Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: Pharmacological inhibition of PIP5Kα attenuates intracellular signaling following stimulation of TRPM3 and Ca v 1.2 Ca 2+ channels. ( a ) Modular structure of TRPM3 showing the interaction sites of phosphatidylinositol 4,5-bisphosphate with the channel (preS1 segment, S4-S5 linker, and TRP domain). ( b ) T-REx-TRPM3 cells containing a Coll.luc reporter gene integrated into the chromatin were serum-starved for 24 h in the presence of tetracycline (1 μg/mL) to induce TRPM3 expression. The serum-starved cells were preincubated with ISA-2011B (10 μM) for 3 h, and then stimulated with pregnenolone sulfate (20 μM) for 24 h in the presence of the inhibitor. Cells were harvested and analyzed as described in the legend to (n = 4; *** p < 0.001). ( c ) Modular structure of Ca v 1.2 voltage-gated Ca 2+ channels, consisting of the α1 subunit, which forms the pore, and the auxiliary subunits α2δ, β, and γ. ( d ) INS-1 832/13 insulinoma cells were infected with a recombinant lentivirus containing the Coll.luc reporter gene. Cells were serum-starved in medium containing 0.5% serum and 2 mM glucose for 24 h. Cells were preincubated in the same medium with ISA-2011B (10 μM) for three hours. Stimulation of the cells was performed with KCl (25 mM) and the voltage-gated Ca 2+ channel activator FPL64176 (2.5 μM) in the presence of the inhibitor for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; *** p < 0.001).

Article Snippet: The stimulation of INS-1 832/13 cells with KCl (PubChem CID: 4873; 55 mM) and FPL64176 (PubChem CID: 3423; 2.5 μM, dissolved in DMSO, a kind gift from Alomone Labs, Israel, Cat #: F-160) was performed in the same medium for 24 h in the presence of the inhibitory compound.

Techniques: Inhibition, Expressing, Infection, Recombinant

Journal: Molecules

Article Title: Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun

doi: 10.3390/molecules29112602

Figure Lengend Snippet: RGS2 expression reduces signal transduction after stimulation of TRPM8 channels or Gαq-coupled designer receptors. ( a ) Modular structure of RGS2. The RGS domain is responsible for binding to Gαq, while the N-terminal domain is required for targeting the protein to the plasma membrane. ( b ) HEK293-M8 cells containing the Coll.luc reporter gene were infected with a recombinant lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were serum-starved for 24 h, and then stimulated with icilin (1 μM) in serum-reduced medium for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3, *** p < 0.001). ( c ) HEK293 cells were infected with a lentivirus encoding the Gαq-coupled designer receptor Rαq. Cells were additionally infected with a lentivirus containing the Coll.luc reporter gene. Furthermore, the cells were infected with a lentivirus encoding either RGS2 or β-galactosidase (mock). We incubated the cells in medium containing 0.05% serum for 24 h. Stimulation of the cells was performed with CNO (1 μM) for 24 h in serum-reduced medium. Cells were harvested and analyzed as described in the legend to (n = 5, *** p < 0.001). ( d ) T-REx-TRPM3 cells containing a chromatin-integrated Coll.luc reporter gene were serum-starved for 24 h in the presence of tetracycline (1 μg/mL). The serum-starved cells were infected with a lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were stimulated with pregnenolone sulfate (20 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; n.s., not significant). ( e ) INS-1 832/13 insulinoma cells were infected with a lentivirus containing the reporter gene Coll.luc. Additionally, we infected the cells with a lentivirus encoding either RGS2 or β-galactosidase (mock). Cells were incubated in medium containing 0.5% serum and 2 mM glucose for 24 h, and then stimulated with KCl (25 mM) and the FPL64176 (2.5 μM) for 24 h. Cells were harvested and analyzed as described in the legend to (n = 3; n.s., not significant).

Article Snippet: The stimulation of INS-1 832/13 cells with KCl (PubChem CID: 4873; 55 mM) and FPL64176 (PubChem CID: 3423; 2.5 μM, dissolved in DMSO, a kind gift from Alomone Labs, Israel, Cat #: F-160) was performed in the same medium for 24 h in the presence of the inhibitory compound.

