Journal: The Journal of Neuroscience

Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain

doi: 10.1523/JNEUROSCI.4528-13.2014

Figure Lengend Snippet: A, TREK2 antibody characterization. Top, GFP fluorescence or interference contrast (IC) shows HEK cells, transfected with plasmids for six rat K2P channels: TREK2, TWIK1, TREK1, TASK3, THIK2, and THIK1 (all subcloned in pCMV-SPORT vector). Bottom, Same fields show immunostaining for TREK2 that was intrinsically very low (GFP alone), increased substantially after transfection with the chimeric TREK2-GFP and with TREK2, but remained very low after transfection with all non-TREK2 K2P channels tested. This antibody therefore selectively recognizes TREK2 and not the other K2P channel tested, including TREK1, structurally the closest related to TREK2. Scale bar, 40 μm. B–D, TREK2 in DRG neurons is selectively expressed in IB4+ neurons. B, Triple fluorescence immunocytochemistry shows TREK2 (blue) to be highly colocalized with IB4 binding (red). RT97 (green) is a marker for neurofilament (against a highly phosphorylated epitope on NF200) that selectively stains DRG somata with A-fibers (i.e., myelinated). TREK2 was only visible in neurofilament-poor (NF-poor, thus C-fiber) neurons, as shown by lack of (green) NF staining in TREK2+ neurons. TREK2 staining is thus in IB4+ DRG neurons with C-fibers. Scale bar, 20 μm. C, D, %intensities refers to cytoplasmic immunointensities expressed as a percentage of maximum staining above background for that marker (see Materials and Methods); gray band(s) show(s) negative staining for TREK2 (<25% %intensity) and IB4 (<20% %intensity). C, TREK2+ neuronal profiles with nuclei were <400 μm2 in cross-sectional area, and mostly <300 μm2. D, TREK2 and IB4 %intensities were positively correlated in small neurons. (Spearman's correlation coefficient, rs, shown) and had a highly significant linear regression (r2 = 0.54, p < 0.0001). There was no significant correlation in medium to large neurons; these were negative for both (data not shown). E, F, TREK2 in DRG neurons of other species. Rat images included for comparison. E, Lumbar DRG cryostat sections, from adult rat and cat (L4) and 2 week female guinea pig (L6), immunostained by ABC against TREK2. As in rat, both cat and guinea pig showed small DRG neurons positively stained for TREK2. TREK2+ neurons in cat (although larger than rat) are consistent with them being small NF-poor neurons. Small NF-poor neurons in adult cat lumbar DRGs have a mean diameter ∼40 μm; maximum ∼50 μm (Perry and Lawson, 1993); in rats, mean diameter ∼18 μm, maximum ∼25 μm. Thus, in three species, a subpopulation of small neurons shows clear TREK2 immunostaining. Scale bar, 40 μm. F, Guinea pig DRG (as above) double-labeled for TREK2 immunocytochemistry and IB4 binding. As with rat, in guinea pig strong TREK2 staining (blue) is present in a subpopulation of small DRG neurons with clear IB4 binding; red dashed lines indicate outlines of large TREK2− neurons for comparison with small TREK2+. Scale bar, 20 μm. G, H, TREK2 and trkA are poorly coexpressed in rat DRG neurons. G, TREK2 (blue) and trkA (red) (double-immunofluorescence, pseudocolored) were not visibly colocalized. White dashed lines indicate outlines of large TREK2− neurons. Scale bar, 20 μm. H, Many neurons were negative for both trkA (<20% %intensity) and TREK2 (see below); others were TREK2+ or trkA+, and few were positive for both. %intensities of neurons of all sizes were weakly but negatively correlated (Spearman's). I, J, TREK2 and Nav1.9 are colocalized in small DRG neurons. Adjacent ABC-immunostained sections were examined because both primary antibodies are from the same species. I, TREK2 and Nav1.9 were strongly colocalized. Scale bar, 25 μm. J, Strong correlation (Spearman's) between their %intensities in all sizes of neuron.

Article Snippet: Rabbit anti-TREK2 (1:500, Alomone Labs) characterized by Alomone Labs by Lin et al. (2004 ) and in this paper (see A ); mouse anti-NF200 (RT97, 1:4000, gift from J.N.

