Journal: Molecular Brain

Article Title: Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells

doi: 10.1186/s13041-015-0162-6

Figure Lengend Snippet: hUBQLN2 co-localized with TDP-43 and P62 but not with NF-κB and IκB-α. Immunofluorescence of Neuro2A cells at 48 h after transfection with ( a ) control plasmid, ( b ) pCMV-hUBQLN2 WT and ( c ) pCMV-hUBQLN2 P497H . Indicated antibodies were used according to Materials and Methods. Microscopy pictures were taken at 63× magnification. d - e Western analysis from three repeated experiments was used for quantification of ( d ) TDP-43 level ( p = 0.0227 for hUBQLN2 WT and p = 0.0032 for hUBQLN2 P497H ) and ( e ) p62 level ( p = 0.0058 for hUBQLN2 WT and p = 0.0130 for hUBQLN2 P497H ) in insoluble fraction. Results were calculated in fold change compared to proteins level in insoluble fraction of control cells. f Neuro2A cells were transfected with control plasmid, pCMV-hUBQLN2 WT or pCMV-hUBQLN2 P497H . Cells were extracted 48 h after transfection using aggregates assay protocol. The results suggested that overexpression of UBQLN2 species increased levels of UBQLN2, TDP-43 and p62 in the insoluble fraction. g Live imaging was performed on Neuro2A pFeGFP-hUBQLN2 P497H and DsRED-TDP-43 co-transfected cells. Microscopy pictures were taken at 24 and 48 h after transfection. h Immunoblot of TDP-43 in cytoplasmic (C) vs nuclear (N) after transfection with control plasmid, pCMV-hUBQLN2 WT or pCMV-hUBQLN2 P497H plasmids. i HEK293 cells and Neuro2A cells were transfected with either control plasmid or pCMV-hUBQLN2 P497H plasmid. Protein levels of hUBQLN2 and mUBQLN2 were measured at 48 h after transfection. Human UBQLN2 levels were similar in control HEK293 and pCMV-hUBQLN2 P497H Neuro2A transfected cells. Scale bar = 25 μm

Article Snippet: Primary antibodies were monoclonal Flag M2 (Sigma-Aldrich, Saint-Louis, 1:100), TARDBP (Proteintech, Chicago, 1:600), NF-κB p65 (Santa-Cruz, 1:200), IκB-α (Santa-Cruz, 1:200) and SQSTM1/P62 (Cell signaling, 1:200).

Techniques: Immunofluorescence, Transfection, Control, Plasmid Preparation, Microscopy, Western Blot, Over Expression, Imaging

Journal: Journal of Neuroinflammation

Article Title: Roseburia intestinalis ameliorates adolescent depression via GPR43‑dependent Treg cell expansion and suppression of neuroinflammation

doi: 10.1186/s12974-026-03755-w

Figure Lengend Snippet: Schematic illustration of the experimental flow in vivo and in vitro experiments. In vivo study, a chronic restraint stress (CRS) mouse model was established and subjected to intervention with R.i. , followed by separate administration of an Free Fatty Acid Receptor 2 (FFAR2) inhibitor (GLPG0974) or a CD25-neutralizing antibody (Anti-mouse CD25 Antibody, PC61.5). Afterward, the inflammatory profiles, levels of short-chain fatty acids (SCFAs), immune and neural parameters in the hippocampus, cortex and colon, as well as intestinal metabolites were evaluated. In vitro experiment, the polarization of microglia was assessed in an LPS+corticosterone (LPS&CORT) induced cellular stress model followed by intervention with R.i. -conditioned medium

Article Snippet: GLPG0974 (HY-12940, MCE), a free fatty acid receptor 2 (FFAR2/GPR43) antagonist, was prepared as a 5 mg/mL working solution and administered intraperitoneally to mice at a dose of 100 μL every three days, for a total of 10 injections throughout the modeling period.

Techniques: In Vivo, In Vitro

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Principle of unbiased correlation using intrinsic landmarks. IF channels are locked and hidden behind nuclear DAPI signal, which is used as only visible fluorescent channel, applied as landmark channel (blue). DAPI signal and SEM nucleus texture are used to achieve unbiased correlation of IF and SEM information. After optimization of the correlation, the channels of interest are made visible, overlaid and regions of interest selected. No further changes of image locations were applied afterwards. IF signal specificity. ( B ) Four consecutive tissue sections with nuclear DAPI (blue), DP-1 (yellow), and PGP9.5 (magenta) labeling showing traceable persistent labeling. Arrowhead indicates PGP9.5 positive nerve fiber and numbers show position of respective nuclei. Nucleus number 2 is ceasing within the z-stack. Scale bar 5 µm. ( C ) Application of secondary antibodies Al488-anti mouse (green), SeTau-647-anti rabbit (yellow), and Cy3-anti guinea pig (red) alone showed no fluorescent signal. DAPI signal (blue) for orientation. Scale bar 10 µm. Abbreviations: Al488, Alexa Fluor 488; Cy3, cyanine 3; DAPI, 4',6-diamidino-2-phenylindole; DP-1, desmoplakin 1; IF, immunofluorescence, PGP9.5, protein product 9.5 SEM, scanning electron microscopy.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Tissue, Labeling, Immunofluorescence, Scanning Electron Microscopy