Techniques: Expressing, Transduction, Binding Assay, Membrane, Infection, Recombinant, Incubation

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet: CD163 + macrophages accumulate in the lung in severe COVID-19 (A) Overview of study design and analyses. CT, computed tomography; BAL, bronchoalveolar lavage; scRNA-seq, single-cell RNA sequencing; snRNA-seq, single-nucleus RNA sequencing; IHC, immunohistochemistry; IF, immunofluorescence microscopy; MELC, multi-epitope ligand cartography; EM, electron microscopy; VCin, inspiratory vital capacity; PBMC, peripheral blood mononuclear cells; IAV, Influenza A virus. (B) Postmortem analysis of consecutive histological sections of non-COVID-19 (left) and COVID-19 autopsy lung samples (right) by hematoxylin and eosin (H&E; top) and CD68 IHC (bottom). Scale bar, 100 μm. (C) IF of CD68 (green) and CD163 (red) in lung tissue autopsy samples of COVID-19 patients and non-COVID-19 controls. Arrows indicate CD68 + CD163 – macrophages, and arrowheads indicate CD68 + CD163 + macrophages. Scale bar, 20 μm. (D) Quantification of CD68 + macrophage density (left) and the proportion of CD163 + macrophages (right) in lung autopsy samples from fifteen donors (as in C). Mann-Whitney test; ∗ p < 0.05. (E) Representative images of consecutive histological sections of lung autopsy samples. H&E (left), CD68 IHC (middle), and SARS-CoV-2 RNA-FISH (right). Arrowheads indicate SARS-CoV-2 RNA-positive macrophages. Scale bars, 50 μm, 25 μm. RNA-FISH, RNA-fluorescence in situ hybridization. (F) Lung autopsy samples of 9 COVID-19 patients were analyzed by MELC with a panel of 22 markers on 19 fields of view (FOVs). Two-dimensional embedding computed by UMAP on 9,684 computationally identified CD45 positive cells (T cells, CD3 + ; B cells, CD20 + ; NK cells, CD56 + ; neutrophils, MRP14 + /CD66b + ; monocytes, MRP14 + /CCR2 + ; macrophages, MRP14 + /HLA-DR + ). (G) Relative proportion (of total CD45 + cells) of cell types in all 19 FOVs (left), and average cell numbers (summary, right).

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Computed Tomography, RNA Sequencing Assay, Immunohistochemistry, Immunofluorescence, Microscopy, Electron Microscopy, MANN-WHITNEY, Fluorescence, In Situ Hybridization

Journal: Cell

Article Title: SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis

doi: 10.1016/j.cell.2021.11.033

Figure Lengend Snippet:

Article Snippet: CD66b-PE , Miltenyi Biotec , Cat# 130-122-922, N/A.

Techniques: Immunohistochemistry, Plasmid Preparation, Recombinant, Staining, Lysis, Protease Inhibitor, Mass Spectrometry, Sequencing, Modification, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: Lung Mammary Metastases but Not Primary Tumors Induce Accumulation of Atypical Large Platelets and Their Chemokine Expression

doi: 10.1016/j.celrep.2019.10.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Goat anti-hamster, AF488 , Jackson ImmunoResearch , Cat# 127-545-160, RRID:AB_2338997.

Techniques: Recombinant, RNA Sequencing, Software

Journal: iScience

Article Title: Prolonged dysbiosis and altered immunity under nutritional intervention in a physiological mouse model of severe acute malnutrition

doi: 10.1016/j.isci.2023.106910

Figure Lengend Snippet:

Article Snippet: Goat anti-Armenian hamster IgG Cy3 , Jackson Immuno-research , Cat #127-165-160; RRID: AB_2338989.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Bacteria, Software, Microscopy

