Journal: Cell Reports

Article Title: Fibroblast-Derived STC-1 Modulates Tumor-Associated Macrophages and Lung Adenocarcinoma Development

doi: 10.1016/j.celrep.2020.107802

Figure Lengend Snippet:

Article Snippet: Rabbit anti-mouse CD204/MSR1 , Sino Biological , Cat# 50129-R004.

Techniques: Plasmid Preparation, Recombinant, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Software, Imaging, Mass Spectrometry

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: Msr1 contributes to VSV pathogenesis in mice (A) Msr1 mRNA expression in various tissues of wild-type (WT, C57BL/6) and Msr1 −/− mice. LOD, limit of detection; N = 3 mice per genotype. (B and C) The survival curves (B) and disease scores (C) of WT and Msr1 −/− mice challenged with 1×10 7 plaque-forming units (PFUs) per mouse of VSV by retro-orbital injection, N = 12 mice/genotype. p = 0.001 (log rank test), ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001, non-parametric Mann-Whitney U test. All the error bars: mean ± standard error of the mean (S.E.M.). (D) The WT mice with paralyzed hindlimbs at day 6 after infection. (E) Immunoblots of Msr1 protein expression in bone-marrow-derived macrophages (BMDMs) of WT, Msr1 −/− , and Msr1 +/− littermates. β-actin is a housekeeping control. (F and G) The survival curves (F) and disease scores (G) of Msr1 −/− and Msr1 +/− littermates challenged with 1 × 10 7 PFU/mouse of VSV by retro-orbital injection, N = 6 mice/genotype. For percent survival, p < 0.01 (log rank test); for disease score, ∗∗p < 0.01, non-parametric Mann-Whitney U test. All the error bars: mean ± S.E.M.

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Expressing, Injection, MANN-WHITNEY, Infection, Western Blot, Derivative Assay, Control

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: Msr1 is dispensable for systemic VSV dissemination and innate immune responses (A) The RNA viremia in the whole blood of WT and Msr1 −/− mice, Msr1 +/− , and Msr1 −/− littermates infected with VSV, assessed by quantitative RT-PCR, N = 6 mice/genotype in each experiment. The VSV RNA is expressed as a fold change over limit of detection (LOD)s. (B) The serum levels of type I IFN and inflammatory cytokines (IFN-α, IFN-γ, TNF-α, IL-10, CXCL-10, IL-1β, IL-6) in Msr1 +/− and Msr1 −/− littermates after VSV infection, assessed by multiplex ELISA, N = 6 mice/genotype. All the data are presented as mean ± S.E.M., and statistical significance is analyzed by non-parametric Mann-Whitney U tests.

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Infection, Quantitative RT-PCR, Multiplex Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: Msr1 is critical for VSV infection in the central nervous system (A–C) Quantitative RT-PCR analyses of VSV loads in (A) different tissues of Msr1 +/− and Msr1 −/− littermates on day 6 after infection (N = 3 mice/genotype), (B) the spinal cords, and (C) the brains of Msr1 +/− and Msr1 −/− littermates on day 6 after infection (N = 6 mice/genotype). The viral RNA load is expressed as fold changes over the limit of detection (LOD). (D) The VSV titers in the spinal cord and brain of Msr1 +/− and Msr1 −/− littermates on day 6 after infection, assessed by a plaque-forming assay (N = 6 mice/genotype). (E) Msr1 mRNA expression (expressed as a fold change over the mock spinal cord group) in different tissues of mock and VSV-infected Msr1 +/− mice on day 6 after infection, N = 3 mice/group. (F–H) (F) Correlation between Msr1 expression and VSV load in various tissues after VSV infection, N = 3 mice/group. The mRNA expression of class A and B scavenger receptors in (G) the spinal cord and (H) the brain of mock and VSV-infected Msr1 +/− and Msr1 −/− littermates on day 6 after virus inoculation, assessed by quantitative RT-PCR and expressed as a fold change over the Msr1 +/- -mock group, N = 3 mice/group. (I) Ldlr mRNA expression in the brain and spinal cord of mock and VSV-infected Msr1 +/− and Msr1 −/− littermates, expressed as a fold change over the brain of Msr1 +/- -mock group, N = 3 mice/group, ∗p < 0.05 vs brain, analyzed by non-parametric Mann-Whitney U test. Mock: no virus infection control. (J) Colocalization of MSR1 with microglia, astrocytes, and neurons in the mouse spinal cord by dual immunofluorescence staining. IBA1: a marker of microglia; GFAP: a marker of astrocyte; MAP2: a marker of neuron; DAPI: nuclei. The yellow regions in the overlay indicate colocalizations. Scale bar represents 10 μm. All the data are presented as mean ± S.E.M., and statistical significances are analyzed by non-parametric Mann-Whitney U test, ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Infection, Quantitative RT-PCR, Expressing, Virus, MANN-WHITNEY, Control, Immunofluorescence, Staining, Marker

