Journal: Cell Death and Differentiation

Article Title: Resolvin D1 promotes the targeting and clearance of necroptotic cells

doi: 10.1038/s41418-019-0370-1

Figure Lengend Snippet: Elevated levels of CD47 on necroptotic cells promotes RhoA-p-MLC signaling in macrophages and result in inefficient clearance of NCs. ACs or NCs were co-cultured with live macrophages in a 3:1 ratio for 1 hr at 37 °C, 5% CO2. a Representative 3D-rendered images from live-cell imaging of macrophages (gray) and AC or NCs (magenta), ingested ACs or NCs (cyan) at 0, 15, and 30 min. Images were captured on a Zeiss LSM 880 microscope and rendered using Imaris software. Scale bar is 10 μm. Results are mean ± sem of n = 4–8 separate cells per group, **p < 0.01, Two-way ANOVA. b CD47 was determined by flow cytometry on live macrophages (Veh), ACs, or NCs and data were analyzed with FlowJo software. Results are represented as fold change and are shown as mean ± sem. Experiments were carried out in triplicate and repeated four separate times, **p < 0.01, One-way ANOVA, Tukey’s multiple comparisons test. c Live-cell confocal imaging was carried out as in panel a. NCs were incubated with 10 μg/mL of IgG or anti-CD47 and then added to macrophages. Images were acquired for 33 min and data was analyzed with Imaris software. Results are mean ± sem, of n = 8 separate cells per group, **p < 0.01 Two-way ANOVA. d Representative maximum intensity projection (MIP) images are shown. Macrophages were transfected with LifeAct-RFP and co-cultured with PKH67 (green) ACs or NCs as above for 40 mins at 37 °C, 5% CO2. Top panel is a merged image with both macrophages (red) and dead cells (green). Bottom panels display only the macrophages (red). Arrows denote the phagocytic synapse. e Quantification of p-MLC in macrophages from experiments carried out as in d. Results are mean ± sem, of n = 6 separate cells per group, **p < 0.01 student’s t-test. f Live-cell confocal imaging was carried out as in c. Results are mean ± sem of n = 8 separate cells per group, **p < 0.01, Two-way ANOVA

Article Snippet: Cross sections of murine aortic root lesion were stained at 4 °C with anti-CD47 antibody (BioXCell as above), p-MLKL (Abcam, as above) p-SHP1 (Abcam, cat#: ab131500), or anti-rat F4/80 (BioRad, CI-A3-1).

Techniques: Cell Culture, Live Cell Imaging, Microscopy, Software, Flow Cytometry, Imaging, Incubation, Transfection

Journal: Cell Death and Differentiation

Article Title: Resolvin D1 promotes the targeting and clearance of necroptotic cells

doi: 10.1038/s41418-019-0370-1

Figure Lengend Snippet: Lesion area, body weight, blood glucose and cholesterol for IgG and anti-CD47 antibody treatment to WD-fed Ldlr −/− mice with established atherosclerosis

Article Snippet: Cross sections of murine aortic root lesion were stained at 4 °C with anti-CD47 antibody (BioXCell as above), p-MLKL (Abcam, as above) p-SHP1 (Abcam, cat#: ab131500), or anti-rat F4/80 (BioRad, CI-A3-1).

Techniques:

Journal: Cell Death and Differentiation

Article Title: Resolvin D1 promotes the targeting and clearance of necroptotic cells

doi: 10.1038/s41418-019-0370-1

Figure Lengend Snippet: Administration of IgG or anti-CD47 to WD-fed Ldlr−/− mice with established atherosclerosis decreases lesional necroptotic cells and increases SPM in plaques. a Aortic roots lesion sections from Ldlr−/− mice were immunostained with an anti-p-MLKL antibody and Hoechst and the percent of p-MLKL+ cells was quantified. b Aortic root lesions were stained with H&E and necrotic areas were quantified with Olympus DP2-BSW software. Necrotic core is abbreviated as NC. Baseline indicates mice that were sacrificed after 10 weeks of WD feeding. Results are mean ± sem, n = 5 for baseline, n = 6 for IgG, and n = 7 for anti-CD47 separate mice per group. *p < 0.05, One-way ANOVA, Tukey’s multiple comparisons test. c–e Aortas were collected and subjected to LC-MS/MS. c An SPM cluster that includes 15R-Lipoxin A4 (15R-LXA4), RvD1, RvD5, Maresin 1 (Mar1), 12-trans Mar1, and 10 S,17S-diHDHA was identified and the MRM chromatogram is shown. d Representative MS/MS spectra for 15R-LXA4, RvD1, Mar-1, and RvD5 are shown. e Quantification of the SPM cluster. Results are mean ± sem, n = 6 separate mice per group, *p < 0.05, student’s t-test

Article Snippet: Cross sections of murine aortic root lesion were stained at 4 °C with anti-CD47 antibody (BioXCell as above), p-MLKL (Abcam, as above) p-SHP1 (Abcam, cat#: ab131500), or anti-rat F4/80 (BioRad, CI-A3-1).

