Journal: eLife

Article Title: Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter

doi: 10.7554/elife.42025

Figure Lengend Snippet: Figure 4. Activation of microglia in the brain white matter of CD47 KO mice. (A) Immunofluorescence staining of coronal brain sections prepared from control (WT) or CD47 KO mice at 19 wks of age with antibodies to Iba1 (red) and CD11c (green). Merged images are shown. The boxed areas in the upper panels are shown at higher magnification in the lower panels. fi, fimbria. Scale bars: 100 mm (upper panels), 50 mm (lower panels). (B) Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.42025 19 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PE conjugated rat mAb to mouse CD172a (SIRPa) (clone P84) eBioscience (Cat# 12-1721-80) RRID:AB_11149864 FCM (1:100) Antibody PE conjugated rat mAbs to CD68 (clone FA-11) BioLegend (Cat# 137013) RRID:AB_10613469 FCM (1:100) Antibody PE conjugated rat mAb to mouse CD14 (clone Sa14-2) BioLegend (Cat# 123309) RRID:AB_940582 FCM (1:100) Antibody PE conjugated recombinant antibody (Ab) to Dectin-1 (REA154) Miltenyi Biotec (Cat# 130-102-987) RRID:AB_2651541 FCM (1:5) Antibody PE conjugated recombinant Ab to CD47 (REA170) Miltenyi Biotec (Cat# 130-103-108) RRID:AB_2659745 FCM (1:10) Antibody Rat mAb to myelin basic protein (MBP) (clone 12) Merck (Cat# MAB386) RRID:AB_94975 IHC (1:500) Antibody Rabbit pAb to Olig2 Immuno-Biological Laboratories (Gunma, Japan) (Cat# 18953) RRID:AB_1630817 IHC (1:400) Antibody Alexa Fluor 488 goat anti-rabbit IgG Molecular Probes (Cat# A11034) RRID:AB_2576217 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-rabbit IgG Jackson Immuno Research (Cat# 111-165-144) RRID:AB_2338006 IHC (1:400) Antibody Cy3-conjugated AffiniPure Goat anti-rat IgG Jackson Immuno Research (Cat# 112-165-167) RRID:AB_2338251 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-mouse IgG Jackson Immuno Research (Cat# 115-165-166) RRID:AB_2338692 IHC (1:400) Antibody Streptavidin, Alexa Fluor 488 conjugate Molecular Probes (Cat# S11223) RRID:AB_2336881 IHC (1:400) Commercial assay or kit Black-Gold II myelin staining kit Merck Cat# AG105 Commercial assay or kit Tyramide Signal Amplification (TSA) Biotin System kit Perkin Elmer Cat# NEL700A001KT Commercial assay or kit RNeasy Mini kit Qiagen Cat# 74106 Commercial assay or kit QuantiTect Reverse Transcription kit Qiagen Cat# 205313 Commercial assay or kit QuantiTect SYBR Green PCR kit Qiagen Cat# 204143 or 24163 Commercial assay or kit GeneChip 3’IVT Express Kit Affymetrix Cat# 901228 or 901229 Commercial assay or kit Ovation Pico WTA system V2 NuGEN Cat# 3302–12/ 60/–A01 Commercial assay or kit Encore Biotin Module NuGEN Cat# 4200–12/ 60/–A01 Chemical compound, drug Tamoxifen Toronto Research Chemicals Inc. Cat# T006000 Chemical compound, drug 4’,6-Diamidino-2phenylindole Nacalai Tesque (Kyoto, Japan) Cat# 11034–56 Continued on next page Sato-Hashimoto et al. eLife 2019;8:e42025.

