n2a mouse neuroblastoma cells  (ATCC)

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Endogenous Grx1 is upregulated in N2a-hTDP-43 cells. (a) Validation of TDP-43 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, TDP-43 was visualized with a TDP-43–specific antibody. Yellow arrows indicate cytoplasmic TDP-43 aggregates. (b, c) Intracellular ROS levels are increased in N2a-hTDP-43 cells. Intracellular ROS levels were measured using CellROX Deep Red and normalized to corresponding cell numbers ( n = 3, unpaired t test). (d, e) Validation of Grx1 antibody specificity. N2a cells were transfected with 20 nM Grx1-specific siRNA (siGrx1) or control siRNA (siCon). Endogenous Grx1 levels were normalized to corresponding cell numbers ( n = 3, unpaired t test). (f, g) Endogenous Grx1 levels are increased in N2a-hTDP-43 cells. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, unpaired t test). Nuclei were stained with DAPI (blue). Scale bars, 25 µm. Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: N2a mouse neuroblastoma cells (CCL-131, ATCC, Manassas, Virginia, USA) were plated at a density of 2 × 10 5 cells/ml and grown in DMEM with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 °C, in a humidified 5% CO 2 incubator.

Techniques: Biomarker Discovery, Over Expression, Transfection, Control, Staining, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 decreases oxidative stress in N2a-hTDP-43 cells. (a, b) Validation of Grx1 overexpression in N2a-hTDP-43 cells; 48 h post-transfection as indicated, cells were simultaneously stained for TDP-43 (red) and Myc-tagged Grx1 (green) ( n = 3, one-way ANOVA). (c, d) Grx1 overexpression suppresses decreases ROS levels in N2a-hTDP-43 cells. Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a relative fold change to control ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; ROS, reactive oxygen species; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: N2a mouse neuroblastoma cells (CCL-131, ATCC, Manassas, Virginia, USA) were plated at a density of 2 × 10 5 cells/ml and grown in DMEM with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 °C, in a humidified 5% CO 2 incubator.

Techniques: Biomarker Discovery, Over Expression, Transfection, Staining, Control, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 prevents TDP-43 aggregation in N2a-hTDP-43 cells. (a, b) Overexpressing Grx1 significantly reduces cytoplasmic TDP-43 aggregates in N2a-hTDP-43 cells. N2a cells were stained for TDP-43 (red) and DAPI (blue). Cells with cytoplasmic TDP-43 aggregates (yellow arrows) were presented as a percentage of cells ( n = 3, one-way ANOVA). Scale bars correspond to 25 µm. (c, d) Overexpressing Grx1 decreases total TDP-43 levels in N2a-hTDP-43 cells; 48 h post-transfection as indicated, total proteins were extracted using SDS-containing RIPA lysis buffer. TDP-43 protein levels were normalized to corresponding GAPDH levels ( n = 3, one-way ANOVA). (e–g) Overexpressing Grx1 decreases both soluble and insoluble TDP-43 levels in N2a-hTDP-43 cells. TDP-43 protein levels were assessed by western blot in Triton X-100 soluble (f) and insoluble fractions (g) and normalized to corresponding GAPDH and Ponceau S levels, respectively ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a-hTDP-43, neuro-2a cells expressing human wild-type TDP-43; TDP-43, transactive response DNA-binding protein 43.

Article Snippet: N2a mouse neuroblastoma cells (CCL-131, ATCC, Manassas, Virginia, USA) were plated at a density of 2 × 10 5 cells/ml and grown in DMEM with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 °C, in a humidified 5% CO 2 incubator.