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Tracking of Cx43 plaques in epidermal layers. First panel illustrates SEM overview of epidermal layers corresponding to five consecutive sections of IF images showing Cx43 signal (red) and nuclei (blue). Asterisks mark examples of traced Cx43 plaques. ( B ) Cx43 plaques at keratinocyte-nerve fiber close contact sites. Nerve fibers processing between keratinocytes in skin samples of two male subjects (M1, M2) and one female subject (F1). Each row represents four consecutive sections of 100 nm thickness. First tile shows keratinocyte cell bodies (orange), nuclei (blue), and nerve fibers (magenta) in pseudo color with Cx43 signal (red). Correlated PGP9.5 labeling (magenta) locates at nerve fibers and Cx43 labeling (yellow) indicates Cx43 plaques. Insets show magnification of contact area. Scale bars: 5 µm ( A ), 1 µm ( B ), magnified insets: 500 nm. Abbreviations: Cx43, connexin 43; IF, Immunofluorescence; PGP9.5, protein gene product-9.5; SEM, scanning electron microscopy.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Labeling, Immunofluorescence, Scanning Electron Microscopy

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Overview of skin section prior to expansion with PGP9.5-labeled IENF (magenta), Cx43 (yellow), and nuclear DAPI (blue); dotted line illustrates epidermis-dermis border and arrow indicates IENF. White rectangle marks inset enlarged in B. ( B ) Enlarged area prior to expansion and ( C ) matched area post-expansion at same magnification with addition of actin marker phalloidin (grey); white rectangles mark inset area of D and E. ( D ) Enlarged IENF area at ×20 magnification and ( E ) at ×63 magnification with arrowheads indicating Cx43 plaque at IENF. Inset in E marks enlarged area in E`-E```. ( E` ) shows z-plane prior to E, ( E`` ) same z-plane as E, and E``` z-plane step after E. White scale bars indicate pre-expansion state, yellow scale bars are corrected for expansion factor. Scale bars: 50 µm ( A ), 5 µm ( B, C ), 2 µm ( D, E ), 500 nm ( E`-E``` ). Z-step size of 1.2 µm translates to approximate 274 nm in expanded gel. Abbreviations: Cx43, connexin 43; IENF, intraepidermal nerve fiber; PGP9.5, protein gene product-9.5.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Labeling, Marker, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Workflow for ensheathment and Cx43 quantification (see also ). ( B ) Magnitude of skin tissue expansion across investigated sections via comparison of epidermal nuclei. Median nucleus size between samples pre-ExM (8.73±0.54 µm) and post ExM (32.46±3.54 µm) denotes a median ExM factor of 3.83±0.36. ( C ) Extracted fiber ensheathment ratio (PGP9.5-positive px without WGA pixel co-localization divided by total PGP9.5 px count) in HC versus SFN patients. ( C ) Extracted fiber ratio (total PGP9.5 px versus PGP9.5 non WGA overlap px) in HC versus SFN patients (n=13 HC and 10 HC ROIs from n=5 HC and n=4 SFN samples; p=0.41; shown as box & whiskers plot with minimum and maximum). ( D ) Cx43 contact ratio (PGP9.5 px with Cx43-WGA double positive px divided by total PGP9.5 px) in HC versus SFN (n=13 HC and 10 SFN ROIs from n=5 HC and n=4 SFN samples; p=0.34; shown as box & whiskers plot with minimum and maximum). Expansion factor corrected scale bars (yellow) of exemplary segmentation: 5 µm. See also . Abbreviations: HC, healthy control; Cx43, connexin 43; NCU, neuro-cutaneous unit; PGP9.5, protein gene product 9.5; px, pixel; SFN, small fiber neuropathy; WGA, wheat germ agglutinin. Figure 5—source data 1. Descriptive data and values for nucleus based calculation of expansion factors. Figure 5—source data 2. Statistical details and input values for .

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Tissue, Comparison, Control, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: ( A ) Confocal overview image of Ctx-positive sensory neurites (green), Cx43 positive (orange) keratinocytes with membrane labeling of keratinocytes via WGA (magenta) and nuclear DAPI (blue). ( B ) Inset of ensheathment area from A. ( C ) Single plane lattice SIM image and respective inset area with 3D visualization of z-stack (2.925 µm depth, 0.196 µm steps) showing nucleus, Cx43, and neurite signal ( C` ) and including WGA ( C`` ). ( D ) Confocal overview image of Ctx-positive sensory neurites (green), Cx43-positive (orange) keratinocytes with membrane labeling of keratinocytes via WGA (magenta), and nuclei (blue). Arrowheads indicate Cx43 - neurite contact areas. ( E ) Single plane lattice SIM image and respective inset area with 3D visualization, numbers represent single Cx43 plaques ( F ) of z-stack (2.925 µm depth, 0.196 µm steps). ( F`-F`` `) detailed neurite - Cx43 contact areas. Co-culture kept in keratinocyte medium. Scale bars: 20 µm ( A, D ), 5 µm ( C-C``, E, F`-F```` ), 1 µm ( F ). Abbreviations: Ctx, cholera toxin subunit B; Cx43, connexin 43; DAPI, 4',6-diamidino-2-phenylindole; SIM, structured illumination microscopy; WGA, wheat germ agglutinin.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Confocal, Membrane, Labeling, Co-culture Assay, Microscopy, Immunofluorescence

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet: Primary and secondary antibodies and marker with specifications.

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Marker

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet:

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques:

Journal: eLife

Article Title: Interaction of human keratinocytes and nerve fiber terminals at the neuro-cutaneous unit

doi: 10.7554/eLife.77761

Figure Lengend Snippet:

Article Snippet: Further, PGP9.5 (Zytomed, Berlin, Germany) and Cx43 (alomone labs, Jerusalem, Israel) antibodies were applied with 0.1% (v/v) Triton X-100 (Thermo Fisher Scientific, MA, USA) instead of saponin.

Techniques: Immunofluorescence