PMCID string | Title string | Sentences string |
|---|---|---|
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Next, we fitted a four-parameter log-logistic model to the dose-response data for each single-agent drug. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | For each dose, we calculated the residual – the difference between the observed percentage cell death and the value predicted by the fitted curve. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Doses with residuals whose absolute value exceeded the Tdefault threshold of 15% were disqualified (Supplementary Fig. 2f). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Finally, we assessed the monotonicity of the cell death response, under the assumption that percentage cell death should increase monotonically with increasing drug concentration. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | We evaluated the differences in cell death between the consecutive doses. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | If the cell death response decreased from one dose to the next by more than the Tdefault threshold of 16% (i.e., a decrease greater than 16%), the corresponding dose was disqualified (Supplementary Fig. 2g). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | For sparse mode data, we additionally evaluated the monotonicity of the observed combination responses along the diagonal of the 10 × 10 matrix – the ten observed combination values corresponding to increasing dose pairs combined at relative 1:1 ratios. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | If any of these combination responses decreased by more than 16% compared to the previous dose pair, that specific combination value was disqualified (Supplementary Fig. 2h). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | After assessing a combination matrix using all three QC metrics, the flagged values were tracked and could be excluded from summary statistics if desired. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | For example, a combination matrix could be summarized by its mean Bliss synergy score (which averages over all 100 Bliss synergy values of the 10 × 10 matrix), or by its “adjusted” Bliss synergy score (which only averages over the non-disqualified Bliss synergy values). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | A pairwise combination screen of 135 drugs was performed in the CHP-134 cells. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Each single-agent plate was replicated six times. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Drug combinations were prioritized for further exploration by first applying a series of filtering criteria:Moran’s I, a measure of spatial autocorrelation, was used to assess the distribution of synergy across each 10 × 10 combination matrix. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Moran’s I quantifies the degree to which similar scores cluster together in space and was applied to all synergy matrices (Supplementary fig. 10). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | A high Moran’s I indicates that similar synergy scores are similarly clustered, forming cohesive patterns, whereas low values suggest a random distribution with no discernible pattern. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | We reasoned that synergy matrices exhibited such localized motifs likely reflect coherent biological effects, while sporadic patterns are more likely to signify noise or spurious results. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | All matrices with a Moran’s I value of <0.6 were filtered out to retain only those with strong patterns of spatial autocorrelation. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | All combination matrices were subjected to a filter based on the quality of the measured values. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | The ten measured values, corresponding to the diagonal of the 10 × 10 matrix, were subjected to our QC pipeline. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | If more than six of these ten measured values were flagged by the QC pipeline as unreliable, the matrices were excluded. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | The mean percentage cell death was also used as a filter for the screening data. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | For all 100 dose pairs per combination, the mean was calculated, and only matrices with ≥20% cell death were retained for further analysis. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | This ensured that the high-ranking synergy was also meaningful in terms of inducing cell death. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Moran’s I, a measure of spatial autocorrelation, was used to assess the distribution of synergy across each 10 × 10 combination matrix. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Moran’s I quantifies the degree to which similar scores cluster together in space and was applied to all synergy matrices (Supplementary fig. 10). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | A high Moran’s I indicates that similar synergy scores are similarly clustered, forming cohesive patterns, whereas low values suggest a random distribution with no discernible pattern. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | We reasoned that synergy matrices exhibited such localized motifs likely reflect coherent biological effects, while sporadic patterns are more likely to signify noise or spurious results. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | All matrices with a Moran’s I value of <0.6 were filtered out to retain only those with strong patterns of spatial autocorrelation. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | All combination matrices were subjected to a filter based on the quality of the measured values. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | The ten measured values, corresponding to the diagonal of the 10 × 10 matrix, were subjected to our QC pipeline. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | If more than six of these ten measured values were flagged by the QC pipeline as unreliable, the matrices were excluded. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | The mean percentage cell death was also used as a filter for the screening data. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | For all 100 dose pairs per combination, the mean was calculated, and only matrices with ≥20% cell death were retained for further analysis. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | This ensured that the high-ranking synergy was also meaningful in terms of inducing cell death. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Once the screening data were filtered, combinations were ranked by their mean Blissadj. ( |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | values. |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | This metric is simply the mean of the 10 measured values per combination after they were subjected to QC (Fig. 4a). |