Techniques: Staining, Transfection, Lysis, Western Blot, Expressing, Binding Assay

Journal: Neuroreport

Article Title: Glutaredoxin-1 attenuates transactive response DNA-binding protein 43–induced neurotoxicity by suppressing oxidative stress and transactive response DNA-binding protein 43 aggregation

doi: 10.1097/WNR.0000000000002266

Figure Lengend Snippet: Increasing Grx1 attenuates neurotoxicity in N2a cells overexpressing hTDP-43; 48 h post-transfection as indicated, N2a cells were stained with cleaved caspase-3–specific antibody and DAPI (blue) (a). Scale bars correspond to 25 µm. Each level was normalized to the corresponding cell number and presented as a percentage of cells with cleaved caspase-3 signal (b) ( n = 3, one-way ANOVA). ANOVA, analysis of variance; Grx1, glutaredoxin-1; N2a, neuro-2a; TDP-43, transactive response DNA-binding protein 43

Article Snippet: N2a mouse neuroblastoma cells (CCL-131, ATCC, Manassas, Virginia, USA) were plated at a density of 2 × 10 5 cells/ml and grown in DMEM with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 °C, in a humidified 5% CO 2 incubator.

Techniques: Transfection, Staining, Binding Assay

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Genomic distribution of SP9 ChIP-seq peaks based on data from ( ; ). Peaks localize predominantly to enhancer-like regions (77%), with smaller fractions at promoters (6.2%) and other regions (16.8%). B , Motif enrichment within SP9 peaks. Promoter-associated peaks (left) are enriched for GC-rich motifs recognized by KLF and SP factors, whereas enhancer-like peaks (right) are enriched for TAATT motifs recognized by DLX homeobox factors. Within each block: motif logos (left), − log 10 𝑃 -value and target/background percentage of peaks containing the motif (middle), and log 2 enrichment (right). C , Schematic of the motif scan-based classification of SP9 peaks. Peaks were grouped based on the presence of an SP motif only (direct binding), a DLX motif only (indirect binding via DLX), or both (cobinding). D , Coarse genomic annotation (enhancer, transcription start site (TSS), or other) of the four SP9 peak categories defined in (C): SP9+DLX (both motifs present), DLX (DLX motif only), Other, and SP9 (SP motif only). E , Fine genomic annotation (exon, intron, intergenic, promoter, UTR, other) of the same four peak categories. F , Intersection of DLX2 and SP9 ChIP-seq peaks from E13.5 mouse GE, based on data from ( ; ; ). Bar plot shows the number of overlapping and non-overlapping peaks per category (DLX2 only, overlap, SP9 only). G , Whole-mount X-Gal staining (blue) of E11.5 transgenic mouse embryos carrying VISTA enhancer reporters hs119 (near Arx ), hs170 (near Fign ), hs883 (near Sox6 ), and hs298 (near Dlx6os1 ) used in the luciferase assays in panels K-N and shown as genome browser tracks in panel H. Images from . H , Genome browser tracks showing ChIP-seq signal for SP9, DLX2, and DLX5 in E13.5 mouse GE at the Sp9 and Six3 promoters (highlighted in grey) and the VISTA enhancers hs119, hs170, hs883, and hs298 (highlighted in blue). Numbers below each track indicate the count of SP and DLX motifs within the selected regulatory element. Data from ( ; ; ). I-N , Luciferase reporter activity in N2A cells transfected with combinations of Sp9 and Dlx2 , Dlx5 , or Dlx6 as indicated, driven by the Sp9 promoter ( I ), Six3 promoter ( J ), or enhancer regions hs119 of Arx ( K ), hs170 of Fign ( L ), hs883 of Sox6 ( M ), and hs298 of Dlx6os1 ( N ). Bars represent mean ± s.e.m. of 9 (I,J,L-N) or 12 (K) replicates from 3 (I,J,L-N) or 4 (K) independent batches performed in triplicate; points indicate batch means. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6.

Article Snippet: Mouse N2A neuroblastoma cells (Neuro2A cells, ECACC, 89121404), human HEK293FT (Thermo Fisher Scientific, R70007), mouse 3T3-L1 (CLS Cell line service, 400103) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, F9665) and containing 1% (v/v) antibiotics (100 Uml −1 penicillin, 100 μgmL −1 streptomycin, Sigma, P0781).