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: MSR1 mediates cellular entry of VSV in primary mouse cells and human cells (A and B) VSV loads in primary neurons of Msr1 +/− and Msr1 −/− littermates at 36 h after inoculation (MOI = 1, multiplicity of infection = 3), as assessed by (A) quantitative RT-PCR (expressed as a fold change over the lowest viral load) and (B) plaque forming assay. Each dot represents an individual mouse. (C) Immunoblots of MSR1 protein expression in WT and Msr1 −/− bone-marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs). (D–F) (D) VSV-G protein expression in BMDMs, (E) virus titer in culture medium, and (F) VSV-GFP fluorescence under microscopy in BMDMs at 48 h after VSV-GFP infection (MOI = 5), N = 3 biological replicates. Objective: 20×, scale bar: 100 μm. (G) Quantitative RT-PCR analyses of VSV virions attached to BMDMs (4°C for 2 h) and entry into BMDMs (37°C for 30 min). The cells were washed 3 times with cold PBS before switching from 4°C to 37°C and after 37°C as well. MOI = 10, N = 3 biological replicates. (H) GFP expression in WT and MSR1 −/− trophoblasts, 24 h after transduction with GFP-encoding VSV-G-pseudotyped lentiviral vectors (VSV-G-LV-GFP) without polybrene. Scale bar: 50 μm. The statistic VSV-G-LV-GFP was acquired with a fluorescence microscope from 9 random fields of three biological replicates. (I) GFP expression in WT and LDLR −/− + RAP trophoblasts, 24 h after transduction with VSV-G-LV-GFP. The LDLR −/− trophoblasts were pre-treated with RAP (200 nM, 30 min, 37°C) followed by VSV-G-LV-GFP transduction. (J and K) The GFP fluorescence (J) and immunoblots of protein level (K) in VSV-GFP-infected WT and LDLR −/− +RAP trophoblasts with or without MSR1 overexpression; the WT and LDLR −/− trophoblasts were transfected with human MSR1 plasmid for 24 h, followed by VSV-GFP infection for 18 h (MOI = 0.5). The LDLR −/− trophoblasts were pre-treated with RAP (200 nM, 30 min, 37°C) before inoculation of VSV-GFP. Scale bar: 50 μm. (L) Immunofluorescence staining for different Myc-tagged MSR1 fragments in human trophoblasts, 24 h after transfection of plasmids. Myc proteins were stained by a mouse anti-Myc antibody, followed by an Alexa Fluor 488 (green)-conjugated secondary antibody. The cell nuclei were stained by DAPI (blue). Scale bar: 10 μm. β-actin is a housekeeping control. Mock: no virus infection control. All the data are presented as mean ± S.E.M., and statistical significances are analyzed by a standard two-tailed unpaired Student's t-test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing, Derivative Assay, Virus, Fluorescence, Microscopy, Transduction, Over Expression, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Control, Two Tailed Test

