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: Current Protocols

Article Title: Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

doi: 10.1002/cpz1.70372

Figure Lengend Snippet: Schematic illustration of 3D co‐culture setup using AEC‐tLgT epithelial cells and MLg mouse fibroblasts. Epithelial cells and fibroblasts are mixed with Matrigel and loaded onto Transwell filters and provided nutrients through medium perfusion from the outside chamber. GFR, growth factor‐reduced; PET, polyester.

Article Snippet: MLg mouse lung fibroblasts (ATCC, CCL‐206, RRID:CVCL_0437) MLF medium (MLg fibroblast culture medium, see recipe) Fmed+ROCKinh culture medium (see recipe) Sterile 1× DPBS (Corning, 21‐031‐CV) 0.05% (w/v) trypsin‐EDTA solution (Gibco, 25300‐062) 0.4% trypan blue staining (Gibco, 15250‐061) AEC‐tLgT immortalized cells (from Basic Protocol ) GFR Matrigel (Corning, 354230) 3D basic medium (see recipe) Accutase (Innovative Cell Technologies, AT‐104) SB‐431542, working stock of 1 mM (Selleck Chemicals, S1067) 15‐ and 50‐ml conical tubes Benchtop centrifuge that can accommodate 15‐ and 50‐ml conical tubes Hemocytometer (Thermo Fisher Scientific, 02‐671‐5) 15‐cm tissue culture dish (Corning, CLS430599) or T‐175 culture flask with vented cap T‐25 culture flask with vented cap 8‐ or 10‐in. length straight specimen forceps, serrated (e.g., VWR, 82027‐436 or ‐434) 24‐well Transwell inserts (Corning, 3470) 24‐well tissue culture plate 1.5‐ml microcentrifuge tubes Bucket or container of ice to bring into the tissue culture hood Sealed 200‐μl pipette filter tips, pre‐cooled at 4°C to be used in the hood (optional) Brightfield microscope with either 4× or 2.5× objective

Techniques: Co-Culture Assay

Journal: Current Protocols

Article Title: Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

doi: 10.1002/cpz1.70372

Figure Lengend Snippet: Representative cytospin immunofluorescence staining results to assess the purity of isolated AT2 cells. This example was from a lung preparation yielding 93% viable EpCAM + cells and 91% AT2 cell purity. CD45, leukocyte cell marker; VIM, vimentin fibroblast marker; CC10, Club cell marker; TUBB4A, ciliated cell marker (airway); KRT5, basal cell marker (airway); MUC5B, goblet cell marker (airway); NKX2‐1, general lung cell marker; HTII‐280 and SPC, AT2 cell markers; mouse IgG, rabbit IgG, and mouse IgM are negative controls. Cytocentrifuged cells were nuclear counterstained with propidium iodide (red). Images were taken at 10× magnification on a Nikon Ti Eclipse fluorescence microscope. Scale bar, 50 µm.

Article Snippet: MLg mouse lung fibroblasts (ATCC, CCL‐206, RRID:CVCL_0437) MLF medium (MLg fibroblast culture medium, see recipe) Fmed+ROCKinh culture medium (see recipe) Sterile 1× DPBS (Corning, 21‐031‐CV) 0.05% (w/v) trypsin‐EDTA solution (Gibco, 25300‐062) 0.4% trypan blue staining (Gibco, 15250‐061) AEC‐tLgT immortalized cells (from Basic Protocol ) GFR Matrigel (Corning, 354230) 3D basic medium (see recipe) Accutase (Innovative Cell Technologies, AT‐104) SB‐431542, working stock of 1 mM (Selleck Chemicals, S1067) 15‐ and 50‐ml conical tubes Benchtop centrifuge that can accommodate 15‐ and 50‐ml conical tubes Hemocytometer (Thermo Fisher Scientific, 02‐671‐5) 15‐cm tissue culture dish (Corning, CLS430599) or T‐175 culture flask with vented cap T‐25 culture flask with vented cap 8‐ or 10‐in. length straight specimen forceps, serrated (e.g., VWR, 82027‐436 or ‐434) 24‐well Transwell inserts (Corning, 3470) 24‐well tissue culture plate 1.5‐ml microcentrifuge tubes Bucket or container of ice to bring into the tissue culture hood Sealed 200‐μl pipette filter tips, pre‐cooled at 4°C to be used in the hood (optional) Brightfield microscope with either 4× or 2.5× objective

Techniques: Immunofluorescence, Staining, Isolation, Marker, Fluorescence, Microscopy

Journal: Current Protocols

Article Title: Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

doi: 10.1002/cpz1.70372

Figure Lengend Snippet: Representative brightfield images of MLg fibroblasts at 50% confluence, the optimal growth pattern to set up 3D co‐cultures with AEC‐tLgT cells. Images taken at 4× magnification (left; scale bar, 900 µm) and 10× magnification (right; scale bar, 360 µm) on an ECHO Revolve R4 fluorescence microscope.

Article Snippet: MLg mouse lung fibroblasts (ATCC, CCL‐206, RRID:CVCL_0437) MLF medium (MLg fibroblast culture medium, see recipe) Fmed+ROCKinh culture medium (see recipe) Sterile 1× DPBS (Corning, 21‐031‐CV) 0.05% (w/v) trypsin‐EDTA solution (Gibco, 25300‐062) 0.4% trypan blue staining (Gibco, 15250‐061) AEC‐tLgT immortalized cells (from Basic Protocol ) GFR Matrigel (Corning, 354230) 3D basic medium (see recipe) Accutase (Innovative Cell Technologies, AT‐104) SB‐431542, working stock of 1 mM (Selleck Chemicals, S1067) 15‐ and 50‐ml conical tubes Benchtop centrifuge that can accommodate 15‐ and 50‐ml conical tubes Hemocytometer (Thermo Fisher Scientific, 02‐671‐5) 15‐cm tissue culture dish (Corning, CLS430599) or T‐175 culture flask with vented cap T‐25 culture flask with vented cap 8‐ or 10‐in. length straight specimen forceps, serrated (e.g., VWR, 82027‐436 or ‐434) 24‐well Transwell inserts (Corning, 3470) 24‐well tissue culture plate 1.5‐ml microcentrifuge tubes Bucket or container of ice to bring into the tissue culture hood Sealed 200‐μl pipette filter tips, pre‐cooled at 4°C to be used in the hood (optional) Brightfield microscope with either 4× or 2.5× objective

Techniques: Fluorescence, Microscopy