Techniques: ChIP-sequencing, Blocking Assay, Binding Assay, Staining, Transgenic Assay, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Schematic of C-terminally V5-tagged SP9 constructs: wild-type (WT) SP9, an N-terminal deletion mutant (NΔSP9), and a zinc finger (ZF) domain deletion mutant (SP9ΔZF). The nine-amino-acid transactivation domain (9aaTAD), the three ZF domains, and the position of the patient-derived SP9*378 variant are indicated. B , Co-immunoprecipitation (CoIP) from nuclear lysates of HEK293FT cells co-transfected with DLX5-FLAG and either SP9-V5 WT, SP9*378-V5, or NΔSP9V5. Left: input fractions probed with anti-SP9 (top) and anti-DLX5 (bottom). Right: CoIP using anti-FLAG beads, probed with anti-SP9 (top) and anti-DLX5 (bottom). C , CoIP from whole-cell lysates of HEK293FT cells co-transfected with DLX5-FLAG and either SP9-V5 WT or SP9ΔZF-V5. Left: input fractions probed with anti-V5 (top) and anti-DLX5 (bottom). Right: CoIP using anti-FLAG beads, probed with anti-V5 (the anti-SP9 epitope lies within the deleted ZF region, requiring V5 detection; top) and anti-DLX5 (bottom). D , Luciferase reporter activity driven by the hs298 enhancer of Dlx6os1 in N2A cells co-transfected with Dlx5 and either Sp9 , Sp9*378 , N Δ Sp9 , or Sp9 Δ ZF . Bars represent mean ± s.e.m. of9replicatesfrom3independentbatchesperformedintriplicate; pointsindicatebatchmeans. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6. E , Label-free quantitative mass spectrometry (MS) of proteins captured by DNA pull-down using a 46-bp fragment of the hs298 enhancer of Dlx6os1 versus the same fragment with the TAATT motifs scrambled, from E14.5 ganglionic eminence (GE) nuclear lysates. Proteins with p ≤ 0.05 and log 2 LFQ FC ≥ 1.5 were considered significantly enriched (n = 25). Transcription factors (TFs) are indicated in black. F , Volcano plot of proteins enriched in anti-SP9 CoIP-MS from E14.5 GE nuclear lysates, relative to IgG controls. Proteins with log 2 FC ≥ 1.5 and p ≤ 0.05 were considered significantly enriched (n = 223). Components of HDAC1/2-containing complexes are highlighted in blue; TFs are indicated in black. 𝑃 -values were calculated using a two-sided Student’s t-test with permutation-based false discovery rate (FDR) correction. G , Gene Ontology (GO) molecular function enrichment analysis of proteins identified by anti-SP9 CoIP-MS. H , Heatmap of ChIP-seq signal intensity across SP9-bound regions for SP9 ( ; ), DLX2, histone modifications (H3K27ac, H3K4me1, H3K4me3) , GTF2I , and NuRD complex subunits (MBD3, CHD4, RBBP4, RBBP7, HDAC1, HDAC2) . Signal is plotted over a ±5 kb window centered on SP9 peak summits and organized by k-means clustering (5 clusters), separating SP9-bound regions by their co-binding patterns with DLX2, NuRD subunits, and active histone marks. All datasets are from mouse E13.5 GE except GTF2I, which is from E13.5 whole brain.

Article Snippet: Mouse N2A neuroblastoma cells (Neuro2A cells, ECACC, 89121404), human HEK293FT (Thermo Fisher Scientific, R70007), mouse 3T3-L1 (CLS Cell line service, 400103) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, F9665) and containing 1% (v/v) antibiotics (100 Uml −1 penicillin, 100 μgmL −1 streptomycin, Sigma, P0781).

