ice-cold PBS and lysed in buffer containing 50 mm MG-101 (Sigma-Aldrich) or 5 μm MG-132 (Sigma-Aldrich) at the start of infection. DMSO (Sigma-Aldrich)-containing medium was used as vehicle control in this experiment. Whole cell lysates were collected 9 h after infection as described above.To block ubiquitin-mediated protein degradation, A/WSN/33 virus-infected (MOI of 2) or mock-infected A549 cells were incubated in fresh medium containing either 40 μm Tris (pH 7.4), 5 mm magnesium chloride, 150 mm sodium chloride, and 0.1% Triton X-100. Permeabilized cells were collected with a rubber policeman and transferred into Eppendorf tubes. After a very brief incubation, supernatant (cytoplasm-enriched) and pellet (nucleus-enriched) fractions were separated upon centrifugation at 20,817 × g for 30 s at 4 °C. The nuclear fraction was then solubilized in buffer containing 25 mm Tris (pH 7.4), 5 mm magnesium chloride, 150 mm sodium chloride, and 1% SDS. DNA shredder was used to process the nuclear fractions to shear the genomic DNA for easier SDS-polyacrylamide gel loading of the samples. Sample loading dye and DTT at a final concentration of 1× and 50 mm, respectively, were added into the lysate before boiling at 95 °C for 10 min.For cell fractionation, A549 cells were infected at an MOI of 2 with influenza A/WSN/33 virus. At 8 h p.i., cells were washed twice with ice-cold PBS and lysed on a dish with ice-cold buffer containing 25 mm MG132 for the duration of infection. 15 h p.i. cells were collected for the immunofluorescence assay.To capture subcellular colocalization between influenza M2 and cyclin D3, A549 cells were transfected with a wild-type cyclin D3 plasmid (pCMV6-CCND3-cherry). 24 h post-transfection, cells were either mock- or virus-infected with A/WSN/33 at an MOI of 2. Cells were maintained in medium supplemented with 5 μTo determine the effect of cyclin D3 on co-localization between influenza M1 and M2, A549 cells were transfected with either an empty vector or cyclin D3 (pCMV6-FLAG-CCND3). 24 h post-transfection, cells were infected with influenza A/WSN/33 at an MOI of 2. 10 h p.i., cells were collected for staining and confocal imaging of influenza M1 and M2.m ammonium chloride for 10 min to quench the autofluorescence of paraformaldehyde before being permeabilized in PBS containing 0.1% Triton X-100 for 10 min. Cyclin D3 was stained with rabbit anti-cyclin D3 mAb (EP463E), followed by incubation with FITC-conjugated goat anti-rabbit IgG. The viral M2 protein was visualized by labeling with mouse anti-M2 mAb (14C2), followed by staining with Texas Red-conjugated goat anti-mouse IgG. The viral M1 protein was visualized by labeling with rabbit anti-M1 pAb, followed by FITC-conjugated goat anti-rabbit IgG. After washing with PBS, coverslips were incubated with DAPI for nucleus staining and mounted in Mowiol on glass slides. Images were acquired with a Zeiss LSM 700 confocal fluorescence microscope at the Faculty Imaging Core Facility of the University of Hong Kong.A549 cells were plated onto glass coverslips 1 day before transfection or infection. Cells were washed with PBS and fixed in 4% paraformaldehyde for 15 min at the end of the experiments. Coverslips were incubated in 50 mm for each gene) by using DharmaFECT 1 transfection reagent according to the manufacturer's protocol. Cells were subcultured 48 h post-transfection to avoid potential cell cycle arrest induced by overconfluence.All gene-specific siRNAs were purchased from Dharmacon (Dharmacon Research). Individual siRNAs against the 5′-AAGGAUCUUUGUGGCCAAGGA-3′ sequence  or 5′-UGTo study the effect of gene knockdown on influenza progeny virus production, siRNA-treated A549 cells or HBECs were infected with influenza viruses 24 h after seeding at an MOI of 0.01 in DMEM containing 0.3% BSA (Sigma-Aldrich), in the presence of 0.1 μg/ml TPCK-treated trypsin (Thermo Scientific). Supernatants were collected at 24 and 48 h p.i., and the progeny virus titers were obtained by a plaque assay as described above. Separate wells without infection were used to determine the knockdown