Techniques: Activation Assay, Immunofluorescence, Staining, Control

Journal: eLife

Article Title: Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter

doi: 10.7554/elife.42025

Figure Lengend Snippet: Figure 5. Microarray transcriptome analyses of the white matter and the brain mononuclear cells of CD47 KO mice. (A,B) The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis with DAVID. Statistically significant (p-value <0.01) KEGG enrichment pathways of up- (A) or downregulated (B) genes in the white matter (optic nerve and optic tract) (upper panels) or in the brain mononuclear cells (lower panels) of CD47 KO mice. Enrichment score is expressed as –Log (p-value). ARVC, Arrhythmogenic right ventricular cardiomyopathy; HCM, Hypertrophic Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.42025 19 of 29 Continued Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody PE conjugated rat mAb to mouse CD172a (SIRPa) (clone P84) eBioscience (Cat# 12-1721-80) RRID:AB_11149864 FCM (1:100) Antibody PE conjugated rat mAbs to CD68 (clone FA-11) BioLegend (Cat# 137013) RRID:AB_10613469 FCM (1:100) Antibody PE conjugated rat mAb to mouse CD14 (clone Sa14-2) BioLegend (Cat# 123309) RRID:AB_940582 FCM (1:100) Antibody PE conjugated recombinant antibody (Ab) to Dectin-1 (REA154) Miltenyi Biotec (Cat# 130-102-987) RRID:AB_2651541 FCM (1:5) Antibody PE conjugated recombinant Ab to CD47 (REA170) Miltenyi Biotec (Cat# 130-103-108) RRID:AB_2659745 FCM (1:10) Antibody Rat mAb to myelin basic protein (MBP) (clone 12) Merck (Cat# MAB386) RRID:AB_94975 IHC (1:500) Antibody Rabbit pAb to Olig2 Immuno-Biological Laboratories (Gunma, Japan) (Cat# 18953) RRID:AB_1630817 IHC (1:400) Antibody Alexa Fluor 488 goat anti-rabbit IgG Molecular Probes (Cat# A11034) RRID:AB_2576217 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-rabbit IgG Jackson Immuno Research (Cat# 111-165-144) RRID:AB_2338006 IHC (1:400) Antibody Cy3-conjugated AffiniPure Goat anti-rat IgG Jackson Immuno Research (Cat# 112-165-167) RRID:AB_2338251 IHC (1:200) Antibody Cy3-conjugated AffiniPure Goat anti-mouse IgG Jackson Immuno Research (Cat# 115-165-166) RRID:AB_2338692 IHC (1:400) Antibody Streptavidin, Alexa Fluor 488 conjugate Molecular Probes (Cat# S11223) RRID:AB_2336881 IHC (1:400) Commercial assay or kit Black-Gold II myelin staining kit Merck Cat# AG105 Commercial assay or kit Tyramide Signal Amplification (TSA) Biotin System kit Perkin Elmer Cat# NEL700A001KT Commercial assay or kit RNeasy Mini kit Qiagen Cat# 74106 Commercial assay or kit QuantiTect Reverse Transcription kit Qiagen Cat# 205313 Commercial assay or kit QuantiTect SYBR Green PCR kit Qiagen Cat# 204143 or 24163 Commercial assay or kit GeneChip 3’IVT Express Kit Affymetrix Cat# 901228 or 901229 Commercial assay or kit Ovation Pico WTA system V2 NuGEN Cat# 3302–12/ 60/–A01 Commercial assay or kit Encore Biotin Module NuGEN Cat# 4200–12/ 60/–A01 Chemical compound, drug Tamoxifen Toronto Research Chemicals Inc. Cat# T006000 Chemical compound, drug 4’,6-Diamidino-2phenylindole Nacalai Tesque (Kyoto, Japan) Cat# 11034–56 Continued on next page Sato-Hashimoto et al. eLife 2019;8:e42025.

Techniques: Microarray

Journal: Frontiers in Immunology

Article Title: IL-27 Derived From Macrophages Facilitates IL-15 Production and T Cell Maintenance Following Allergic Hypersensitivity Responses

doi: 10.3389/fimmu.2021.713304

Figure Lengend Snippet: CD3, CD47, and CD172a expression in human ACD clusters. (A–C) Immunofluorescence staining of CD3 (green), CD47 (red), CD172a (purple), CD14 [green, a serial slide section with the staining of CD172a (purple)], and Hoechst (blue) in human donor-matched patch-test negative control and patch-test (+) ACD skin. White dashed lines mark the epidermal-dermal junction. Data are representative of 3 patient samples per tested condition. (A) Scale bars are 200 µm (left) and 100 µm (right). (B, C) Scale bars are 20 µm.

