Journal: The Journal of Clinical Investigation

Article Title: Wdr26 insufficiency causes Skraban-Deardorff syndrome–like neurodevelopmental deficits in mice

doi: 10.1172/JCI195537

Figure Lengend Snippet: ( A ) mPFC tissues from Wdr26 +/+ and Wdr26 +/– mice were collected for Western blotting. ( B ) Quantitative analysis of RUNX1T1 protein levels ( n = 5) from A . ( C ) N2a cells transfected with si- Wdr26 for 36 hours were tested for RUNX1T1 and WDR26 protein levels by Western blotting. ( D ) Quantitative analysis of WDR26 protein levels from C ( n = 3). ( E ) Quantitative analysis of RUNX1T1 protein levels from C ( n = 3). ( F ) N2a cells were treated with CHX, MG132, bafilomycin A1 (Baf A1), and Z-VAD-FMK (Z-V-F) for 12 hours, and RUNX1T1 protein levels were measured by Western blotting. ( G ) Quantitative analysis of RUNX1T1 protein levels from F ( n = 3). ( H ) N2a cells were treated with CHX and MG132 for 2, 4, and 8 hours and RUNX1T1 protein levels were measured by Western blotting. ( I ) Quantitative analysis of RUNX1T1 protein levels from H ( n = 3). ( J and K ). Immunoprecipitation analysis of WDR26 and RUNX1T1 interactions in the brains of Wdr26 +/+ mice. Data are representative of 3 independent experiments. ( L ) Immunoprecipitation analysis of ubiquitination of RUNX1T1 in the brains of Wdr26 +/+ and Wdr26 +/– mouse embryos. ( M ) Quantitative analysis of ubiquitination levels from L ( n = 3). ( N ) MAP2 levels in the mPFC of Wdr26 +/+ and Wdr26 +/– mice at P0.5 were measured by Western blotting ( n = 3). Data are presented as the mean ± SEM. * P < 0.05 and ** P < 0.01, by unpaired, 2-tailed Student’s t test ( B , D , E , G , and M ) and 2-way ANOVA with Tukey’s post hoc test for multiple comparisons ( I ).

Article Snippet: N2a cells were obtained from the American Type Culture Collection (ATCC) (catalog CCL-131).

Techniques: Western Blot, Transfection, Immunoprecipitation, Ubiquitin Proteomics

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: Discovery of a CI-994 derivative as a dual modulator of class I HDACs and Wnt/β-catenin signaling for Alzheimer’s disease therapy

doi: 10.64898/2026.04.30.721954

Figure Lengend Snippet: N2a cells were treated with CI-994, W2A-28, W2A-42 and W2A-43 at 8 μM for 24 h, and the level of β-catenin was examined by Western blotting. Data represent mean ± SEM from four independent experiments. P values were calculated using one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: HEK293 cells (CRL-1573) and N2a neuroblastoma cells (CCL-131) were obtained from ATCC.

Techniques: Western Blot, Comparison

Journal: bioRxiv

Article Title: CRISPR activation of PIKFYVE as potential therapy for FIG4 deficiency

doi: 10.64898/2026.04.24.718784

Figure Lengend Snippet: (A) Fourteen sgRNA sequences were designed up to - 300 base pairs upstream of the Pikfyve transcriptional start site. (B) Upregulation of Pikfyve in N2A cells was evaluated using Pikfyve Taqman assay and normalized with Tata binding protein Taqman assay. The asterisk labels the highest expressing sgRNA used in later experiments. (C) Combinations of sgRNAs

Article Snippet: Neuro-2a (N2A) cells (CCL-131) were obtained from The American Type Culture Collection.

Techniques: TaqMan Assay, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Neuron-specific epigenetic repression of Cdk5 impairs hippocampal-dependent memory in male and female mice

doi: 10.64898/2026.04.08.716689

Figure Lengend Snippet: (a) Schematic of targeted histone deacetylation at the Cdk5 promoter using CRISPR/dCas9-HDAC3. (b) CUT&Tag bigwig-normalized signal and called peak for H3K9/14ac in mouse hippocampus (top), and positions of designed sgRNAs targeting 62–1107 bp upstream of the Cdk5 TSS (bottom). (c) Cdk5 mRNA expression in N2a cells treated with dCas9-HDAC3 paired with each designed sgRNA, or dCas9-AM tag with Cdk5 -sgRNA ( n = 3). One-way ANOVA: main effect of sgRNA, F(6, 14) = 13.35, p < 0.0001; post-hoc NT vs. Cdk5 -sgRNA, p = 0.0049; dCas9-AM tag vs. NT, p = 0.0907 (ns). (d) Intra-NAc injection of the neuronal hSyn-dCas9-HDAC3 with NT (left hemisphere) or Cdk5 -sgRNA (right hemisphere) reduces Cdk5 mRNA at days 5 and 6 post-injection ( n = 9-10). Two-way ANOVA: main effect of sgRNA, F (1, 84) = 12.91, p = 0.0005; post-hoc NT vs. Cdk5 -sgRNA at day 5, p = 0.0276; day 6, p = 0.0034. (e) Intra-dHPC injection of hSyn-dCas9-HDAC3 with NT (left hemisphere) or Cdk5 -sgRNA (right hemisphere) reduces Cdk5 mRNA at day 6 post-injection in males (left; paired t-test, p = 0.0395, n = 3) and females (right; paired t-test, p = 0.0425; n = 4). (f) hSyn-dCas9-HDAC3 with Cdk5 -sgRNA reduces Cdk5 protein levels at day 6 PI in the dorsal hippocampus of a mixed-sex cohort (paired Wilcoxon test, p = 0.0312; n = 5). (g) hSyn-dCas9-HDAC3 with Cdk5 -sgRNA reduces phosphorylation of Tau at Serine 396 in the dorsal hippocampus of a mixed-sex cohort (paired Wilcoxon test, p = 0.0292; n = 4). All data are presented as mean ± SEM.

Article Snippet: Murine Neuro-2a (N2a) cells (ATCC, #CCL-131) were maintained in EMEM (ATCC, #30-2003) supplemented with 10% fetal bovine serum (FBS; ATCC #30-2020).

Techniques: CRISPR, Expressing, Injection, Phospho-proteomics