sheep anti cd38 polyclonal antibody  (R&D Systems)

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, mRNA levels of CD38 in human peripheral blood monocytes (PBMC)-derived macrophages treated with recombinant human IL-4 (M2) or LPS (M1) for 18 hours. Representative data from one of three patient samples. (n = 4 independent biological experiments) b, Immunofluorescence of human PBMC derived macrophages stimulated as described above using an anti-human CD38 antibody (Green) and nuclei with DAPI (Blue). Scale bars represents 10μm. Analyzed in PBMCs derived from one patient. c, NADase activity in human PBMC-derived macrophages treated with recombinant human IL-4 (M2) or LPS (M1) for 18 hours. Shown is the mean of two separate experiments from different donors with 2 replicates each. d, Schematic of the de novo NAD synthesis pathway. e, mRNA levels of de novo NAD synthesis pathway enzymes. f, Quantification of tryptophan metabolites measured by LC-MS in M0, M2 and M1 mouse BMDMs activated for 24 hours. ND=not detected. Data shows the mean ± SEM n=3 independent experiments except in A and B. Statistical significance indicated as *P<0.05, **P<0.01, and ***P<0.001; two-sided Student’s t-test.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Expressing, Derivative Assay, Recombinant, Immunofluorescence, Activity Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, Flow cytometry results comparing CD38 surface staining in naive (M0) WT and Cd38 KO BMDMs or BMDMs treated with IL-4 (M2) and LPS (M1) for 16 hours. b-c, NADase activity measured with non-cell permeable εNAD in intact M0, M2, and M1 WT and Cd38 KO BMDMs activated for 16 hours relative to cell number (B) and protein content (C). d, mRNA levels of Cd157 in M0 and M1 WT and Cd38 KO BMDMs treated for 16 hours. e, LC-MS quantification of NR in M0 and M1 WT and Cd38 KO BMDMs treated for 16 hours. f, mRNA levels of anti-oxidant genes in WT and Cd38 KO BMDMs treated with IL-4 (M2) and LPS (M1) for the indicated intervals. g, Western analysis of PARP activity (PARylation) and DNA damage (γH2AX) in WT and Cd38 KO BMDMs treated with IL-4 (M2) and LPS (M1) for the indicated intervals compared to WT MO macrophage treated with 1 mM H2O2 for 10 minutes. h, Western analysis of PARP activity (PARylation) and DNA damage (γH2AX) in WT and Cd38 KO BMDMs treated with IL-4 (M2) and LPS (M1) for 8 hours prior to treatment with 1 mM H2O2 for 10 minutes. Data show the mean ± SEM. (n= at least 3 independent experiments). Statistical significance indicated as *P<0.05, **P<0.01, and ***P<0.001; two-sided Student’s t-test. Unless noted with a bar, statistical comparisons are relative to the appropriate MO WT or Cd38 KO sample of the same genotype.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Activity Assay, Flow Cytometry, Staining, Liquid Chromatography with Mass Spectroscopy, Western Blot