Article Snippet: Mouse IgG1 isotype control (MOPC-21) (Tonbo Biosciences), Goat IgG isotype control (R&D Systems), Sheep IgG isotype control (R&D Systems), Rabbit isotype control (Southern Biotech, Birmingham, AL), anti-human CD14 (61D3, Tonbo Biosciences), anti-human iNOS (polyclonal, Thermo Fisher Scientific), anti-human CD8 (MCD8, Santa Cruz Biotechnology, Dallas, TX), and IL27R (polyclonal, R&D Systems), anti-human IL-27 (polyclonal, R&D Systems), anti-human CD86 (IT2.2, Biolegend), anti-human CD3 (SP7, Abcam, Cambridge, England), anti-human CD47 (polyclonal, R&D Systems), anti-human SIRP alpha (CD172a) (OTI7B3, Origene), anti-human IL-15 (polyclonal, R&D systems), anti-human BCL2 (clone 100, BioLegend), anti-mouse CD3 (17A2, Tonbo Biosciences), and anti-mouse CD8 (YTS 105.18, Novus Biologicals, Littleton, CO) followed by reaction with Cy3, Alexa Fluor 555, Alexa Fluor 647, Alexa Fluor 488, or FITC-conjugated secondary antibodies (Thermo Fisher Scientific).

Techniques: Expressing, Immunofluorescence, Staining, Negative Control

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: SARS-CoV-2 infection is associated with increased CD47 levels. A) TF protein abundance in uninfected (control) and SARS-CoV-2-infected (virus) Caco-2 cells (data derived from . P-values were determined by two-sided Student’s t-test. B) CD47 and SARS-CoV-2 N protein levels and virus titres (genomic RNA determined by PCR) in SARS-CoV-2 strain FFM7 (MOI 1)-infected air-liquid interface cultures of primary human bronchial epithelial (HBE) cells and SARS-CoV-2 strain FFM7 (MOI 0.1)-infected Calu-3 cells. Uncropped blots are provided in Suppl. Figure 1. C) CD47 mRNA levels in post mortem samples from COVID-19 patients (data derived from ). P-values were determined by two-sided Student’s t-test.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Infection, Quantitative Proteomics, Control, Virus, Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 aging” (A) and “CD47 hypertension” (B). C) Overview figure of the data derived from the literature searches. Age-related increased CD47 levels may contribute to pathogenic conditions associated with severe COVID-19.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: CD47 as a potential biomarker for the early diagnosis of severe COVID-19

doi: 10.1101/2021.03.01.433404

Figure Lengend Snippet: Results of the PubMed ( https://pubmed.ncbi.nlm.nih.gov ) literature search for “CD47 diabetes” (A). B) Overview figure of the data derived from the literature search. Hyperglycaemia- and diabetes-induced increased CD47 levels may contribute to immune escape of SARS-CoV-2-infected cells.

Article Snippet: Detection occurred by using specific antibodies against CD47 (1:100 dilution, CD47 Antibody, anti-human, Biotin, REAfinityTM, # 130-101-343, Miltenyi Biotec), SARS-CoV-2 N (1:1000 dilution, SARS-CoV-2 Nucleocapsid Antibody, Rabbit MAb, #40143-R019, Sino Biological), and GAPDH (1:1000 dilution, Anti-G3PDH Human Polyclonal Antibody, #2275-PC-100, Trevigen).