Journal: Frontiers in Immunology

Article Title: Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

doi: 10.3389/fimmu.2023.1264179

Figure Lengend Snippet: Biochemical characterization of hSIRPα Nbs. (A) Amino acid (aa) sequences of the complementarity-determining region (CDR) 3 from 14 unique hSIRPα Nbs (complete sequences shown in Supplementary Table 1 ) identified by a bidirectional screening strategy. Nbs S7 to S36 were selected against full-length hSIRPα and Nbs S41 to 45 against domain 1 of hSIRPα (hSIRPαD1). (B) Recombinant expression and purification of hSIRPα Nbs using immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Coomassie staining of purified Nbs is shown. (C) Stability analysis using nano–differential scanning fluorimetry (nanoDSF) displaying fluorescence ratio (350 nm/330 nm) and light intensity loss due to scattering shown as first derivative exemplarily shown for Nb S36 (upper panel). Data are shown as mean value of three technical replicates. BLI-based affinity measurements exemplarily shown for Nb S36 (bottom panel). Biotinylated hSIRPα was immobilized on streptavidin biosensors. Kinetic measurements were performed using four concentrations of purified Nbs ranging from 0.625 nM to 5 nM (displayed with gradually darker shades of color). The binding affinity (K D ) was calculated from global 1:1 fits shown as dashed lines. (D) Summary table of stability and affinity analysis of selected hSIRPα Nbs. Melting temperature (T M ) and aggregation temperature (T Agg ) determined by nanoDSF shown as mean ± SD of three technical replicates. Affinities (K D ), association constants ( k on ), and dissociation constants ( k off ) determined by BLI using four concentrations of purified Nbs shown as mean ± SD. (E) Representative images of hSIRPα and GFP-coexpressing U2OS cells stained with hSIRPα Nbs of three technical replicates. Images show individual Nb staining detected with anti-VHH-Cy5 (red), intracellular IRES-derived GFP signal (green), nuclei staining (Hoechst, blue), and merged signals; scale bar, 50 µm.

Article Snippet: Unlabeled hSIRPα Nbs (1 nM to 100 nM) in combination with anti-VHH secondary Cy5 AffiniPure Goat Anti-Alpaca IgG (2.5 μg/mL; Jackson Immuno Research) were added and incubated for 1 h at 37°C.

Techniques: Recombinant, Expressing, Purification, Affinity Chromatography, Size-exclusion Chromatography, Staining, Nano Differential Scanning Fluorimetry, Fluorescence, Binding Assay, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

doi: 10.3389/fimmu.2023.1264179

Figure Lengend Snippet: Epitope characterization of hSIRPα Nbs. (A) Domain mapping analysis by immunofluorescence staining with hSIRPα Nbs on U2OS cells displaying human hSIRPα domain 1 (D1), domain 2 (D2), or domain 3 (D3) at their surface. Representative images of live cells stained with individual Nbs in combination with Cy5-labeled anti-VHH of three technical replicates are shown; scale bar, 50 µm. (B) Epitope binning analysis of hSIRPα Nbs by BLI. Graphical summary of epitope binning analysis on the different hSIRPα domains (left panel). Representative sensograms (n = 1) of combinatorial Nb binding to recombinant hSIRPα on sharing/overlapping epitopes or on different epitopes (right panel).

Article Snippet: Unlabeled hSIRPα Nbs (1 nM to 100 nM) in combination with anti-VHH secondary Cy5 AffiniPure Goat Anti-Alpaca IgG (2.5 μg/mL; Jackson Immuno Research) were added and incubated for 1 h at 37°C.

Techniques: Immunofluorescence, Staining, Labeling, Binding Assay, Recombinant

Journal: Frontiers in Immunology

Article Title: Two birds with one stone: human SIRPα nanobodies for functional modulation and in vivo imaging of myeloid cells

doi: 10.3389/fimmu.2023.1264179

Figure Lengend Snippet: Cross-reactivity and binding specificity of hSIRPα Nbs. (A) Cross-reactivity analysis of hSIRPα Nbs by immunofluorescence staining on U2OS cells displaying hSIRPα-V1, hSIRPα-V2, hSIRPβ1, hSIRPγ, or mouse SIRPα at their surface. Representative images of live cells stained with individual Nbs in combination with Cy5-labeled anti-VHH are shown of three technical replicates; scale bar, 50 µm. (B) Flow cytometry analysis of human peripheral blood mononuclear cells (PBMCs) stained with fluorescently labeled hSIRPα Nbs (AlexaFluor 647, AF647). Flow cytometry plots of Nb S36 staining on CD14 + and CD3 + PBMC populations derived from human donor K1 are shown as an example. (C) Flow cytometry analysis of hSIRPα Nbs staining CD14 + PBMCs of three different human donors (K1, K2, and K3). As control, PBMCs were stained with a Pep Nb (Control-Nb) and a SIRPα-antibody (positive control). Data are presented as mean ± SD of three technical replicates.

Article Snippet: Unlabeled hSIRPα Nbs (1 nM to 100 nM) in combination with anti-VHH secondary Cy5 AffiniPure Goat Anti-Alpaca IgG (2.5 μg/mL; Jackson Immuno Research) were added and incubated for 1 h at 37°C.

Techniques: Binding Assay, Immunofluorescence, Staining, Labeling, Flow Cytometry, Derivative Assay, Control, Positive Control