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, Representative flow-cytometry plots comparing CD38 surface staining in naive (M0) WT and Cd38 KO BMDMs or BMDMs treated with IL-4 (M2) and LPS (M1) for 16 h. b, Western analysis of NADase enzymes in M0, M1 and M2 WT and Cd38 KO BMDMs for the indicated times. c, NADase rates measured in WT and Cd38 KO M0, M2 and M1 BMDMs activated for 16 h. d, Quantification of the NADase activity rate. e, LC-MS was used to quantify NAD and NAD-related metabolites in M0, M2 and M1 WT and Cd38 KO BMDMs activated for 16 h. f, NAD/NAM ratios from LC-MS data in e. g, Western analysis of CD38 and CD157 in M0 and M1 BMDMs from WT, Cd38 KO, Cd157 KO and Cd38/Cd157 DKO mice stimulated for 16 h. h, NADase rates measured in M0 and M1 BMDMs from WT, Cd38 KO, Cd157 KO and Cd38/Cd157 DKO mice stimulated for 16 h. Data are shown as the mean ± s.e.m. (n = 3 independent biological experiments, but n = 4 in d). *P < 0.05, **P < 0.01, ***P < 0.001; two-sided Student’s t-test. Unless noted with a bar, statistical comparisons are relative to the appropriate M0 WT or Cd38 KO sample of the same genotype.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Flow Cytometry, Staining, Western Blot, Activity Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, LC-MS was used to quantify NAD and NADP in visceral epididymal white adipose tissue (eWAT) from 6- and 25-month-old WT male mice. NAD and NADP concentrations are shown as pmol mg−1 of tissue (young n = 7 mice per group, old n = 10 mice per group). b, mRNA levels of senescence markers, inflammatory genes, macrophage marker Cd68 and M2 genes in eWAT from young (6 months old) and old (25 months old) WT male mice (young n = 7 mice per group, old n = 9 mice per group). c, Western analysis of the indicated proteins in eWAT from young (3 months old) and old (30 months old) WT male mice. Each lane represents one mouse (young n = 7 mice per group, old n = 4 mice per group). d, Quantification of CD38 protein levels in c, relative to actin levels, in eWAT from young (3 months old) and old (30 months old) WT mice (young n = 7 mice per group, old n = 4 mice per group). e, Quantification of total macrophages, CD38+ resident macrophages and CD38+ non-resident macrophages isolated from eWAT of WT male mice at the indicated ages (2 months n = 6 mice per group, 6 months n = 5 mice per group, 12 months n = 5 mice per group, 18 months n = 5 mice per group, 25+ months n = 7 mice per group). f, IF of the macrophage marker/ antigen F4/80 (magenta) and DAPI-stained nuclei (blue) in eWAT in young (4 months old) and old (26 months old) male and female WT mice. Scale bars, 10 μm. Images are representative of 4-5 mice per group. g, Results from trained neural network analysis of IF images, quantifying the mean number of macrophages (F4/80 colocalized to DAPI) per slide, and mean F4/80+ region size for old and young eWAT, graphed as mean cell count or region size (arbitrary units) per slide. Each column is based on two mice per group, except young/male which has three mice. Multiple images were taken from each mouse, with each image/slide represented as a dot; 9,11,9,13 images/slides for old/f, old/m, young/f, young/m, respectively. h, Analysis of CD38 and other macrophage markers in eWAT from single-cell transcriptome data using the Tabula Muris database (https://tabula-muris.ds.czbiohub.org). Data from individual mice are shown for in vivo experiments. t-SNE, t-distributed stochastic neighbour embedding. Data are shown as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; two-sided Student’s t-test except, for one-sided t-test in a.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Liquid Chromatography with Mass Spectroscopy, Marker, Western Blot, Isolation, Staining, Cell Counting, In Vivo