Techniques: Construct, Mutagenesis, Derivative Assay, Variant Assay, Immunoprecipitation, Transfection, Luciferase, Activity Assay, Mass Spectrometry, ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Stoichiometric transcription factor partnerships specify GABAergic neuron subtype identity

doi: 10.64898/2026.05.25.727662

Figure Lengend Snippet: A , Schematic of the transcription factor (TF) overexpression strategy in N2A cells. Cells were transfected with SP9 alone or co-expressed with WT DLX5 (overexpression experiment 1, OE1) or with the N-terminal deletion mutant NΔDLX5 (OE2), followed by CUT&RUN profiling. B , Domain organization of SP9, DLX5, and NΔDLX5, highlighting the zinc finger (ZF) and homeobox domains. C , Heatmaps of SP9 CUT&RUN signal (anti-V5) centered on SP9 peak regions (±5 kb), clustered into four groups (C1-C4) by k-means. Conditions: SP9 alone, SP9+DLX5 (OE1), SP9+NΔDLX5 (OE2). D , Aggregate signal plots for each cluster (C1-C4) showing normalized SP9 binding profiles at peak centers under the three conditions in (C). E , Top enriched motifs identified by motif analysis of peaks in each cluster, with associated 𝑃 -values. F , Genomic annotation of SP9-bound peaks across clusters. Left: distribution across promoter, exon, intron, intergenic, and other regions. Right: distance to transcription start sites (TSS). G , Genome browser tracks at three representative loci showing SP9 binding under OE1 and OE2 conditions, illustrating loss of SP9 occupancy upon DLX5 coexpression at SP-motif-containing sites and rescue with NΔDLX5, as well as gain of SP9 occupancy at DLX-motif-containing sites with WT DLX5. H , Luciferase reporter activity driven by the Six3 promoter in N2A cells transfected with combinations of Sp9 , Dlx5 , and N Δ Dlx5 as indicated. I , Volcano plot of co-immunoprecipitation followed by mass spectrometry (CoIP-MS) using anti-V5 in N2A cells co-transfected with Sp9-V5 and Dlx5-FLAG , relative to IgG controls. Proteins with log 2 FC > 1.5 and p < 0.05 were considered significantly enriched (n = 2254). NuRD complex components are highlighted in blue, transcription factors (TFs) in black, and AP-1 family TFs in green. J , Volcano plot comparing the anti-V5 CoIP-MS interactomes between SP9+DLX5 and SP9+NΔDLX5 conditions in N2A cells, using the same cutoffs as in (I). I,J , 𝑃 -values were calculated using a two-sided Student’s t-test with permutation-based false discovery rate (FDR) correction. K , Luciferase reporter activity driven by the Six3 promoter in N2A cells transfected with varying amounts (ng plasmid DNA) of SP9 (S) and DLX5 (D), showing dose-dependent transcriptional activation. H-K , Bars represent mean ± s.e.m. of 9 replicates from 3 independent batches performed in triplicate; points indicate batch means. Statistical significance was assessed by two-way ANOVA with Tukey’s honestly significant difference (HSD) post hoc test. Exact 𝑃 -values are provided in Table S6. L , Proposed mechanism. When SP9 levels exceed DLX (SP9≫DLX), SP9 binds directly at GC-rich SP motifs. When SP9 and DLX are comparable or DLX is in excess (SP9≈DLX or SP9 < DLX), DLX5 sequesters SP9 to TAATT-containing DLX motifs through the DLX5 N-terminal domain, away from its direct SP-motif binding sites. NΔDLX5, which cannot interact with SP9, fails to redirect SP9 to DLX motifs, restoring SP9 binding at SP motifs.

Article Snippet: Mouse N2A neuroblastoma cells (Neuro2A cells, ECACC, 89121404), human HEK293FT (Thermo Fisher Scientific, R70007), mouse 3T3-L1 (CLS Cell line service, 400103) were cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma, F9665) and containing 1% (v/v) antibiotics (100 Uml −1 penicillin, 100 μgmL −1 streptomycin, Sigma, P0781).