efficiency by Western blotting.The effect of siRNA treatment on influenza viral protein expression was investigated in cells prepared as described above. Cells were infected with A/WSN/33 virus at an MOI of 2 and supplied with fresh cell culture medium containing 10% FBS and 100 units/ml penicillin and 100 μg/ml streptomycin after removing the virus inoculums. Whole cell lysates were prepared from individual wells at 3, 6, 9, and 12 h p.i. for Western blotting analysis.Cyclin D3-specific siRNA-treated and control A549 cells were infected at an MOI of 1 with A/WSN/33 virus to study the effect of cyclin D3 reduction on M2 plasma membrane expression. To synchronize viral entry, cells were incubated with virus at 4 °C for 1 h and replaced with prewarmed fresh cell culture medium. Virus-infected cells were collected and fixed at 6 and 8 h p.i. for surface M2 expression detection using anti-M2 mAb (Thermo Scientific) followed by FITC-conjugated secondary antibody without permeabilization. Cells were then immediately analyzed on a BD LSR II flow cytometer (BD Immunocytometry Systems). Approximately 30,000 cells were acquired for each sample, and data were analyzed using FlowJo software (Treestar).5 HBECs and pneumocytes using an RNeasy Plus kit in combination with QIAshredder (Qiagen). cDNA was then generated from the total RNA using the SuperScript III first-strand synthesis system (Invitrogen) according to the manufacturer's recommendation. The cDNA product was used undiluted for HBECs or 20-fold diluted for pneumocytes. Serial dilutions of plasmids of cyclin D1, D2, D3, and E1 with known gene copy numbers were used to generate standard curves for absolute quantification of the desired gene expression. The real-time PCRs were performed in 25-μl reactions containing fluorigenic Taqman probe at a final concentration of 400 nm together with an 800 nm concentration of each primer and 12.5 μl of 2× Probe Master (Roche Applied Science). The assays were performed on the LightCycler 480 II (Roche Applied Science) with thermal cycling conditions of 10 min at 95 °C followed by 40 cycles of 95 °C for 10 s, 60 °C for 1 min, and 72 °C for 1 s. Each sample was quantified in triplicate for each experiment. Primers and probes were as follows: 5′-CTGTCCTACTACCGCCTCACA-3′ (forward), 5′-GCCTTGGGGTCCATGTTCT-3′ (reverse), and 5′-(FAM)CCAGAGTGATCAAGTGTGACCCAGACTGCC(TAMRA)-3′ (Taqman probe) for cyclin D1 (Gene expression patterns were determined by two-step RT-PCRs. Briefly, total RNA was isolated from around 1 × 10yclin D1 ; 5′-GGGAyclin D1 ; 5′-TCCGyclin D1 ; 5′-GAAGyclin D1 .t test or two-way ANOVA, with a 0.05 significance level.Results are shown as mean ± S.D. of the indicated number of experiments. Statistical difference between groups was determined by unpaired Student's Y. F. designed, performed, and analyzed the experiments. C. K. P. M. performed the H5N1 experiment in


Given that mutated p53  is over-expressed in many cancers, restoration of mutant p53 to its wild type biological function has been sought after as cancer therapy. The conformational flexibility has allowed to restore the normal biological function of mutant p53 by short peptides and small molecule compounds. Recently, studies have focused on physiological mechanisms such as acetylation of lysine residues to rescue the wild type activity of mutant p53. Using p53 null prostate cancer cell line we show that ID4 dependent acetylation promotes mutant p53 DNA-binding capabilities to its wild type consensus sequence, thus regulating p53-dependent target genes leading to subsequent cell cycle arrest and apoptosis. Specifically, by using wild type, mutant , acetylation mimics (K320Q and K373Q) and non-acetylation mimics (K320R and K373R) of p53, we identify that ID4 promotes acetylation of K373 and to a lesser extent K320, in turn restoring p53-dependent biological activities. Together, our data provides a molecular understanding of ID4 dependent acetylation that suggests a strategy of enhancing p53 acetylation at sites K373 and K320 that may serve as a viable mechanism of physiological restoration