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, LC-MS quantification of NAD in the liver from young (4 months old) and old (26 months old) WT male and WT female mice (old n = 6 male and 6 female mice per group, young n = 6 male and 6 female mice per group), and Cd38 KO young (3 months old) and old (26 months old) male and female mice (old Cd38 KO n = 5 male and 5 female mice per group, young Cd38 KO n = 5 male and 5 female mice per group) NAD concentrations are shown as pmol per mg of tissue. b, IF images of the macrophage marker/antigen F4/80 (red), CD38 (green) and nuclei with DAPI (blue) in liver from WT young (4 months old) and WT old (26 months old) male mice, and young (3 months old) Cd38 KO male mice. Scale bars, 10 μm. Representative of 7-8 mice per group. c, Analysis of IF images above (a trained neural network to identify macrophage regions and colocalization of F4/80 and CD38, measured by Pearson correlation) for WT old and young liver slides (each dot represents 1 slide), n = 20 slides per mouse (young n = 8 mice per group, old n = 7 mice per group). d, t-SNE plot of annotated cell populations found in the livers of old and young male and female mice using single-cell transcriptome data from the Tabula Muris database (https://tabula-muris-senis.ds.czbiohub.org; used for d-i). e, t-SNE plot of CD38 expression in liver-cell populations in aged mice. f, t-SNE plot of liver cells annotated on the basis of mouse age. Note: Kupffer cells cluster by age. g, Dot plot of the indicated genes in Kupffer cells, with data sorted by mouse age. Logarithmic axes, base 10. h, Heatmap of the indicated genes in Kupffer cells, with data sorted by mouse age. Logarithmic axes, base 10. i, Percentage of total CD38+ Kupffer cells per total amount of cells per age group. j, SA-Bgal staining in liver sections from young (3 months old) and old (19 months old) WT male mice. Representative images of two out of four mice per group. k, mRNA levels of the p16+ senescent-cell reporter mRFP, senescent-cell markers (Cdkn2a (p16Ink4a) and Cdkn1a (p21Cip1)), inflammatory cytokines (I1b and Il6) and Cd38 in liver from 4- to 6-month-old p16-3MR male mice treated with PBS (vehicle); n = 7 mice per group, doxo (vehicle) n = 4 mice per group, and doxo (GCV) n = 4 mice per group. Data from individual mice are shown for in vivo experiments. Data are shown as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; two-sided Student’s t-test, except one-tailed t-test in a.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Liquid Chromatography with Mass Spectroscopy, Marker, Expressing, Staining, In Vivo, One-tailed Test

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, LC-MS quantification of NAD in eWAT from WT young male mice (6 month) n=7 mice/group, Cd38 KO young male mice (3 month) n=5 mice/group, WT old male mice (25 month) n=10 mice/group, and Cd38 KO old male mice (26 month) n=5 mice/group. NAD concentrations are shown as pmol/mg of tissue. (same WT data from Fig. 4a). b, mRNA levels of Il-1α and IL-10 in eWAT from 6 and 25 month-old WT male mice. (WT young male mice (6 month) n=7 mice/group, WT old male mice (25 month) n=9 mice/group) c, Western analysis of adipose tissue from young (3 Month) and old (19 month) WT male mice to detect PARP activity (PARylation) and DNA damage (γH2AX). Each lane represents one mouse (young n=4 mice/group, old n=4 mice/group). d, mRNA levels of Cd38 in visceral adipose tissue, the stromal vascular fraction, and adipocyte fraction from young (3 month) and old (19 month) WT male mice. (young n=4 mice/group, old n=4 mice/group). e, Flow cytometry gating strategy to identify CD45+ immune cells from the stromal vascular fraction of eWAT. Cells were first gated on forward scatter (FSCA) vs side scatter (SSCA) to discard cell debris and dead or dying cells. Next FSCH (height) vs FSCA (Area) was used to select single cells. Single cells were then gated for auto-fluorescent using the Empty(E) BV421 vs BV711 channels (not used as antibody fluorophores) to discard cells that showed auto-fluorescence in these channels. Then CD45+ cells were selected and analyzed for CD38 and macrophage markers. Flow cytometry gating strategy to identify resident and non-resident macrophages from the stromal vascular fraction of eWAT, showing representative flow plots and histograms for the indicated ages of mice. f, Flow cytometry quantification of CD38- (low) resident macrophages, CD38- non-resident macrophages, and CD38+ (high) non-macrophage immune cells from eWAT of WT male mice for the ages shown. (2 months n=6 mice/group, 6 months n=5 mice/group, 12 months n=5 mice/group, 18 months n=5 mice/group, 25+ months n=7 mice/group) For in vivo experiments, data from individual mice are shown. Statistical significance indicated as *P<0.05, **P<0.01, and ***P<0.001; two-sided Student’s t-test.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Liquid Chromatography with Mass Spectroscopy, Western Blot, Activity Assay, Flow Cytometry, Fluorescence, In Vivo