PMC12705714 | An open-source screening platform accelerates discovery of drug combinations | Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Background: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, remains a leading cause of viral encephalitis. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Current management is largely supportive, with no specific antivirals. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | This study evaluated the antiviral efficacy and mechanism of action of CC-90009 against JEV in vitro and in vivo. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Methods: Five targeted protein degraders (TPDs) were screened for anti-JEV activity in the human neuroblastoma cell line SH-SY5Y. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Time-of-addition, binding, and endocytosis assays were used to delineate the phase of action of CC-90009, a cereblon (CRBN) E3 ligase modulator (CELMoD) and molecular glue degrader. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Small interfering RNA knockdown and co-immunoprecipitation (Co-IP) confirmed targets essential for its antiviral effects. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The broad-spectrum activity of CC-90009 against other mosquito-borne viruses was also evaluated. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In vivo efficacy was tested in a murine JEV model. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Results: Of the five TPDs tested, only CC-90009 significantly inhibited JEV infection in SH-SY5Y cells, acting during both viral entry and post-entry phases without reducing adsorbed or internalised virions. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 reduced JEV RNA and non-structural protein accumulation. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Knockdown of G1-to-S phase transition 1 (GSPT1), a key target of CC-90009, suppressed JEV infection and translation; Co-IP confirmed GSPT1 interaction with JEV non-structural protein 5 (NS5). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 disrupted JEV translation and replication by inducing proteasomal degradation of the GSPT1/NS5 complex, further demonstrating its broad-spectrum antiviral activity through the effective inhibition of West Nile virus and chikungunya virus. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | In vivo, it protected mice from JEV-induced mortality, reducing viral load, antigen levels, and brain pathology. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Conclusions: CC-90009 exerts potent anti-JEV activity both in vitro and in vivo by inducing proteasomal degradation of the GSPT1/NS5 complex, thereby disrupting viral translation and replication. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | This targeted protein degradation strategy represents a novel host-directed antiviral approach with promising therapeutic potential against mosquito-borne viral encephalitis. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Japanese encephalitis (JE) is an acute infectious disorder of the central nervous system resulting from infection with Japanese encephalitis virus (JEV), which belongs to the Flaviviridae family . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | JEV is transmitted between animal and human hosts via mosquitoes of the Culex genus . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | As a significant human-pathogenic virus, it is responsible for approximately 100,000 cases of acute encephalitis annually. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Among confirmed cases, the case fatality rate ranges from 25% to 40%, while persistent neurological complications are observed in as many as half of all survivors . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Despite the availability of vaccines, JEV remains a significant public health concern and retains the potential for global dissemination . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | At present, no specific antiviral medications are available for JE, and clinical management is centred on symptomatic treatment. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | As a result, there is an urgent requirement to discover new antiviral candidates with optimal safety and therapeutic efficacy against JEV infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | JEV is a single-stranded positive-sense RNA virus with a genome of approximately 11 kilobases . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | It encodes 10 viral proteins, comprising three structural proteins (core, premembrane, and envelope) and seven non-structural proteins (NS1, NS2A–B, NS3, NS4A–B, and NS5) . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The process of JEV infection in nerve cells involves multiple stages. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Initially, the virus attaches to and enters host cells via unidentified host receptors, followed by caveolin-mediated endocytosis . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Once in the cytoplasm, the viral RNA commences translation and replication . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | A key function of the non-structural proteins is to form the viral replication complex, facilitating critical processes such as genome replication, virion assembly, and evasion of host immune responses through interactions with host cell proteins . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | JEV NS5 is a multifunctional, evolutionarily conserved protein, and its RNA-dependent RNA polymerase (RdRp) activity is crucial for replication of the viral genome . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Consequently, NS5 has emerged as a pivotal antiviral target for therapeutic development. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Emerging targeted protein degradation (TPD) technologies, notably proteolysis-targeting chimeras (PROTACs) and molecular glue degraders, act by binding to protein–protein interaction interfaces, inducing spatial proximity to drive target protein degradation via the ubiquitin–proteasome system . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Clinical-stage compounds, including cereblon (CRBN) E3 ligase modulators (CELMoDs)—a class of molecular glue degraders—such as CC-92480 , NVP-DKY709 , and CC-90009 , as well as PROTACs such as ARV-110 and ARV-471 , have shown promising antitumour efficacy. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Their mechanisms of action against mosquito-borne viral infections, including JEV, however, remain poorly characterized. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Unlike conventional small-molecule inhibitors, which fail to regulate target protein abundance, struggle with “undruggable” pocketless targets, require high doses, and frequently induce resistance, TPD leverages the host’s protein degradation machinery for selective clearance of pathogenic proteins, offering advantages in reducing toxicity, overcoming resistance, and targeting challenging substrates . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 is a CELMoD that functions as a molecular glue degrader. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | It binds to CRBN, the substrate receptor of the Cullin4-RING E3 ubiquitin ligase complex (CRL4), thereby targeting G1-to-S phase transition 1 (GSPT1) for ubiquitination and subsequent degradation via the proteasome . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 is in Phase I trials to assess its efficacy and safety in patients with acute myeloid leukaemia. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Recent studies have indicated that CC-90009 exhibits antiviral activity against both Lassa virus (LASV) and Ebola virus (EBOV) without significant cytotoxicity. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Prompted by these findings, we sought to evaluate the antiviral potential of CC-90009 against JEV using the human neuroblastoma cell line SH-SY5Y and a suckling mouse model of infection. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Our findings demonstrate that CC-90009 markedly suppresses JEV infection in vitro and confers protection in vivo against JEV-induced mortality, supporting its potential for repurposing as a broad-spectrum antiviral agent against mosquito-borne viral infections. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | SH-SY5Y (ATCC CRL-2266), baby hamster kidney cell line BHK-21 (ATCC CCL-10), African green monkey kidney Vero cells (ATCC CCL-81), and human embryonic kidney cell line HEK-293T (ATCC CRL-11268) were maintained in Dulbecco’s modified eagle medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% foetal bovine serum (GIBCO, Waltham, MA, USA) at 37 °C in a 5% CO2-humidified incubator. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The JEV strain SA14 (GenBank accession no. U14163.1) was propagated and titrated on BHK-21 cells, with viral titres quantified using a plaque assay. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Chikungunya virus (CHIKV) infectious clone of Ross isolate (GenBank accession no. AF490259) and West Nile virus (WNV) infectious clone of NY2000 strain (GenBank accession no. AF404756) were synthesised in this laboratory. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CHIKV and WNV were propagated in Vero cells. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | CC-90009 (HY-130800), CC-92480 (HY-129395), NVP-DKY709 (HY-144998), ARV-110 (HY-138641), ARV-471 (HY-138642), chloroquine (CQ, HY-17589A), heparin (HY-17567), nystatin (HY-17409), and MG132 (HY-13259) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The chemical structure and pharmacological profile of CC-90009 are provided in the Supplementary Materials (Figure S1). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The Cell Counting Kit-8 was obtained from Invitrogen (Carlsbad, CA, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Antibodies against JEV NS1 (GTX633820), NS2B (GTX125972), NS3 (GTX125868), NS4A (GTX132028), NS4B (GTX125865), NS5 (GTX131359), as well as CHIKV nsP4 (GTX638958) and WNV NS5 (GTX131961), were purchased from GeneTex (Irvine, CA, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Anti-GSPT1 protein (ab49878) and CRBN (ab315344) antibodies were obtained from Abcam (Cambridge, UK). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Anti-Ubiquitin antibody (#43124) was obtained from Cell Signaling Technology (Danvers, MA, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Alexa Fluor 488 secondary antibody was purchased from Invitrogen (Carlsbad, CA, USA). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Transfection assays were conducted as previously described . |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Cells (50–60% confluence) were transfected with 80 nM siRNA or 800 ng plasmid and incubated for 48 h at 37 °C. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | The siRNAs targeting GSPT1 and CRBN were purchased from GenePharma (Shanghai, China). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Plasmids encoding human GSPT1, JEV NS5, WNV NS5 and CHIKV nsP4 were obtained from GeneChem (Shanghai, China). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Following methanol fixation, infected cells were incubated in a blocking solution containing 3% bovine serum albumin for 2 h. Immunofluorescence staining was performed using the following primary antibodies, incubated overnight at 4 °C: a rabbit anti-JEV NS3 antibody (GTX125868), a rabbit anti-CHIKV nsP4 antibody (GTX638958), and a rabbit anti-WNV NS5 antibody (GTX131961). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Subsequently, the cells were incubated with an Alexa Fluor™ 488-conjugated goat anti-rabbit immunoglobulin G (IgG) secondary antibody for 1 h at 25 °C. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | Finally, cells were stained with phosphate-buffered saline containing 0.1 µg/mL 4′,6-diamidino-2-phenylindole for 10 min before imaging. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | All immunofluorescence results were acquired using the BioTek Lionheart FX Imaging Reader (Agilent, Santa Clara, CA, USA), and the number of positive cells across the entire well was quantified. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | To quantify viral RNA levels, total RNA was extracted from treated SH-SY5Y cells or mouse brains using TRIzol reagent (TaKaRa, Shiga, Japan). |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | First-strand cDNA synthesis was performed using PrimeScript RT Master Mix (TaKaRa) according to the manufacturer’s instructions. |
PMC12736548 | CC-90009, a Cereblon E3 Ligase Modulator, Exhibits Antiviral Efficacy Against JEV In Vitro and In Vivo via Targeted Degradation of GSPT1 and Viral NS5 Protein | PCR amplification of JEV RNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; reference gene) was performed with SYBR Premix Ex Taq (TaKaRa) using respective primer pairs in an ABI 7300 system (Applied Biosystems, Foster City, CA, USA). |
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