of mutant p53 to its wild type biological function. The somatic p53 missense mutations observed in almost half of all human cancers is a critical step in the oncogenic process , 2. The Mutant p53 can be classified into two groups: type I mutations, which affect amino acid residues directly involved in the DNA interaction , and class II mutations involving residues responsible for stabilization of the three-dimensional structure of p53 , 11. StrStructurally, wild type p53 consists of unfolded regions with high tendency for aggregation , 13NumerStudies have shown that the all p53 mutations are not functionally equivalent . The mutWe and others have shown that in the case of wild type and mutant forms of p53, the interaction with acetylases and subsequent acetylation of p53 itself is indispensable for DNA binding and transcriptional activity such as the ability of p53 to trigger cell cycle arrest or apoptosis , 27, 28.Here, we demonstrate that ID4, can promote p53-dependent apoptosis and senescence in prostate cancer cells by specifically modifying the acetylation of p53, which increases its transcriptional activity and promotes the expression of pro-apoptotic and cell cycle regulatory genes. Furthermore, using acetylation mimics of mutant p53, we identify lysine 373 and to a lesser extent lysine 320 as critical ID4 dependent acetylation sites that aid in p53 biological activities. Together, the results presented herein suggest that ID4 dependent p53 acetylation can be used as model to re-activate mutant p53 to wild type biological activity.The PCa cell line PC3 was used to investigate ID4-p53 cross-talk. Specifically, we focused on the role of ID4 in regulating wild type and mutant p53 dependent apoptosis, senescence and transcriptional activity. The PC3 cell line is null for p53 and has low endogenous levels of ID4 Figure . Wild tyStable Silencing of ID4 and Expression of wild type and mutant P53 in PC3 cells: ID4 expression Figure  was undeP < 0.001), the decrease in apoptosis of VF and PL was only marginal (75.3% and 81.4% respectively) as compared to wild type p53 (set to 100%). Thus the two mutant forms of p53 (VFPL) expressed together (as expressed in DU145 cells) were less effective in promoting apoptosis (50.8%) as compared to wild type p53 or VF and PL mutants expressed separately  Figure  < 0.001.y Figure . These rP < 0.05). Interestingly, a significantly lower apoptotic index was observed in PC3(-)ID4 cells expressing PL and VF mutants  as compared to PC3(-)ID4 cells with wild type p53 (100%) and as compared to PC3 cells with PL and VF mutants (75.3% and 81.4% respectively)  was higher compared to the mutants expressed individually in PC3(-)ID4. The results suggested that loss of ID4 leads to a decrease in apoptosis overall but has a significantly stronger effect on the apoptosis induced by the two mutant alone (VF and PL) as compared to the two mutants together (VFPL). Thus, the results indicate that p53 promotes apoptosis in an ID4 dependent manner in PC3 cells.The apoptotic index of PC3(-)ID4 with wild type p53 (64%) was significantly lower as compared to PC3 cells with wild type p53  Figure .The percThe expression of key downstream targets of p53 were used to investigate the mechanism by which wild type and mutant p53 regulates apoptosis in the presence or absence of ID4.The expression of cyclin dependent kinase inhibitor CDKN1A (p21), BAX and PUMA, which are well characterized p53 target genes increased in the presence of wild type and mutant p53 as compared to cells transfected with EV Figure  29]. In. In29]. Loss of ID4 resulted in a significant decrease in the expression of all three p53 target genes in PC3(-)ID4 cells transfected with either wild type or mutant p53. These results suggested that ID4 expression may be required to promote the activation of p53 downstream targets, irrespective of the p53 mutations Figure .in vitro p53 DNA binding activity assay (using p53 response element immobilized on a 96 well plate followed by detection with p53 specific antibody) using nuclear extracts, a significant increase