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: MSR1 facilitates cellular entry of VSV through its extracellular domains (A–C) WT and MSR1 −/− trophoblasts were examined at 12 h after VSV-GFP inoculation, (A) immunoblots of VSV-G protein level, (B) VSV-GFP fluorescence intensity, and (C) VSV titers in the cell culture medium, MOI = 0.5. N = 3 biological replicates. (D–F) WT and MSR1-overexpressed trophoblasts were examined at 12 h after VSV-GFP inoculation, (D) MSR1 protein expression, VSV-G protein level, (E) VSV-GFP fluorescence intensity, and (F) VSV titers in the culture medium, MOI = 0.5. N = 3 biological replicates. The VSV-GFP fluorescence intensity in (B) and (E) was acquired with a fluorescence microscope from 9 random fields of three biological replicates and quantified by ImageJ. Objective: 4x (top) and 20x, scale bar: 100 μm. (G) Immunoblots of VSV-G protein level in MSR1 −/− trophoblasts with epichromosomal complementation of an empty vector or FLAG-MSR1 expression plasmid at 12 h after VSV infection, MOI=0.5. (H) The protein expression of different FLAG-tagged MSR1 fragments at 24 h after transfection of plasmids in trophoblasts. IB: immunoblotting. Amino residues 1-50 (cytoplasmic N-tail), 1-80 (cytoplasmic N-tail plus transmembrane), 51-end (extracellular domains with transmembrane), and 1-end (full length). (I) VSV-G protein level and (J) VSV-GFP fluorescence intensity in trophoblasts overexpressing FLAG-MSR1 fragments at 12 h after VSV-GFP infection at an MOI = 0.5. The GFP fluorescence intensity was acquired with a fluorescence microscope from random regions of three biological replicates (N = 3). β-actin is a housekeeping control. Mock: no virus infection control. Scale bar: 100 μm. All the data are presented as mean ± S.E.M., and statistical significances are analyzed by a standard two-tailed unpaired Student's t-test, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, compare with WT or vector.

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Western Blot, Fluorescence, Cell Culture, Expressing, Microscopy, Plasmid Preparation, Infection, Transfection, Control, Virus, Two Tailed Test

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet: MSR1 interacts with VSV glycoprotein G via its extracellular domains (A–C) Binding of MSR1 to VSV virions. FLAG-MSR1 (human, 1-end) and its fragments (aa1-80, 51-end) were expressed in HEK293T cells and immunoprecipitated (IP) using anti-FLAG antibody-coated magnetic beads, which were then incubated with intact VSV-GFP virions (4°C for 2 h). The bound virions were eluted for (A) immunoblotting (IB) with a mouse monoclonal anti-FLAG and rabbit anti-VSV-G antibody (B and C) Re-infection in Vero cells for 12 h followed by detection of (B) VSV-GFP by fluorescence microscopy and (C) VSV-G protein level by immunoblotting. Mock: no virion control. (D) co-IP of FLAG-MSR1 and its fragments with VSV-G in HEK293T cells transfected with plasmids. WCE: whole-cell extraction. Arrows point to the faint protein bands.

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Binding Assay, Immunoprecipitation, Magnetic Beads, Incubation, Western Blot, Infection, Fluorescence, Microscopy, Control, Co-Immunoprecipitation Assay, Transfection, Extraction

Journal: iScience

Article Title: A critical role for MSR1 in vesicular stomatitis virus infection of the central nervous system

doi: 10.1016/j.isci.2021.102678

Figure Lengend Snippet:

Article Snippet: The mouse anti-human MSR1 (Cat# MAB2708) antibody was obtained from R&D Systems (Minneapolis, MN 55413, USA).

Techniques: Virus, Recombinant, Software

Journal: International immunology

Article Title: Role of scavenger receptors as damage-associated molecular pattern receptors in Toll-like receptor activation.