Techniques: Derivative Assay, Infection

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 regulates mucosal wound healing in vivo. Utilizing a miniature video endoscope and biopsy scissors, 5–7 wounds were created in the dorsal aspect of the descending colon mucosa of anesthetized mice. a Digital measurement of wound surface area at 24 and 72 h post wounding revealed a striking impairment in wound closure in Cd47 −/− mice. Points represent the mean value within all wounds from individual mice. Data are representative of three independent experiments with five mice per group and are expressed as means ± SEM. *** p < 0.001; two-tailed Student’s t test. b In total, 10 µg of control antibody (IgG) or anti-CD47 antibody (miap301 or miap410) were injected into wound beds of wounds created 24 h previously in C57Bl/6 mice, resulting in substantial reduction of wound closure upon blockade of CD47. c Mice treated locally or systemically with anti-CD47 monoclonal antibodies miap301 or miap410 experienced less wound area reduction in comparison with IgG-treated controls. Points represent mean value within all wounds from an individual mouse. Data are representative of two independent experiments with five mice per group. Date are means ± SEM. *** p < 0.001; one-way ANOVA. Scale bars: 50 mm. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: In Vivo, Two Tailed Test, Control, Injection, Bioprocessing, Comparison

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: Loss of CD47 in IEC does not induce immune-mediated mucosal damage. a – c Naive Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice housed in pathogen-free conditions were analyzed for intestinal epithelial CD47 expression. Cd47 ERΔIEC mice were treated with tamoxifen to induce acute CD47 deletion in intestinal epithelial cells, and analyzed 2 weeks later. a Tissue sections from naive Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice were stained with anti-CD47 antibodies (green) with DAPI counterstain (blue). CD47 expression is absent in the epithelium but retained in the lamina propria and submucosa. Scale bars = 100 μm upper panels, 50 μm lower panels. b IECs were isolated from the terminal ileum of naive mice. Protein lysates were analyzed by SDS–PAGE and immunoblot for CD47. c IECs were isolated from Cd47 ERΔIEC mice treated with vehicle (corn oil) or tamoxifen, and analyzed as in b . Results are representative of three independent experiments with 3–5 mice per group. d Paraffin-embedded colon tissue sections from Cd47 ΔIEC mice were stained with Hematoxylin and Eosin counterstain for histological examination. Gross mucosal architecture is intact in the absence of epithelial CD47 expression. Scale bars = 50 μm upper panels, 100 μm lower panels. e Colon tissue digests from naive Cd47 ΔIEC mice were stained and analyzed by flow cytometry, showing no significant differences in major lamina propria immune cell populations. Points represent individual samples each containing two mice ( n = 4 mice per group). Data are means ± SEM and are representative of three independent experiments. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Expressing, Staining, Isolation, SDS Page, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: IEC-specific deletion of CD47 results in impaired mucosal healing. Utilizing a miniature video endoscope and biopsy scissors, 5–7 wounds were created in the dorsal aspect of the descending colon mucosa of anesthetized mice. Cd47 ERΔIEC mice were wounded 2 weeks after tamoxifen treatment. a Digital measurement of wound surface area at 24 and 72 h post wounding revealed a striking impairment in wound closure in Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice. Points represent mean value within all wounds from an individual mouse. Data are representative of two independent experiments with 5–6 mice per group. Data are means ± SEM. *** p < 0.001; one-way ANOVA. b Tissue sections taken from day 3 wounds were stained with the epithelial-specific marker E-Cadherin (green), plus DAPI counterstain (blue). Re-epithelialization of the wound is disorganized in the absence of CD47 ( Cd47 ΔIEC ) compared with control Cd47 f/f . c Tissue sections taken from day 3 wounds were stained with the epithelial-specific marker E-Cadherin (green), brush border protein Villin (magenta), and DAPI (blue). Insets of epithelial cells on top of the wound bed show polarized wound-associated epithelial cells (WAE) expressing Villin (arrows) in Cd47 f/f wounds while cells are not polarized in Cd47 ΔIEC wounds. Scale bars = 100 μm. d Ki67 staining of frozen sections of wounded colon mucosa 3 days post-wounding (red) revealed similar proliferation rates in crypt epithelial cells immediately adjacent to wounds in the absence of epithelial CD47. Sections were counterstained with E-Cadherin (green) and DAPI (blue). Scale bars = 50 μm. Points represent the average number of Ki67-positive cells for four crypts adjacent to wounds for each individual mouse. Data are means ± SEM and are representative of two independent experiments with 4–6 mice per group. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Staining, Marker, Control, Expressing