in p53 DNA binding activity in the PC3+p53 (wild type and mutant p53) as compared the PC3(-)ID4 was observed (p < 0.001)  Figure  with corWe next performed a functional assay to further confirm the p53 transcriptional activity in PC3 cells. Using a p53 response element (wild type-p53RE) luciferase reporter plasmid assay, the relative p53 luciferase activity decreased significantly in PC3(-)ID4+p53 (wild type and mutant) as compared to PC3+p53 (wild type and mutant) cells Figure . SurprisHaving established that ID4 plays a critical role in modulating wild type and mutant p53 associated apoptosis, through the induction of a cell cycle checkpoint p21 and apoptosis (BAX and PUMA), we next investigated whether p53 also regulates cellular senescence in an ID4 dependent or independent manner. Senescence in normal cells is associated with a flattened, enlarged morphology with > 2-fold increase in cellular diameter compared to non-senescent cells [P < 0.001). A varying degree in SA-βgal staining was also observed in the PC3+PL/ VF or VFPL. Interestingly, senescence was not significantly different in PC3+PL but a disproportionate increase in cells with low-moderate senescence was observed in VF cells  with a corresponding decrease in cells with high senescence  as compared to PC3+wild type p53 cells. The senescence in VFPL cells was not significantly different as compared to the PC3 cells transfected with EV  and those with light (Low-Moderate) blue staining were quantitated Figure . Over 60V Figure .A significant increase in senescent cells was also observed in PC3(-)ID4 cells transfected with either wild type or mutant p53. Interestingly cells with high senescence were not observed in PC3(-)ID4+wild type p53 cells but almost all cells showed low/moderate senescence. This is in stark contrast to PC3+wild type p53 cells in which a significant fraction of cells was highly senescent. The PL or VF mutants in PC3(-)ID4 also demonstrated low/ moderate senescence that was not significantly different from PC3(-)ID4+wild type p53 Figure . A decrep < 0.001) as compared to vector only control (27.5%) but was significantly lower (p < 0.001) as compared to PC3+wild type p53 (apoptotic index 100%). The apoptotic index of the R273H mutant (41%) was not significantly different from the vector only control.p53 hotspot mutants are known to not only lose their tumor-suppressor function but also acquire oncogenic gain of function (GOF) . StudiesP < 0.01) as compared to the corresponding PC3 cells with the same mutant (56.6%) and PC3(-)ID4 cells with wild type p53 . However there was not a significant decrease in apoptosis between PC3 and PC3(-)ID4 cells with R273H mutant probably due to R273H classified as a DNA contact mutant.Interestingly, a significant decrease in apoptosis was observed in PC3(-)ID4 cells with R175H mutant  whereas the relative luciferase activity of R173H (p < 0.01) and R273H (p < 0.001) was lower as compared to the relative luciferase activity of the wild type p53 (used as a positive control).We next investigated whether ID4 promotes DNA binding of the hotspot mutants in a p53 response element driven luciferase reporter assay. As shown in Figure p < 0.001), R173H (P < 0.001) and R273H (P < 0.001) was significantly lower as compared to relative luciferase activity observed for the wild type p53 in PC3 cells. Furthermore the relative luciferase activity of R173H and R273H in PC3(-)ID4 was significantly lower (p < 0.001) as compared to the respective mutants in PC3 cells ID4 cells, the relative luciferase activity of wild type ID4 cell lines Figure . WhereasNext, we investigated the specific acetylation of p53 within its C-terminal region that is known to regulate its stability and transcriptional activity, thereby facilitating active transcription of cell-cycle arrest, survival, or apoptotic gene programs . HoweverCurrent understanding of the complex biological processes regulated by the p53 tumor suppressor is still not clear , 34. A nPrevious reports have characterized p53 mutations in prostate cancer cell lines DU145 and PC3 , 36, 37.et