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, mRNA levels of Cd38 in WT BMDMs treated with the indicated TLR ligands for 16 h (n = 3 independent biological experiments). LMW, low molecular weight; HMW, high molecular weight. b, Treatment summary for WT male mice (2 months old) treated with 0.25 mg per kg (body weight) LPS or PBS for 4 weeks. c,d, Quantification of total and CD38+ macrophages in the spleen of 2-month-old WT male mice treated with LPS or PBS as above, and Cd38 KO mice, by flow cytometry. PBS n = 10 mice per group, LPS n = 9 mice per group. e, mRNA levels in eWAT from 3-month-old WT male mice i.p. injected with PBS or LPS for 4 weeks (PBS n = 4 mice per group, LPS n = 5 mice per group). f, LC-MS quantification of NAD and NADP in eWAT from 3-month-old WT male mice i.p. injected with PBS or LPS for 4 weeks. NAD and NADP concentrations are shown as pmol per mg of tissue (PBS n = 5 mice per group, LPS n = 5 mice per group). g, Cd38 mRNA levels in visceral adipose tissue (VAT)/eWAT and liver of WT male mice (4 months old) treated with 1 mg per kg (body weight) LPS or PBS over a 24-h period (PBS n = 3 mice per group, LPS n = 3 mice per group). h, Treatment summary for WT and Cd38 KO male mice (4 months old) treated with 1 mg per kg (body weight) LPS or PBS for 12 h. i, mRNA levels in eWAT from 4-month-old WT and Cd38 KO male mice injected with PBS or LPS for 12 h (n = 10 mice per group). j, LC-MS quantification of NAD and other metabolites in VAT/eWAT from 4-month-old WT and Cd38 KO male mice injected with PBS or LPS for 12 h (n = 10 mice per group). k, mRNA levels in livers from 4-month-old WT and Cd38 KO male mice injected with PBS or LPS for 12 h (n = 10 mice per group). l, LC-MS was used to quantify NAD and other metabolites in livers from 4-month-old WT and Cd38 KO male mice injected with PBS or LPS for 12 h (n = 10 mice per group). m, Diagram showing how excess NAM, derived from CD38, is methylated by NNMT and shunted away from the NAM-salvage pathway. Data from individual mice are shown for in vivo experiments. Data are shown as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001; two-sided Student’s t-test, except for one-tailed t-test in j and l.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Molecular Weight, Flow Cytometry, Injection, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Methylation, In Vivo, One-tailed Test

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, SA-Bgal staining in young (3 months old) and old (19 months old) eWAT from WT male mice. n = 4 mice per group. b, IF images of the macrophage marker F4/80 (magenta) and DAPI-stained nuclei (blue) in eWAT in young (4 months old) and old (26 months old) WT male mice. Scale bars, 10 μm. Representative of 4-5 mice/group. c, mRNA levels in eWAT from 6-month-old WT male mice, which were intraperitoneally (i.p.) injected with PBS or doxo, of senescence markers, inflammatory cytokines, macrophage markers Cd68 and Cd38 in total tissue and isolated macrophages (PBS n = 8 mice per group, doxo n = 7 mice per group). d, Flow-cytometry analysis and quantification of CD38+ macrophages isolated from 6-month-old WT male mice i.p. injected with PBS or doxo (PBS n = 8 mice per group, doxo n = 8 mice per group). e, Conditioned medium (CM) was isolated from non-senescent control mouse dermal fibroblasts (CTRL-MDF), doxo-treated senescent MDFs (sen(doxo)-MDF) or irradiated senescent MDFs (sen(IR)-MDF) at 10 d following treatment, and then was used to stimulate BMDMs for 24 h. f, mRNA levels of Cd38 and those encoding other NAD-consuming enzymes in BMDMs treated for 24 h with CM from CTRL-MDFs, sen(doxo)-MDFs or sen(IR)-MDFs. g, Results from flow cytometry of EdU+ BMDMs treated with sen(IR)-MDF CM or CM from CTRL-MDFs for 24 h. h, Representative bright-field microscopy image of BMDMs treated with CTRL-MDF CM or sen(IR)-MDF CM for 24 h. i, SA-Bgal staining in control (CTRL-PA) or irradiated senescent primary mouse preadipocytes (sen(IR)-PA). j, mRNA levels of the indicated genes in CTRL-PA or sen(IR)-PA. k, mRNA levels of Cd38 and other NAD-consuming enzymes in BMDMs treated with CM from CTRL-PA sen(IR)-PA for 24 h. l, Model showing that inflammatory cytokines (SASP) derived from senescent cells can promote macrophage expression of CD38. Data from individual mice are shown for in vivo experiments. Data are shown as the mean ± s.e.m. (n = at least 3 independent biological experiments). *P < 0.05, **P < 0.01, ***P < 0.001; two-sided Student’s t-test.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Staining, Marker, Injection, Isolation, Flow Cytometry, Irradiation, Microscopy, Derivative Assay, Expressing, In Vivo