Techniques: Staining, Software, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Cell Death and Differentiation

Article Title: Resolvin D1 promotes the targeting and clearance of necroptotic cells

doi: 10.1038/s41418-019-0370-1

Figure Lengend Snippet: Administration of RvD1 to WD-fed Ldlr−/− mice with established atherosclerosis decreases lesional CD47 and necrosis. Ldlr−/− mice were fed a WD for 12 weeks, after which Veh or 100 ng/mouse of RvD1 (3x/week i.p.) was administered for 3 weeks. Aortic root lesions were immunostained with anti-CD47 (a) or anti-p-SHP1 (b) antibody and counterstained with Hoechst. The mean fluorescence intensity of CD47 was quantified. c Lesional necrosis was quantified with Olympus DP2-BSW software. All results are mean ± sem, n = 8 separate mice per group. *p < 0.05, Student’s t-test

Article Snippet: Cross sections of murine aortic root lesion were stained at 4 °C with anti-CD47 antibody (BioXCell as above), p-MLKL (Abcam, as above) p-SHP1 (Abcam, cat#: ab131500), or anti-rat F4/80 (BioRad, CI-A3-1).

Techniques: Fluorescence, Software

Journal: Cell Death and Differentiation

Article Title: Resolvin D1 promotes the targeting and clearance of necroptotic cells

doi: 10.1038/s41418-019-0370-1

Figure Lengend Snippet: CBC counts from IgG and anti-CD47 antibody treatment to WD-fed Ldlr −/− mice with established atherosclerosis

Article Snippet: Cross sections of murine aortic root lesion were stained at 4 °C with anti-CD47 antibody (BioXCell as above), p-MLKL (Abcam, as above) p-SHP1 (Abcam, cat#: ab131500), or anti-rat F4/80 (BioRad, CI-A3-1).

Techniques:

Journal: Cell Death and Differentiation

Article Title: Resolvin D1 promotes the targeting and clearance of necroptotic cells

doi: 10.1038/s41418-019-0370-1

Figure Lengend Snippet: Overall scheme. NCs have high levels of CD47 that are clustered on the cell surface. Macrophages nibble the NCs in regions devoid of CD47 in a RhoA dependent manner. The uptake of NC enhances the biosynthesis of SPM. SPM, like RvD1 then enhances the engulfment of NCs in a CDC42 dependent manner. Additionally, RvD1 stimulates the release of ER proteins like calreticulin to target and label the NCs for swift engulfment in a CDC42 dependent manner

Article Snippet: Cross sections of murine aortic root lesion were stained at 4 °C with anti-CD47 antibody (BioXCell as above), p-MLKL (Abcam, as above) p-SHP1 (Abcam, cat#: ab131500), or anti-rat F4/80 (BioRad, CI-A3-1).

Techniques:

Journal: Cancer cell

Article Title: Liver Cancer Initiation Requires p53 Inhibition by CD44-Enhanced Growth Factor Signaling

doi: 10.1016/j.ccell.2018.05.003

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibody details are provided in the . table ft1 table-wrap mode="anchored" t5 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-CD44pan (DF1485) Santa Cruz Biotechnology Cat# SC-7297; RRID: AB_627065 anti-CD44pan (IM7) ABDSerotec (Biorad) Cat# MCA4703; RRID: AB_2076194 anti-CD44v6 ABD Serotec (Biorad) Cat# MCA1967; RRID: AB_323213 anti-AFP R&D Systems Cat# AF5369; RRID: AB_2258018 anti-Ki67 Genetex Cat# GTX16667; RRID: AB_422351 anti-F4/80 (Clone: A3-1) Caltag/Thermo Fisher Cat# MA1-91124; RRID: AB_2277854 anti-Clvd.

Techniques: Recombinant, Blocking Assay, In Situ, TUNEL Assay, Plasmid Preparation, Amplification, Staining, Labeling, In Situ Hybridization, RNAscope 2.5 HD Assay, SYBR Green Assay, Negative Control, Software