doi: 10.1093/intimm/dxx010

Figure Lengend Snippet: Fig. 2. Internalization of HMGB1 through class A scavenger receptors. (A) Confocal microscopy images of RAW264.7 cells, variant RAW264.7 cell line (#03) and #03 cells expressing Msr1 after incubation with DyLight 650-conjugated HMGB1 (F-HMGB1) for 1 h. DNA was labeled with Hoechst dye. Phase-contrast images are also shown. (B) The fluorescence intensities of internalized F-HMGB1 in the variant RAW264.7 cell line (#03) and #03 cells expressing Msr1, Marco and CD36 by lentiviral vectors. (C) The effect of M-BSA on the internalization of F-HMGB1 by RAW264.7 cells. RAW264.7 cells were treated with or without M-BSA or AGEs-BSA (300 μg ml−1) for 1 h before incubation with 50 nM F-HMGB1. Scale bar = 10 μm. (D) Left panel: CD11b+ cells from BMs of WT or Msr1/Marco-deficient mice were incubated with F-HMGB1 for 1 h in the presence or absence of M-BSA. Representative data from three independent experiments. Right panel: the mean fluorescence intensities (MFI) of internalized DyLight 488-conjugated GST (F-GST) or F-HMGB1 in GM-BMM cells from WT or Msr1/Marco-deficient mice. The fourth and fifth bars show the intensity of F-HMGB1 in the presence of M-BSA or AGEs-BSA (300 μg ml−1). **P < 0.01 versus F-HMGB1-treated GM-BMM cells.

Article Snippet: Cells were incubated overnight with a primary antibody pair of different species directed to HMGB1 (rabbit, ab18256; Abcam, Cambridge, UK), to MSR1 (rabbit, NBP1-00092; Novus, Littleton, CO, USA) and to TLR4 (mouse, 76B357.1; Thermo Fisher Scientific).

Techniques: Confocal Microscopy, Variant Assay, Expressing, Incubation, Labeling, Fluorescence

Journal: International immunology

Article Title: Role of scavenger receptors as damage-associated molecular pattern receptors in Toll-like receptor activation.

doi: 10.1093/intimm/dxx010

Figure Lengend Snippet: Fig. 4. HMGB1 activates TLR signaling through class A scavenger receptors. (A) Schematic representation of HMGB1 constructs. (B) The fluorescence intensities of internalized full-length or C-terminal tail-deleted HMGB1 molecules fused with GST. RAW264.7 cells were incubated with DyLight 488-conjugated recombinant proteins for 1 h in the presence or absence of 300 µg ml−1 M-BSA. (C) ELISA analysis of TNFα and IL-6 in supernatants of RAW264.7 cells stimulated with 10 µM GST, GST-HMGB11–215 or GST-HMGB11–181 for 15 h. (D) ELISA analysis of TNFα and IL-6 levels in the supernatants from 1 µg ml−1 HMGB1-stimulated parental RAW264.7 cells, variant #03 cells and #03 cells expressing Msr1 for 12 h. (E) Immunoblotting of p65 and ERK in GM-BMM cells treated with 0.3 µg ml−1 HMGB1 or 100 ng ml−1 LPS. M-BSA (300 µg ml−1) was added to the culture medium 1 h before HMGB1 or LPS stimulation. (F) Immunoblotting of p65, JNK, ERK, p38 and IκBα in RAW264.7 cells or variant RAW264.7 cell line (#03) treated with 0.3 µg ml−1 HMGB1 or 100 ng ml−1 LPS. *P < 0.05 versus GST-HMGB11–215-treated RAW264.7 cells (C). *P < 0.05, ***P < 0.001 versus variant #03 cells (D).

Article Snippet: Cells were incubated overnight with a primary antibody pair of different species directed to HMGB1 (rabbit, ab18256; Abcam, Cambridge, UK), to MSR1 (rabbit, NBP1-00092; Novus, Littleton, CO, USA) and to TLR4 (mouse, 76B357.1; Thermo Fisher Scientific).