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, mRNA levels of Il-1α, Cxcl1, and IL-10 in eWAT from 6 month-old WT male mice i.p. injected with Doxo or PBS. (PBS n=8 mice/group, Doxo n=7 mice/group) b, Quantification of CD38-low resident macrophages, and CD38-low non-resident macrophages from eWAT of 6 month-old WT male mice injected with Doxo or PBS. (PBS n=8 mice/group, Doxo n=8 mice/group). c, CD38 mRNA levels in WT and Cd38 KO BMDMs co-cultured (10:1) with non-senescent control mouse dermal fibroblasts (CTRL-MDF) or irradiated senescent MDF (Sen(IR)-MDF) for 24 hours. (n=4 independent biological experiments per condition) d, mRNA levels of Cd38 in WT BMDMs treated with the indicated DAMPs for 16 hours. (n=3 independent biological experiments per condition) e, mRNA levels of inflammatory genes in CTRL-MDF and Sen(IR)-MDF. (n=4 independent biological experiments per condition) f, mRNA levels of Cd38 in BMDMs treated with the indicated concentrations (ng/ml) of recombinant mouse cytokines for 24 hours. (n=3 independent biological experiments per condition) g, Heatmap of significantly upregulated proteins identified by mass spectrometry in conditioned media from CTRL-MDF and Sen(IR)-MDF. (n=4-6 independent biological experiments per condition). For in vivo experiments, data from individual mice are shown. Data show the mean ± SEM. (n= at least 3 independent experiments). Statistical significance indicated as *P<0.05, **P<0.01, and ***P<0.001; two-sided Student’s t-test.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Expressing, Injection, Cell Culture, Irradiation, Recombinant, Mass Spectrometry, In Vivo

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: a, Representative gating for the splenic leukocyte populations quantified in Fig. 7c, ​,dd and Extended Data Fig. 7a. Left six panels show gating for identification of B cells and different myeloid cells, as indicated, as well as gating for CD38-positive cells in all populations. Right six panels show gating for T cell subsets, as indicated. Red arrows indicate sequential gating, pointing from parent plots towards child plots. b, Quantification of immune cell populations and CD38+ immune cells in the spleen of 3 month-old WT male mice i.p. injected with PBS or LPS for 4 weeks, and analyzed by flow cytometry. (PBS n=10 mice/group, LPS n=9 mice/group) c, Western analysis of CD38, CD157, CD68, and NAMPT in eWAT of 3 month-old WT male mice injected with PBS or LPS for 4 weeks and Image J quantification of CD38 protein levels relative to NAMPT. Each lane represents one mouse (PBS n=4 mice/group, LPS n=5 mice/group) d, mRNA levels of NAD consuming enzymes in eWAT from 3 month-old WT male mice injected with PBS or LPS for 4 weeks. (PBS n=4 mice/group, LPS n=5 mice/group) e, mRNA levels of the indicated genes in whole eWAT from 4 month-old WT and Cd38 KO male mice injected with PBS or LPS for 12 hours. (n=10 mice/group) f, Western analysis of eWAT from 4 month-old WT and Cd38 KO male mice injected with PBS or LPS for 12 hours (n=3 mice/group). g, LC-MS quantification of NAD-related metabolites in eWAT from 4 month-old WT and Cd38 KO male mice IP injected with PBS or LPS for 12 hours. (n=10 mice/group) h, LC-MS quantification of NAD-related metabolites in liver from 4 month-old WT and Cd38 KO male mice IP injected with PBS or LPS for 12 hours. (n=10 mice/group) Data from individual mice are shown for in vivo experiments. Data show the mean ± SEM. Statistical significance indicated as *P<0.05, **P<0.01, and ***P<0.001; two-sided Student’s t-test except for 7 g and 7 h one-tailed t-test was used.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: Injection, Flow Cytometry, Western Blot, Liquid Chromatography with Mass Spectroscopy, In Vivo, One-tailed Test