Techniques: Fluorescence, Transfection, Plasmid Preparation, Immunostaining, Immunocytochemistry, Binding Assay, Marker, Staining, Negative Staining, Labeling, Immunofluorescence

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: NHNE cells from five healthy volunteers were inoculated with human nasal S. epidermidis at an MOI of 0.25. A FemA mRNA levels, normalized to cellular GAPDH transcript levels, were monitored by real-time PCR. B S. epidermidis CFUs were determined at 1 dpi in the supernatant of S. epidermidis -inoculated NHNE cells. C Dot plot visualization of enriched GO terms in S. epidermidis– inoculated NHNE cells at 1 dpi. D Scatterplots indicating enriched genes correlated with serine-type peptidase inhibitors. E Expressions of Serpine1 and Serpine2 from scRNA-seq of NHNE cells. Dot size represents the proportion of Serpines RNA expression within the respective cell type expressing the gene, and dot color represent the average Serpines RNA expression level in the particular cell type. F Serpine1 and Serpine2 mRNA levels, normalized to cellular GAPDH transcript levels, were monitored by real-time PCR (blue bar: Serpine1; red bar: Serpine2). G The intracellular protein level of Serpine1 was measured in the cell lysate of S. epidermidis– inoculated NHNE cells using western blot analysis. H The secreted protein level of Serpine1 was measured in the supernatant of S. epidermidis -inoculated NHNE cells using ELISA. I Heatmap depicting the expression levels of genes related with serine-type peptidase inhibitor activity differentially expressed in S. epidermidis -inoculated NHNE cells depending on the cellular subset. J Serpine1 mRNA level, normalized to cellular GAPDH transcript levels, was monitored by real-time PCR over 1 day after IAV infection following S. epidermidis inoculation. K , L The neutralizing antibody for Serpine1 was administered to NHNE cells one hour before S. epidermidis inoculation, and then the cells were infected with IAV for 1 day. IAV mRNA and NPs of IAV were compared in IAV-infected NHNE cells after S. epidermidis inoculation depending upon the neutralization of Serpine1. Results are presented as mean ± SD values from five independent experiments. * p < 0.05 vs. mock-infected NHNE cells.

Article Snippet: In addition, a human Serpine1 antibody (molecular weight 45 kDa, primary antibody 1:500) was purchased from Abcam (Waltham, MA, USA, cat #ab66705) and mouse Serpine1 blocking peptide from MyBioSource (San Diego, CA, USA, cat #MBS8309331).

Techniques: Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Infection, Neutralization

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: Differential expression gene (DEG) linked with serine-type peptidase inhibitors in S. epidermidis -inoculated NHNE cells.

Article Snippet: In addition, a human Serpine1 antibody (molecular weight 45 kDa, primary antibody 1:500) was purchased from Abcam (Waltham, MA, USA, cat #ab66705) and mouse Serpine1 blocking peptide from MyBioSource (San Diego, CA, USA, cat #MBS8309331).

Techniques: Quantitative Proteomics

Journal: NPJ Biofilms and Microbiomes

Article Title: Nasal symbiont Staphylococcus epidermidis restricts the cellular entry of influenza virus into the nasal epithelium

doi: 10.1038/s41522-022-00290-3

Figure Lengend Snippet: A Scatterplots indicating enriched genes related with serine-type peptidase. B PPI network of DEGs linked with serine-type peptidase indicates the protein – protein interaction with Serpine1 and Serpine2 (PP protein – protein). C Heatmap depicting the expression levels of genes related with serine-type peptidase inhibitor activity differentially expressed in S. epidermidis– inoculated NHNE cells depending on a cellular subset. D Serpine1 mRNA and E protein levels were monitored by real-time PCR and western blot analysis following S. epidermidis inoculation at 1 dpi with transfection with cont shRNA and uPA shRNA. F Serpine1 (blue bar) and PLAU mRNAs (red bar) were compared in NHNE cells after S. epidermidis inoculation. G ELISA results showed the secreted protein levels of Serpine1 (blue bar) and uPA (red bar) in S. epidermidis -inoculated NHNE cells. H The intracellular protein levels of HAT and Serpine1 were measured in the cell lysate of S. epidermidis– inoculated NHNE cells at 4, 8, and 24 h using western blot analysis. I The mRNA levels of proteases, such as TMPRSS2, TMPRSS11E, TMPRSS11F, and KLK5, which were targeted by Serpine1, were measured using the cell lysate of S. epidermidis -inoculated NHNE cells at 4, 8, and 24 h. Western blot results are representative of five independent experiments. Real-time PCR and plaque assay results are presented as mean ± SD values from five independent experiments. * p < 0.05 vs. mock-infected NHNE cells.

Article Snippet: In addition, a human Serpine1 antibody (molecular weight 45 kDa, primary antibody 1:500) was purchased from Abcam (Waltham, MA, USA, cat #ab66705) and mouse Serpine1 blocking peptide from MyBioSource (San Diego, CA, USA, cat #MBS8309331).

Techniques: Expressing, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Plaque Assay, Infection