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: 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