al. [Our studies suggest that the p53-mediated processes of V274F to be similar to that of the hotspot p53 mutants (R175H and R273H), suggesting that V274F more likely shares properties of “real” cancer-derived mutants as shown by Gurova et al. . The V27et al. , 37. Impet al. . These fet al. , 32.In our earlier studies, we proposed that ID4 dependent activation of mutant p53 in DU145 cells is mediated via acetyl-transferases such as CBP/p300 or by other post-translational modifications that occur in an acetylation dependent manner. It has been well established that several C-terminal lysines  of p53 Studies have shown that acetylation of p53 is usually activating but may also be repressive, that is largely cell type dependent , 41 . InOur results show ID4 dependent acetylation of p53 as activating, consequently the DU145 and hotspot p53 mutants transfected into PC3 gains trans-activation potential and DNA binding activity as compared to PC3 cells that lack ID4. The gain of transactivation potential of mutant p53 in PC3 cells that express ID4 is supported by the observations that show increased p53 dependent luciferase reporter activity, direct DNA binding of mutant p53 and up-regulation of p53 target genes such as p21, BAX, and PUMA. Interestingly, BAX and in some cases p21 is not activated by most rumor derived p53 mutants . HoweverTo elucidate the mechanism by which ID4 promotes p53-mediated transcriptional activity in PC3 cells we focused on ID4 dependent acetylation of p53. The results suggested an increased global p53 lysine and site-specific lysine K373) acetylation in wild type and mutant p53 in an ID4 dependent manner, largely supporting our earlier studies in DU145 cells . Moreove3 acetylaet al. [Acetyl-mimics were used as a model to study the effects of p53 acetylation and de-acylation (acetyl-mimics: K320Q/R and K373Q/R) by studying of site-specific residues critical to the regulation of p53 activity. By overexpressing a collection of acetylation-mimic on the p53-R175H backbone mutants in PC3 cells, we show that specific acetylation at K373, and to a lesser extent at K320, are sufficient for inducing p53 target gene dependent transactivation, apoptosis and restoration of DNA-binding capabilities in an ID4 dependent manner providing further evidence on the role of ID4 in modulating acetylation of p53. Interestingly, the inhibitory effect of deacetylation/non-acetylation mimics (373R and 320R) on p53 induced apoptosis and DNA-binding capability seemingly disrupts the global transcriptional program of p53 by decreasing these activities. Moreover, ID4 may be preferentially modulating apoptosis and DNA-binding capabilities at higher rate via acetyl-mimic K373Q as compared to K320Q suggesting the K373 is critical for ID4 dependent p53 acetylation. Although not significant we did observe lower relative luciferase activity and apoptosis in K320R and K373R (deacytlation mimics) as compared to K320Q and K373Q acetyl-mimics in PC3 cells. Interestingly, Liu et al.  found siet al. , which cApoptosis and to a lesser extent cell cycle arrest and senescence are largely regulated by acetylated p53 . SeveralCell type differences also influence the role of acetylation in apoptosis. In HCT116 colorectal cells, loss of CBP/p300 dependent p53 acetylation leads to increased expression of PUMA and apoptosis following DNA damage, . The aceAs noted, studies have shown that the p53 mutations are indeed not all functionally equivalent, hence reports of p53 mutants with differential effects on the cell . In the In conclusion, our present study finds that ID4 plays a pivotal role in modulation of acetylation and preferentially acts to acetylate K373 for transactivation of p53 in context of both DU145 specific p53 mutants and hotspot mutants overexpressed in PC3 cells. This phenomenon provides clear evidence that ID4 may serve as a physiological agent to restore p53 wild type function to various mutant forms of p53 in a non-cell line/non-mutation specific manner. While this data is intriguing, it is of great interest to determine if this phenomenon of p53 