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: Loss of CD47 in IEC results in impaired recovery from DSS-induced colitis. Age- and sex-matched Cd47 ΔIEC and tamoxifen-treated Cd47 ERΔIEC mice were treated with three consecutive cycles of 2.5% DSS in drinking water for either 4 days ( Cd47 ΔIEC ) or 3 days ( Cd47 ERΔIEC ), followed by 5 days of water recovery. Cd47 ERΔIEC mice were treated with DSS 2 weeks after tamoxifen treatment. a – b Disease activity index scores are represented as an average of scores 0–4 for percent weight loss, stool consistency, and presence of blood in stools. Cyclical treatment of a Cd47 ΔIEC and b tamoxifen-treated Cd47 ERΔIEC mice with 2.5% DSS in drinking water, followed by a plain water recovery period, induced greater DAI scores in the absence of epithelial CD47. Data are representative of two independent experiments with 5–6 mice per group and are expressed as means ± SEM. a * p = 0.02, b * p = 0.036 by two-way ANOVA. c Histological scoring of hematoxylin and eosin (H&E)-stained tissue sections of colonic mucosa: percentage of injury/ulceration represents a ratio of the length of injured/ulcerated areas (≥ 50% crypt loss) relative to the entire colon length, as assessed in Swiss roll mounts of the entire colon. Results indicate greater damage in the absence of epithelial CD47. Points represent individual mice. Data are representative of two independent experiments with 5–6 mice per group and are expressed as means ± SEM. Significance determined by two-tailed Student’s t test. * p = 0.016, *** p = 0.001. d , e Representative H&E staining of colon tissue sections after three cycles of DSS/water revealed extensive crypt destruction in distal colon of Cd47 ΔIEC and Cd47 ERΔIEC mice. Large areas of ulcerated mucosa with granulocytic infiltrates were present in the mid colon in Cd47 ΔIEC and Cd47 ERΔIEC mice, in comparison with littermate controls. Scale bars = 100 μm upper panels, 300 μm lower panels. Results are representative of at least two independent experiments with 5–6 mice per group. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Activity Assay, Staining, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 is required for wound repair in cultures of primary epithelial monolayers. a Primary epithelial cell monolayers derived from CD47-expressing (CD47( + )) or CD47-deficient (CD47(−)) murine enteroids were scratch-wounded and monitored for closure. CD47(−) epithelial monolayers showed significant impairment in reduction of scratch-wound surface area at 24 h post scratch. Edges of scratch wounds are indicated by dashed lines. Scale bars = 50 μm. b Primary epithelial cell monolayers derived from human stem cell-derived colonoids were scratch-wounded and treated with 10 µg/ml of either IgG control antibody, function-blocking anti-CD47 antibody (clone B6H12), or non-blocking anti-CD47 antibody (clone 2D3), resulting in the inhibition of cell migration upon blockade of CD47. a – b Results are representative of three independent experiments with three replicates per treatment group. Data are means ± SEM. Significance determined by two-way ANOVA, ** p ≤ 0.01, *** p ≤ 0.001. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Derivative Assay, Expressing, Control, Blocking Assay, Inhibition, Migration