Techniques: Construct, Fluorescence, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Variant Assay, Expressing, Western Blot

Journal: International immunology

Article Title: Role of scavenger receptors as damage-associated molecular pattern receptors in Toll-like receptor activation.

doi: 10.1093/intimm/dxx010

Figure Lengend Snippet: Fig. 5. Class A scavenger receptor is required for the interaction of HMGB1 with TLR4. (A) The mean fluorescence intensities of surface TLR4/ MD2 in GM-BMM cells after incubation with 2.5 μg ml−1 HMGB1 and/or 300 μg ml−1 M-BSA for the indicated periods. (B) GM-BMM cells were subjected to in situ PLA (see Methods), using primary antibody pairs of anti-HMGB1 and anti-TLR4 antibodies after 30 min treatment with 2.5 μg ml−1 HMGB1 in the presence or absence of 300 μg ml−1 M-BSA at 37°C. The percentage of cells with ≧ 5 red PLA spots was determined for 50–80 individual cells from three fields. (C) RAW264.7 cells or variant RAW264.7 cell line (#03 and #03-Msr1) were subjected to PLA, using primary antibody pairs of anti-MSR1 and anti-TLR4 antibodies. *P < 0.05 versus HMGB1-treated GM-BMM cells (B). **P < 0.01 versus variant #03 cells (C).

Article Snippet: Cells were incubated overnight with a primary antibody pair of different species directed to HMGB1 (rabbit, ab18256; Abcam, Cambridge, UK), to MSR1 (rabbit, NBP1-00092; Novus, Littleton, CO, USA) and to TLR4 (mouse, 76B357.1; Thermo Fisher Scientific).

Techniques: Fluorescence, Incubation, In Situ, Variant Assay

Journal: Journal of Neuro-Oncology

Article Title: Exploring the prognostic value of BRMS1 + microglia based on single-cell anoikis regulator patterns in the immunologic microenvironment of GBM

doi: 10.1007/s11060-024-04781-5

Figure Lengend Snippet: Cell communication between BRMS1 + microglia and malignant cells in spatial transcriptomics. ( A ) Spatial map showing 11 clusters identified by stRNA-seq. ( B ) Bubble chart presenting the expression of ARGs in different clusters. Red represents high expression, and blue represents low expression. ( C ) Spatial map showing the expression of BRMS1 in GBM. ( D , E ) Identification of cell types and proportions in each spot through deconvolution methods, with spatial maps showing the proportion and expression of different cell types, especially BRMS1 + and BRMS1- microglia. ( F , G ) Heatmaps and network diagrams calculate the communication intensity between different cell types based on the stlearn method

Article Snippet: The primary antibodies used in this experiment were: GAPDH (dilution 1:3000, Proteintech, Wuhan, product number 60004–1-Ig); BRMS1 (dilution 1:1000, Proteintech, Wuhan, product number 16096–1-AP); CD204 (dilution 1:1000, Abcam, Shanghai, product number ab123946); CD209 (dilution 1:800, Proteintech, Wuhan, product number 25404–1-AP); TGF-β1 (dilution 1:1000, Abcam, Shanghai, product number ab315254); SPP1 (dilution 1:1000, Proteintech, Wuhan, product number 80912–4-RR); CD44 (dilution 1:1000, Proteintech, Wuhan, product number 15675–1-AP); Phosphorylated AKT ( p -AKT, dilution 1:1000, Proteintech, Wuhan, product number 66444–1-Ig); PI3K (dilution 1:800, Abcam, Shanghai, product number ab283852); AKT (dilution 1:800, Abcam, Shanghai, product number ab283852); Cleaved-Caspase3 (dilution 1:800, Abcam, Shanghai, product number ab214430); Bax (dilution 1:800, Abcam, Shanghai, product number ab53154); Bcl-2 (dilution 1:1000, Abcam, Shanghai, product number ab194583).

Techniques: Expressing

Journal: Journal of Neuro-Oncology

Article Title: Exploring the prognostic value of BRMS1 + microglia based on single-cell anoikis regulator patterns in the immunologic microenvironment of GBM