Journal: Nature metabolism

Article Title: Senescent cells promote tissue NAD + decline during ageing via the activation of CD38 + macrophages

doi: 10.1038/s42255-020-00305-3

Figure Lengend Snippet: Cellular stressors such as DNA damage lead to an accumulation of senescent cells over time. Using in vivo and cell-culture models, we show that the accumulation of senescent cells and accompanying inflammatory cytokines of the SASP is necessary and sufficient to promote CD38 expression and proliferation in macrophages. In addition, increased intestinal permeability occurs during ageing, increasing serum levels of endotoxins and other PAMPS, which activate innate immune cells. Chronic and acute exposure to LPS promotes CD38 expression in macrophages in the eWAT and liver, and decreases tissue NAD levels. Collectively, the SASP and PAMPs promote an inflammatory state associated with increased expression of CD38 by tissue-resident M1-like macrophages, and hence enhanced NADase activity.

Article Snippet: The sheep anti-CD38 polyclonal antibody (R&D Systems, cat. no. AF4947, diluted 1:100) and rabbit anti-F4/80 monoclonal antibody (Cell Signaling, cat. no. 70076, diluted 1:100) were used for both single and costaining.

Techniques: In Vivo, Cell Culture, Expressing, Permeability, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: A cytosolic chaperone complex controls folding and degradation of type III CD38

doi: 10.1074/jbc.RA118.005844

Figure Lengend Snippet: Hip interacts with CD38 and regulates its protein levels. a and b, HEK-293T cells stably expressing EGFP-tagged sCD38 (a, sCD38-cells in short; note: sCD38 in the following studies were all EGFP-tagged and labeled as sCD38 for simplicity.) and mutCD38 (b, mutCD38-cells in short) were transfected with HA-Hip or vector (Vec) and the whole cell lysates were subjected to IP with anti-CD38 (left panel) or anti-HA (right panel), followed by blotting with anti-HA or anti-CD38. c, visualization of intracellular association between Hip (Hip-VC155) and soluble (VN173-sCD38, upper panel) or type III CD38 (mutCD38-VN173, lower panel) by BiFC technique. BiFC signal, yellow; immunostaining of CD38, red; Hip, purple; nuclear staining (DAPI), blue. The merged image showed superposition of BiFC, CD38, Hip, and DAPI signals. d, scatter-plot pixels of sCD38 and BiFC signals shown in the images in c. e, colocalization parameters including Pearson's correlation coefficient (upper chart) and Manders overlap coefficient (lower chart) were analyzed with JACoP. M1 is defined as the fraction of mutCD38 (blue dots) or sCD38 (red dots) overlapping BiFC signal; M2 is defined conversely. Mean ± S.D.; n = 21 (mutCD38); n = 29 (sCD38). f and g, sCD38- or mutCD38-cells were transfected with scramble or Hip-specific siRNAs. The whole lysates were blotted with anti-CD38 and anti-Hip, with anti-GAPDH as a loading control. Lower panel, one representative blot; upper panel, quantification of the protein levels in four independent experiments. The abundance of CD38 or Hip in one sample was firstly normalized with the corresponding GAPDH level to eliminate loading unevenness and further normalized with the expression from the scramble siRNA controls. *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001 by Student's t test (n = 4).