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 associates with β1 integrin and promotes focal adhesion formation. a In situ proximity ligation assay utilizing antibodies against CD47 and β1 integrin indicating close association between CD47 and β1 integrin in the colonic epithelium. Positive PLA signals are shown in green, beta-catenin in magenta and DAPI/nuclei in blue. Crypts are indicated by dashed lines. Arrowheads and asterisks (*) indicate positive PLA signals on IECs and immune cells in the lamina propria, respectively. Strong PLA signals are detected in IECs that are mainly concentrated at the base of the crypts in Cd47 f/f colon in contrast to very few PLA signals are observed in crypts IECs in Cd47 ΔIEC colons. Scale bars = 20 μm. The specificity of the PLA signals was evaluated in Supplementary figure demonstrating no PLA signals in Cd47 −/− colons. b Whole-cell lysates from freshly isolated intestinal epithelial cells from Cd47 f/f and Cd47 ΔIEC mice were subjected to SDS–PAGE and immunoblot for signaling molecules downstream of β1 integrin-dependent cell adhesion, revealing decreased β1 integrin protein expression and reduced phosphorylation of Src Y416 , FAK Y397/Y861 , and p130CAS Y410 in cells from Cd47 ΔIEC mice. Results are representative of three independent experiments. c Murine enteroid-derived primary epithelial cell monolayers were scratch-wounded and harvested at the indicated time points, then analyzed by SDS–PAGE and immunoblot. CD47-deficient epithelial cells show a reduction in phosphorylated Src Y416 , FAK Y397/Y861 , and p130CAS Y410 upon wounding, whereas maintaining reduced β1 integrin protein baseline expression. d Epithelial cells immediately adjacent to wounds from Cd47 f/f and Cd47 ΔIEC mice were imaged by confocal microscopy, showing disrupted basal co-staining for phosphorylated FAK Y861 and β1 integrin (apical surface indicated by dashed line). Arrows indicate reduced colocalization of phospho-FAK Y861 and β1 integrin in the absence of CD47 expression. Scale bars = 10 μm. Results are representative of three independent experiments with three mice per treatment group. e Lamellipodia of CD47(−) cells exhibited fewer phosphorylated FAK Y861 -positive focal adhesions in comparison with CD47-expressing cells (insets). Scale bars = 10 μm. Results are representative of three independent experiments with two independently derived enteroid culture lines. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: In Situ, Proximity Ligation Assay, Isolation, SDS Page, Western Blot, Expressing, Phospho-proteomics, Derivative Assay, Confocal Microscopy, Staining, Comparison

Journal: Nature Communications

Article Title: Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

doi: 10.1038/s41467-019-12968-y

Figure Lengend Snippet: CD47 regulates thrombospondin-1, TGF-β1, and collagen deposition after injury. a Whole cell lysates from freshly isolated intestinal epithelial cells from Cd47 f/f and Cd47 ΔIEC mice were subjected to SDS–PAGE and immunoblot for thrombospondin-1/TSP-1, TGF-β1, and phosphorylated SMAD2 and SMAD3. Results are representative of three independent experiments. b Representative Masson’s trichrome staining of wounds beds and chronic DSS-colitis colons from Cd47 f/f and Cd47 ΔIEC mice. Scale bars = 50 μm. Results representative of three independent experiments with 5–7 mice per group. Source data are provided as a Source Data file

Article Snippet: From R&D systems: Anti-mouse CD47 (AF1866; WB:1/2000, IF:1/100), Anti-mouse E-Cadherin (AF748; IF:1/100), Rat anti- mouse Integrin beta 1 (MAB2405; IF:1/100).

Techniques: Isolation, SDS Page, Western Blot, Staining

Journal: Journal of nanobiotechnology

Article Title: Thrombolytic therapy based on lyophilized platelet-derived nanocarriers for ischemic stroke.

doi: 10.1186/s12951-023-02206-5

Figure Lengend Snippet: Fig. 1 A Schematic representation of the preparation of rtPA-loaded CSM derived from platelets. B Representative mean hydrodynamic diameter of CSM and CSM@rtPA before and after lyophilization (CSM@rtPA/L) process measured by DLS. Data represent mean ± SEM (n = 3, independent samples). C Representative mean diameter of CSM and CSM@rtPA before and after lyophilization process (CSM@rtPA/L) measured by NTA. Data represent mean ± SEM (n = 3, independent samples). D Representative STEM-in SEM images of CSM@rtPA negatively stained with uranyl acetate. Scale bars: 200 nm. E Loading capacity of rtPA encapsulated in CSM samples. Data represent mean ± SEM (n = 3, independent samples). F Representative density plots and quantitative analysis of CSM and CSM@rtPA measured by FC. Scatter density plots of Green fluorescence signal (Green-B channel, rtPA channel) versus Red fluorescence signal (Red-R channel, CellMask DeepRed channel) for CSM, CSM@rtPA, and CSM@rtPA sample after labeling with CellMask Deep Red for lipid staining. Mean fluorescence intensities (MFI) for Green-B channel (D) and Red-R channel. Data represent mean ± SEM (n = 3, independent samples). G. Schematic representation of platelets and platelet-derived CSM surface proteins studied by APC-fluorescently labeled antibodies: anti-hCD47 Ab and anti-hCD42b/GPlba Ab. In vitro Ab binding to platelets and CSM@rtPA before and after lyophilization process. Representative MFI histogram of Red-R channel (APC signal) for platelets, CSM@rtPA and CSM@rtPA/L samples after incubation with APC-anti-CD47 and APC-anti-CD42b/GPIbα antibodies