Techniques: Over Expression, Transfection, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Mass Spectrometry, Plasmid Preparation, Activation Assay

Journal: eLife

Article Title: An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging mouse neurons

doi: 10.7554/eLife.101533

Figure Lengend Snippet: ( A ) Protein sequence alignment of mouse and human POLK, with the 133–310 amino acid epitope for sc-166667 anti-POLK antibody showing complete homology between species. ( B ) qPCR of Polk transcript shows a 35% reduction upon siRNA against Polk but not scrambled control siRNA. Polh mRNA levels were not affected by siRNA against Polk. ( C ) Western blot immunostained with SC-166667 antiPOLK-HRP antibody of whole cell lysates of two biological replicates of mouse primary cortical neurons treated with four individual shPOLK lentivirus, shows a decrease in 99 and 120 kDa POLK bands upon treatment with shPOLK#C. ( D ) Western blot immunostained with SC-166667 antiPOLK-HRP antibody of whole cell lysates of two biological replicates of mouse Neuro-2A cells treated with either siPOLK or shPOLK lentivirus, showed a decrease in 99 kDa POLK band. ( E ) Western blot immunostained with SC-166667 antiPOLK-HRP antibody of whole cell lysates of three biological replicates of mouse 4T1 cells treated with siPOLK showed a decrease in 99 and 120 kDa POLK bands. ( F ) Immunofluorescence staining of wild-type 18-month-old mouse, from brain cortical area S1 shows a similar pattern and distribution of POLK nuclear speckles (arrowheads) and cytoplasmic granules (arrows) using sc-166667 and A12052. The bottom row is a crop of the boxed area in the corresponding top image. ( G ) Full western blot showing all six replicates nucleus and cytoplasmic fractions from 7- and 22-month-old whole brain unsorted cells. Quantitation of the 99 kDa POLK band did not show an increase, possibly due to inability to extract POLK associated with lysosomal and stress granules (as observed in ). ( H ) Representative low (×20) and high magnification (×63) images of REV1 and POLI expression using IF from S1 and M1 cortical regions in ages 1, 10, and 18 months. REV1 (green) and Nissl depicting all cells (purple) (scale bar = 10 µm in ×20 image). Arrowheads point to nuclear speckles and arrows indicate cytoplasmic granules. Wild-type mouse brain cortical areas S1 and M1 show REV1 and POLI punctate nuclear expression resembling speckles and progressive cytoplasmic accumulation with age at 10 and 18 months. Figure 1—figure supplement 1—source data 1. Annotated original blots corresponding to . Figure 1—figure supplement 1—source data 2. Raw scans of original blots to .

Article Snippet: Mouse Neuro2a (N2a) (CCL-131; ATCC, USA) and mouse 4T1 (CRL-2539; ATCC, USA) cell lines were obtained from and authenticated by ATCC, tested for mycoplasma using MycoAlert Mycoplasma Detection Kit (Lonza, Catalog #LT07-218), cultured in DMEM (Dulbecco’s Modified Eagle Medium) and RPMI 1640 medium, respectively, both supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml) (Cat No. 30-002-CI, Corning) in a humidified incubator at 37°C with 5% CO 2 .

Techniques: Sequencing, Control, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Expressing