Article Snippet: Sheep polyclonal anti-CD38 antibody (for immunostaining in c ) was purchased from R&D Systems and rabbit anti-CD38 were made by Absea.

Techniques: Stable Transfection, Expressing, Labeling, Transfection, Plasmid Preparation, Immunostaining, Staining, Control

Journal: The Journal of Biological Chemistry

Article Title: A cytosolic chaperone complex controls folding and degradation of type III CD38

doi: 10.1074/jbc.RA118.005844

Figure Lengend Snippet: Cytoplasmic Hsp70s, Hsp90s, Hsp40s, chaperonins, and Hip were immunoprecipitated together with the soluble CD38. a, HEK-293T cells stably expressing FLAG-sCD38, or FLAG-EGFP as a negative control, were permeabilized by 100 μm digitonin and the cytosolic fractions were subjected to IP with anti-FLAG M2 Magnetic Beads, eluted by 3×FLAG peptide and analyzed by SDS-PAGE. b, the information of the chaperones identified by LC-MS/MS. Family: name of the protein family; Protein: reference name of protein documented in GenBank, NCBI; Accession: accession number in UniProt; Protein NM: molecular mass of the proteins; Coverage: percentage of the protein sequence that was covered by identified peptides; No. of peptides: number of peptide sequences unique to the identified proteins. c, Western blot analysis of both the immunoprecipitation (left panel) and input (right panel) by the antibodies labeled.

Article Snippet: Sheep polyclonal anti-CD38 antibody (for immunostaining in c ) was purchased from R&D Systems and rabbit anti-CD38 were made by Absea.

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Negative Control, Magnetic Beads, SDS Page, Liquid Chromatography with Mass Spectroscopy, Sequencing, Western Blot, Labeling

Journal: The Journal of Biological Chemistry

Article Title: A cytosolic chaperone complex controls folding and degradation of type III CD38

doi: 10.1074/jbc.RA118.005844

Figure Lengend Snippet: Chaperones regulate the folding and proteostasis of cytosolic soluble CD38. a, sCD38-cells were treated with a series of concentrations of VER, a specific inhibitor of Hsp70s for 24 h. The protein levels of CD38, together with a housekeeping protein GAPDH were assayed by Western blotting and the cellular cADPR contents were measured by cycling assay. The representative blots (lower panel) are shown under the chart quantifying the relative expression of CD38 in at least three separate experiments (middle panel); the corresponding cADPR results are shown in the upper panel. b, sCD38-cells were transfected with Hsc70-specific or scramble siRNAs and the proteins or mRNA were analyzed by primers specific for Hsc70 or antibodies against CD38 or GAPDH. c, sCD38-cells were treated with different concentrations of geldanamycin (GA), an Hsp90 inhibitor for 3 h. The relative expression levels of CD38 were analyzed and presented as panel a. d–g, sCD38-cells were transfected with siRNAs specific for Hsp90α/β (d), CCT8 (e), DNAJA1 (f), or DNAJA2 (g) or scramble siRNA. The knockdown efficiency was evaluated by quantifying the target genes by qRT-PCR or Western blotting, and the relative expression levels of CD38 were analyzed and presented as panel a. Mean ± S.D.; n = 3 or 4; Student's t test, *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001. All the protein or mRNA levels were normalized with the housekeeping genes such as GAPDH, tubulin, or β-actin and the relative levels were calculated by dividing the normalized levels by those from the control groups as described in Fig. 1.

Article Snippet: Sheep polyclonal anti-CD38 antibody (for immunostaining in c ) was purchased from R&D Systems and rabbit anti-CD38 were made by Absea.