Article Snippet: Then, fluorescently antibodies, human anti-CD47 APC conjugated Antibody (FAB4670A, R&D Systems) and human antiCD42b/GPlbα APC conjugated Antibody (FAB4067A, R&D systems) were incubated with platelets and with CSMs (10 μL of antibody stock solution /106 cells) and the samples were analyzed by flow cytometer.

Techniques: Derivative Assay, Lyophilization, Staining, Fluorescence, Labeling, In Vitro, Binding Assay, Incubation

Journal: Nature Communications

Article Title: Phagocytosis-initiated tumor hybrid cells acquire a c-Myc-mediated quasi-polarization state for immunoevasion and distant dissemination

doi: 10.1038/s41467-023-42303-5

Figure Lengend Snippet: a CD47 expression levels in benign prostatic hyperplasia, primary, and metastatic tumors analyzed from three published prostate cancer datasets – . b Biochemical recurrence (BCR)-free survival of the prostate cancer patients in the TCGA cohort. c Western blotting analysis of CD47 expression levels in three human prostate cancer cell lines. d A representative example of flow cytometry analysis of THCs in human primary macrophage and C4-2 cell co-culture model. Some CD86 + macrophages acquire Tag-it-Violet (TiV) dyes through engulfing cancer cells in co-culture. The cells maintained EGFP expression in this TiV + /CD86 + population were further identified as THCs. e Percentage of THCs in different co-culture models at different time points. f , g Percentage of THCs in 3-day co-cultures of U937 macrophages and CD47 overexpressed ( e ) or knockdown ( g ) cells. h – j Flow cytometry histograms and the corresponding violin plots showing BCL-2 expression in U937 macrophages and C4-2 ( h ), 22Rv1 ( i ), or DU145 ( j ) cells co-culture model. k Left: schematic diagram of proximity-ligation assay (PLA). Right: PLA signals of C4-2 cells with or without SIRPα stimulation. The dots represent mean values of total PLA counts per cell ( n = 7 images/group, 180 cells analyzed). l – n Effects of C4-2 cells unstimulated or stimulated by TNFα or TNFα combined with SIRPα. Capillary Western immunoassay (WES) showing BCL-2 and Lamin A/C expression ( l ), violin plots showing Annexin V expression ( m ), and bar graph showing cell viability ( n each dot represents the mean for each independent experiment). o Schematic diagram showing strong “don’t-eat-me” signals effectively inhibit phagocytosis. p Schematic diagram showing low “don’t-eat-me” signals allow macrophages to engulf cancer cells but partially increase the pro-survival function of cancer cells to escape from complete phagocytosis, subsequently resulting in THC formation. Data are the mean ± SD. P -values were determined using a two-sided unpaired t -test ( a , c , e – l , n ), a log-rank test ( b ), and a one-way ANOVA followed by Tukey test ( m ). Three independent experiments were carried out in ( c , e – g , l , n ). Source data for c , e – n are provided as a Source Data file.

Article Snippet: For CD47 anti-apoptosis function, cancer cells were treated with 20 ng/mL TNFα (Novus Biologicals, 210-TA/CF) or 20 ng/mL TNFα combined with 20 μg/mL SIRPα (R&D Systems, 9378-SA) for three days.

Techniques: Expressing, Western Blot, Flow Cytometry, Co-Culture Assay, Knockdown, Proximity Ligation Assay