restoration via ID4 is effective in other organ systems (outside prostate cancer) as well as with a variety of other p53 mutations. Continued studies elucidating the mechanism of action will help to unravel the complex biological processes of ID4 and p53 in cancer. The strong anti-cancer effect of ID4 previously reported in prostate cancer and cross-talk with p53 will provide a strong framework of study in other systems, which eventually could serve as an attractive therapeutic approach . ID4 proPC3 prostate cancer cell line was purchased from ATCC and cultured as per ATCC recommendations. ID4 was stably silenced in PC3 cells using gene specific shRNA retroviral vector as described earlier .Mutagenesis of p53: The retroviral p53 vector previously described  was maniForward: 5′-CTA TGA GCC GCC TGA GGT TGG CTC TGReverse: 5′-CAG AGC CAA CCT CAA GCG CAT AGPrimers for V274F mutant:Forward: 5′ AGG TGC GTG TTT GTG CCT GTC CTG GReverse: 5′ CCA GGA CAG GCA CAA AAA CGC ACC TThe following primer pair was used for reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of p53 for measuring expression levels and verifying the mutants by sequencing the PCR product:P53 wild typeForward 5′-GCT CAG ATA GCG ATG GTC TGReverse: 5′ TCT TCT TTG GCT GGG GAG AGHRP) coupled secondary antibody against rabbit IgG and visualized using the Super Signal West Dura Extended Duration Substrate (Thermo Scientific) on Fuji Film LAS-3000 Imager.Western Blot Analysis: 30 ug of total protein, extracted from cultured prostate cancer cell lines using M-PER (Thermo Scientific) was size fractionated on 4–20% SDS-polyacrylamide gel. The fractionated proteins were transferred onto a nitrocellulose membrane (Whatman) and subjected to western blot analysis using respective protein specific antibodies. After washing with 1x PBS, 0.5% Tween 20, the membranes were incubated with horseradish peroxidase (Apoptosis was quantified using Propidium Iodide and Alexa Fluor 488 conjugated Annexin V (Molecular Probes) as described previously .The senescence associated-β-galactosidase assay was performed essentially as described earlier . Brieflyp53 DNA binding activity and quantitation on nuclear extracts was performed as described previously .PC3 cells cultured in 96-well plates (70–80% confluency) were transiently transfected with reporter plasmids by FuGENE HD transfection reagent (Promega) as described earlier . BrieflyCo-immunoprecipitations were performed to detect the protein-protein interactions. Briefly, protein specific IgG (anti-p53 or –ID4) was first immobilized to Protein A Mag beads by incubating overnight at 4C as per manufacturer's instructions . The co-elution of IgG following immuno-precipitation was minimized by cross linking, IgG on protein A mag beads as described in the kit. To detect protein interactions, the cross-linked protein specific IgG-protein A-Mag beads were incubated overnight (4C) with freshly extracted total cellular proteins (500 μg/ml). The complex was then eluted with 0.1M Glycine buffer (pH 2–3) after appropriate washing with PBS and neutralized by adding neutralization buffer .Plasmids containing sequences for human p53-R175H DNA-binding mutants: K320R+K373R, K320Q+K373Q, K320R+K373Q, and K320Q+K373R were commercially synthesized (GenScript). The following primers were used:Lysine 373 Non-Acetylation MimicForward: K373R 5′-CAC CTG AAG TCC AAA AGG GGT CAG TCT ACC TCCReverse: K373R 5′-GGA GGT AGA CTG ACC CCT TTT GGA CTT CAG GTGLysine 373 Acetylation MimicForward: K373Q 5′-CAC CTG AAG TCC AAA GAG GGT CAG TCT ACC TCCReverse: K373Q 5′-GGA GGT AGA CTG ACC CTC TTT GGA CTT CAG GTGLysine 320 Non-Acetylation MimicForward: K320R 5′-TCT CCC CAG CCA AAG AGG AAA CCA CTG GAT GGAReverse: K320R 5′-TCC ATC CAG TGG TTT CCT CTT TGG CTG GGG AGALysine 320 Acetylation MimicForward: K320Q 5′-TCT CCC CAG CCA AAG GAG AAA CCA CTG GAT GGAReverse: K320Q 5′-TCC ATC CAG TGG TTT CTC CTT TGG CTG GGG AGAp-value of < 0.05 was considered statistically significant.Data was analyzed by SPSS 13.0 statistics software. Experimental data is presented as means ± standard error of the mean (SEM). A