doi: 10.1007/s11060-024-04781-5

Figure Lengend Snippet: BRMS1 promotes M2 polarization of microglia and activates the PI3K/AKT signaling pathway in GBM cells. ( A ) Western blot analysis was used to measure the protein levels of CD209, CD204, TGF-β1, BRMS1, and SPP1 in HMC3 cells after co-culture with U118/U251 cells. ( B ) RT-qPCR was performed to detect the RNA expression of BRMS1 in HMC3 cells from different groups. ( C ) ELISA was employed to assess the activity levels of CXCL2, IL-10, and TGF-β1 in the supernatant of HMC3 cell cultures. ( D ) Western blot analysis was conducted to evaluate the expression levels of CD44, PI3K, p-AKT, Bax, Bcl2, and cleaved-caspase 3 in U118/U251 cells following co-culture with HMC3 cells. Group I: Untreated HMC3 cells; Group II: HMC3 cells co-cultured with U118/U251 cells; Group III: HMC3 cells transfected with Flag-BRMS1 and co-cultured with U118/U251 cells. a: Untreated U118/U251 cells; b: U118/U251 cells co-cultured with HMC3 cells; c: U118/U251 cells co-cultured with Flag-BRMS1-transfected HMC3 cells. All data are represented as the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 for comparisons between Group II and Group I. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 for comparisons between Group III and Group II

Article Snippet: The primary antibodies used in this experiment were: GAPDH (dilution 1:3000, Proteintech, Wuhan, product number 60004–1-Ig); BRMS1 (dilution 1:1000, Proteintech, Wuhan, product number 16096–1-AP); CD204 (dilution 1:1000, Abcam, Shanghai, product number ab123946); CD209 (dilution 1:800, Proteintech, Wuhan, product number 25404–1-AP); TGF-β1 (dilution 1:1000, Abcam, Shanghai, product number ab315254); SPP1 (dilution 1:1000, Proteintech, Wuhan, product number 80912–4-RR); CD44 (dilution 1:1000, Proteintech, Wuhan, product number 15675–1-AP); Phosphorylated AKT ( p -AKT, dilution 1:1000, Proteintech, Wuhan, product number 66444–1-Ig); PI3K (dilution 1:800, Abcam, Shanghai, product number ab283852); AKT (dilution 1:800, Abcam, Shanghai, product number ab283852); Cleaved-Caspase3 (dilution 1:800, Abcam, Shanghai, product number ab214430); Bax (dilution 1:800, Abcam, Shanghai, product number ab53154); Bcl-2 (dilution 1:1000, Abcam, Shanghai, product number ab194583).

Techniques: Western Blot, Co-Culture Assay, Quantitative RT-PCR, RNA Expression, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Cell Culture, Transfection

Journal: iScience

Article Title: Targeting endometrial inflammation in intrauterine adhesion ameliorates endometrial fibrosis by priming MSCs to secrete C1INH

doi: 10.1016/j.isci.2023.107201

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-MSR1 , Cell Signaling Technology , Cat# 98215; RRID: AB_2798781.

Techniques: Staining, Recombinant, Cell Counting, Enzyme-linked Immunosorbent Assay, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse

doi: 10.3390/ijms25105118

Figure Lengend Snippet: Murine and human osteoblasts express genes involved in S1P production. ( A ) Clustered murine early osteoprogenitors, pre-osteoblasts (Pre-Ob), and mature osteoblasts (Mature Ob) were analysed for expression of S1P production genes. Dotplot of Sphk1 , Sphk2 , and Spns2 expression in each murine osteoblast subset, where circle size represents percentage of cells expressing the gene and colour indicates average expression value. ( B ) Clustered human MSCs and osteoblasts (Ob) were analysed for expression of S1P receptor genes. Dotplot of SPHK1 , SPHK2 , and SPNS2 expression in each human osteoblast subset. ( C – E ) Calvarial osteoblasts were cultured in control (Pre-Ob, blue, n = 8) or osteogenic media (Ob, black, n = 8) for 8 days and gene expression for ( C ) Sphk1 , ( D ) Sphk2 , and ( E ) Spns2 was normalised as 2 −ΔCt to β 2 m . ( F – H ) Human MSCs (blue, n = 3) and human primary osteoblasts (Ob, black, n = 11) were analysed for expression of ( F ) SPHK1 , ( G ) SPHK2 , and ( H ) SPNS2 and data were normalised as 2 −ΔCt to β 2 M . Data are mean ± SEM, with each point representing an individual experiment/donor. ( C – H ) * = p < 0.05; ** = p < 0.01 by unpaired t -test.