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV infection activates the innate immune response. ( A ) Viral load in subcultures harvested at different time points post-infection was quantified by qRT-PCR targeting the viral N gene. All the experiments were performed in triplicate. ( B ) IFN-β levels in PHEV-infected samples were determined by ELISA. Cells infected with VSV (MOI = 1) or treated with Poly(I:C) (20 μM, 24 h) served as positive controls. ( C ) QRT-PCR analysis of IFNA and IFNB1 mRNA expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( D ) QRT-PCR analysis of ISGs (Mx, OAS, GBP, STAT) expression in N2a cells at 24 and 48 h post-PHEV infection. ( E ) QRT-PCR analysis of RIG-I, MDA5, IRF3, and IRF7 expression in N2a cells at various time points (0–48 h) post-PHEV infection. ( F ) WB analysis of RIG-I, IRF7, MAVS, and viral N protein levels in N2a cells at indicated times (0–48 h) after PHEV infection. ( G ) Detection of PHEV, RIG-I, IRF3, IRF7, and IFNB1 in brain tissues from mice at 5 days post-PHEV infection. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: Mouse neuroblastoma (N2a) (ATCC, CCL-131), PK15 cells (ATCC, CCL-33), and HEK293T (ATCC, CRL-11268) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, U.S.) with 10% fetal bovine serum (Biological Industries, Israel), and 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV dsRNA serves as a prerequisite for RIG-I-dependent induction of type I IFN. ( A ) Schematic illustration of the proposed mechanisms by which Remdesivir (RDV) and Lopinavir (LPV) suppress PHEV replication. RdRp, RNA-dependent RNA polymerase; 3CLpro, 3C-like protease. ( B ) RDV and LPV suppress PHEV replication. N2a cells were infected with PHEV for 1 h, treated with RDV or LPV (20 μM), and harvested at 24 h post-infection for qRT-PCR analysis of PHEV mRNA levels. ( C ) QRT-PCR analysis of IFNA and IFNB1 mRNA expression as described in panel B . ( D ) QRT-PCR analysis of I RIG-I, IRF3, and IRF7 mRNA expression as described in panel B . ( E ) WB analysis of RIG-I, phosphorylated IRF3 (p-IRF3), p-IRF7, and PHEV N protein as described in panel B . ( F ) Immunostaining assay. N2a cells as indicated in panel B were harvested and immunostained with anti-dsRNA (red) and anti-RIG-I (green) antibodies, DAPI (blue). Scale bar, 10 µm. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: Mouse neuroblastoma (N2a) (ATCC, CCL-131), PK15 cells (ATCC, CCL-33), and HEK293T (ATCC, CRL-11268) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, U.S.) with 10% fetal bovine serum (Biological Industries, Israel), and 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Quantitative RT-PCR, Expressing, Immunostaining, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: IRF3 initiates the interferon response to suppress viral replication. ( A ) Inhibition of PHEV replication by recombinant IFN-β. WB analysis of viral protein levels to evaluate the suppression of PHEV replication by recombinant IFN-β (0.5 or 1 μg/mL). ( B ) QRT-PCR analysis of IRF3 mRNA in N2a cells overexpressing Myc-tagged IRF3 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( C ) QRT-PCR analysis of IRF7 mRNA in N2a cells overexpressing Myc-tagged IRF7 (1, 2, or 4 μg) for 24 h, followed by 24 h PHEV infection. ( D ) QRT-PCR analysis of PHEV mRNA expression as described in panels B and C . ( E ) WB analysis of PHEV N protein as described in panels B and C . ( F ) QRT-PCR analysis of IFNB1 mRNA expression as described in panels B and C . Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test). ns, not significant.

Article Snippet: Mouse neuroblastoma (N2a) (ATCC, CCL-131), PK15 cells (ATCC, CCL-33), and HEK293T (ATCC, CRL-11268) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, U.S.) with 10% fetal bovine serum (Biological Industries, Israel), and 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Inhibition, Recombinant, Quantitative RT-PCR, Infection, Expressing, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV N protein targets IRF3 to suppress IFN production. ( A ) Schematic illustration of the experimental design for the dual-luciferase assay using HT1080 cells with a stably integrated IFN-β promoter reporter. ( B ) PHEV N protein inhibits the IFN-β promoter. HT1080 cells were transfected with PHEV proteins, cultured for 24 h, and then stimulated with Poly(I:C) (20 μM) for another 24 h to induce IFN-β promoter activity. IFN-β-Luc reporter activity is normalized to that of Renilla luciferase and shown. Detection of viral protein expression by WB. ( C ) WB analysis demonstrated that the protein levels of RIG-I and IRF3 in N2a cells were unaltered by transfection with a gradient of N protein (0.5–4 μg). ( D ) Nuclear-cytoplasmic fractionation. N2a cells were transfected with 2 μg GFP-N recombinant plasmid for 24 h, and then treated with Poly(I:C) (20 μM) for 24 h or infected with VSV (MOI = 1) for 12 h. Cells were collected for cytoplasmic and nuclear isolation. WB analysis of IRF3 nuclear translocation using cytoplasmic and nuclear fractions prepared from harvested cells. ( E ) N2a cells were infected with PHEV for 24 h (with Poly(I:C) treatment (20 μM, 24 h) as a positive control), followed by immunostaining with anti-N (red) and anti-IRF3 (green) antibodies. Nuclei were counterstained with Hoechst (blue). Scale bar, 10 μm. Data represent mean ± SD (** P < 0.01 and *** P < 0.001 by unpaired two-tailed Student’s t-test).