Techniques: Western Blot, Expressing, Transfection, Knockdown, Quantitative RT-PCR, Control

Journal: The Journal of Biological Chemistry

Article Title: A cytosolic chaperone complex controls folding and degradation of type III CD38

doi: 10.1074/jbc.RA118.005844

Figure Lengend Snippet: Chaperones regulate the folding and proteostasis of the artificial type III CD38, mutCD38. a, lysates from mutCD38-cells, with WT HEK-293T cells as a negative control, were subjected to IP with anti-FLAG M2 Magnetic Beads, followed by Western blotting with antibodies labeled. b and e, mutCD38-cells were treated with a series of concentrations of VER for 24 h (b) or GA for 3 h (e) and the expression levels of mutCD38 were assayed and presented as Fig. 3, a and c. c and f–h, the effects of knocking down different chaperones to the expression of mutCD38 were assayed and presented by the same methods as Fig. 3, b, d, f, and g. d, mutCD38-cells were transfected with siRNAs against Hsc70 (KD-2) for 24 h and treated with 10 μg/ml cycloheximide for 15 or 30 min. The cells were harvested and analyzed by Western blotting (for CD38 and tubulin) or qRT-PCR (for Hsc70) and analyzed as described in Fig. 1. All the above experiments were repeated at least three times and statistical analysis were done by GraphPad. Mean ± S.D.; n = 3; Student's t test, *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001.

Article Snippet: Sheep polyclonal anti-CD38 antibody (for immunostaining in c ) was purchased from R&D Systems and rabbit anti-CD38 were made by Absea.

Techniques: Negative Control, Magnetic Beads, Western Blot, Labeling, Expressing, Transfection, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: A cytosolic chaperone complex controls folding and degradation of type III CD38

doi: 10.1074/jbc.RA118.005844

Figure Lengend Snippet: Chaperone-mediated lysosomal degradation of type III CD38. a, mutCD38-cells (left) or sCD38-cells (right) were treated with 1 μm Baf-A1 or 5 μm MG-132 for 6 h. The protein levels of CD38, together with a housekeeping protein tubulin or GAPDH were assayed by Western blotting. The relative expression levels of three independent experiments were analyzed and plotted by GraphPad as above. b, mutCD38-cells were cultured in zero-serum medium for 6, 12, or 24 h and the CD38 levels were analyzed as panel a. c, mutCD38-expressing cells were treated with 10 μm GA for 3 h, or zero-serum medium for 24 h and stained with anti-CD38 (purple), anti-Lamp1 (green), and DAPI (blue, nucleus). Scale bar: 10 μm. d, Pearson's correlation coefficient between CD38 and Lamp1 signals in the images from the same experiments shown in panel c by NIS-Elements AR analysis software. Signals from more than 20 cells were analyzed. e and g, HEK-293T cells stably expressing mutCD38 (e) or mutCD38-dC19 (g) were transfected with Lamp2A-specific siRNAs or scramble siRNA. The relative expression levels were analyzed as panel a. f, the cADPR levels and protein expression of mutCD38 (FL) and mutCD38-dC19–expressing cells were measured by cycling assay and Western blotting. h, mutCD38 (FL) and mutCD38-dC19–expressing cells were lysed and immunoprecipitated with anti-FLAG beads. The associated CD38, chaperones, and GAPDH were blotted with the specific antibodies. The relative amount of chaperones were calculated by normalization to the amount of mutCD38 or dC19 in the immunoprecipitates and the results were plotted in the right chart. All the above experiments were repeated at least three times and statistical analysis was done by GraphPad. Mean ± S.D.; n = 3; Student's t test, *, p <0.05; **, p <0.01; ***, p <0.001; ****, p <0.0001; ns, not significant.

Article Snippet: Sheep polyclonal anti-CD38 antibody (for immunostaining in c ) was purchased from R&D Systems and rabbit anti-CD38 were made by Absea.

Techniques: Western Blot, Expressing, Cell Culture, Staining, Software, Stable Transfection, Transfection, Immunoprecipitation