Article Snippet: Gene-specific Assay on Demand TaqMan FAM labelled primers ( β 2 M –Mm00437762, Acp5 –Mm00475698, Ctsk –Mm00484039, Msr1 –Mm00446214, S1pr1 –Mm00514644, S1pr3 –Mm00515669, S1pr4 –Mm00468695, Sphk1 –Mm00448841, Sphk2 –Mm00445021, Spns2 –Mm01249328, ACP5 –Hs00356261, CTSK–Hs00166156, MSR1 –Hs00234007, S1PR1 –Hs00173499, S1PR3 –Hs01015603, S1PR4 –Hs02330084, SPHK1 –Hs01116530, SPHK2 –Hs01016543, IL6 –Hs00174131, CXCL9 –Hs00171065, CXCL10 –Hs00171042) were diluted 1:10 in a master mix (both Applied Biosystems), before plating into 384 LightCycler plates along with 2.75 μL of diluted (1:5) cDNA in diH 2 O.

Techniques: Expressing, Cell Culture, Control, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: The Species Effect: Differential Sphingosine-1-Phosphate Responses in the Bone in Human Versus Mouse

doi: 10.3390/ijms25105118

Figure Lengend Snippet: Kinases involved in S1P production vary over osteoclast differentiation and species. ( A , B ) Analysis of publicly available single cell sequencing datasets GSE147174 (mouse) and GSE162454 (Human) and looking at S1P producer ( Sphk1 and Sphk2 ) and transporter (Spns2). ( A ) Dotplot of clustered murine monocytes, macrophages, pre-osteoclasts, and osteoclasts, showing expression of Sphk1 , Sphk2 , and Spns2 . ( B ) Dotplot of clustered human monocytes, macrophages, pre-osteoclasts, and osteoclasts, showing expression of SPHK1 , SPHK2 , and SPNS2 . ( C – F ) Primary mouse bone marrow-derived monocytes cultured (8 days) with or without M-CSF and RANKL. Undifferentiated monocytes (mono, blue) or osteoclasts (Oc, black), analysed for ( C ) Sphk1 (n = 3) and ( D ) Sphk2 (n = 3) gene expression and ( E ) mSPHK1 and ( F ) mSPHK2 protein expression. ( G – J ) Human monocytes derived from PBMCs cultured (8 days) with or without M-CSF and RANKL. Undifferentiated monocytes (blue) or osteoclasts (black), analysed for ( G ) SPHK1 (n = 5) and ( H ) SPHK2 (n = 5) gene expression and ( I ) hSPHK1 and ( J ) hSPHK2 protein expression. ( K , L ) Primary mouse bone marrow-derived osteoclasts ( K ) or primary human osteoclasts ( L ) were treated with or without S1P and analysed for ( K ) mSPHK2 and ( L ) hSPHK1 protein expression. Gene expression data are displayed as 2 −ΔCt normalised to β 2 m or β 2 M . Protein expression data normalised to human or mouse β-actin. Data are mean ± SEM. ** = p < 0.01 by unpaired t -test.

Article Snippet: Gene-specific Assay on Demand TaqMan FAM labelled primers ( β 2 M –Mm00437762, Acp5 –Mm00475698, Ctsk –Mm00484039, Msr1 –Mm00446214, S1pr1 –Mm00514644, S1pr3 –Mm00515669, S1pr4 –Mm00468695, Sphk1 –Mm00448841, Sphk2 –Mm00445021, Spns2 –Mm01249328, ACP5 –Hs00356261, CTSK–Hs00166156, MSR1 –Hs00234007, S1PR1 –Hs00173499, S1PR3 –Hs01015603, S1PR4 –Hs02330084, SPHK1 –Hs01116530, SPHK2 –Hs01016543, IL6 –Hs00174131, CXCL9 –Hs00171065, CXCL10 –Hs00171042) were diluted 1:10 in a master mix (both Applied Biosystems), before plating into 384 LightCycler plates along with 2.75 μL of diluted (1:5) cDNA in diH 2 O.

Techniques: Sequencing, Expressing, Derivative Assay, Cell Culture, Gene Expression