Article Snippet: Mouse neuroblastoma (N2a) (ATCC, CCL-131), PK15 cells (ATCC, CCL-33), and HEK293T (ATCC, CRL-11268) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, U.S.) with 10% fetal bovine serum (Biological Industries, Israel), and 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Luciferase, Stable Transfection, Transfection, Cell Culture, Activity Assay, Expressing, Fractionation, Recombinant, Plasmid Preparation, Infection, Isolation, Translocation Assay, Positive Control, Immunostaining, Two Tailed Test

Journal: Journal of Virology

Article Title: Porcine hemagglutinating encephalomyelitis virus nucleocapsid protein targets RIG-I and IRF3 to evade IFN immunity

doi: 10.1128/jvi.02112-25

Figure Lengend Snippet: PHEV N protein interrupts the K63-linked polyubiquitination. ( A ) N2a cells were infected with PHEV (MOI = 1) after overexpression of RIG-I for 24 h, and ubiquitination was evaluated by Western blotting. ( B ) N2a cells were co-transfected with Myc-RIG-I and HA-tagged K63 (HA-K63) for 24 h and infected with PHEV for 24 h. Precipitation was performed with Myc antibody and detected by Western blotting. ( C ) N2a cells were transfected with the indicated plasmids for 24 h, and then treated with 20 μM MG132 for 12 h. Cell lysates were collected, and ubiquitination level was detected by Western blotting. ( D ) Effects of N protein on the conjugation of diverse polyubiquitin linkages to RIG-I under viral stimulation. Plasmids encoding HA-Ub (K63, K48), together with expressing vectors for Myc-RIG-I and Flag-N, were co-transfected into HEK293T cells. After 24 h, these cells were infected with VSV for 12 h and then subjected to immunoprecipitation using anti-Myc beads. ( E ) HEK293T cells that were pretransfected with Flag-N-expressing vectors (0.5–4 μg) for 24 h were infected with VSV for 12 h and then subjected to immunoprecipitation using anti-Myc beads. ( F ) HEK293T cells were co-transfected with plasmids encoding Myc-RIG-I, Flag-TRIM25, and V5-N for 48 h. Cell lysates were immunoprecipitated with an anti-Flag antibody and immunoblotted with indicated antibodies. ( G ) Immunostaining assay. White arrows indicate reduced fluorescence signal of RIG-I and TRIM25 co-localization in cells with high N protein expression. Anti-Myc (red), anti-Flag (purple), anti-GFP (green) antibodies, Hoechst (blue). Scale bar, 10 µm. ( H ) Wild type (WT) or TRIM25 knockout (KO) N2a cells were co-transfected with Myc-RIG-I and GFP-N plasmid (2 μg) for 24 h, followed by treatment with MG132 for 12 h. Cell lysates were analyzed by Western blotting.

Article Snippet: Mouse neuroblastoma (N2a) (ATCC, CCL-131), PK15 cells (ATCC, CCL-33), and HEK293T (ATCC, CRL-11268) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (Gibco, U.S.) with 10% fetal bovine serum (Biological Industries, Israel), and 100 U/mL penicillin, and 100 μg/mL streptomycin.

Techniques: Infection, Over Expression, Ubiquitin Proteomics, Western Blot, Transfection, Conjugation Assay, Expressing, Immunoprecipitation, Immunostaining, Fluorescence, Knock-Out, Plasmid Preparation