Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Mesenchymal stem cell culture and characterization. (A) Phase contrast image of BM derived MSCs at Day 0 and Day 21(Bar = 100 μm). (B) CD13 expression in WT-MSCs by fluorescence immunostaining (Bar = 20 μm) and protein expression of CD13 in WT-MSC. (C) RT PCR analysis of stem cell expression profiles. (D) Flow cytometric analysis of MSCs. Cells were characteristically positive for CD29, CD49e and negative for CD19, CD31, and CD34. Unstained;WT-MSC; KO-MSC–. (E) Both WT-MSC and KO-MSC expressed transcription factor OCT3/4 (Bar = 20 μm). (F) Confluent MSCs were transferred to adipogenic and osteogenic medium for 3 weeks. Adipocytes were detected by oil red O staining and osteoblasts by alizarin red staining (Bar = 200 μm). (G) 1 × 10 5 cells were seeded on Matrigel coated 6-well plates and incubated for 12 h. cells isolated from CD13 KO mice are unable to form capillary networks and form fewer branches (Bar = 200 μm).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Stem Cell Culture, Derivative Assay, Expressing, Fluorescence, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Staining, Incubation, Isolation

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Lack of CD13 impairs MSC adhesion, proliferation, migration, and invasion. (A) Adhesion assay: Cells (1 × 10 4 ) were seeded in 96 well plates coated separately with fibronectin, Matrigel, or gelatin and allowed to adhere for 1 h at 37°C. After PBS wash, adherent cells were detected by MTT assay. n = 6, ** P < 0.01. (B,C) Proliferation assay: Cells (0.5 × 10 4 ) were seeded in 96 well plate and cell proliferation detected by MTT assay at the indicated time points. n = 6, * P < 0.05, ** P < 0.01. (D) Migration assay: 1 × 10 4 cells were seeded in FluoroBlok chambers. After 4 h. incubation the cells were stained with DAPI and counted. n = 4, ** P < 0.01. (E) Invasion assay: 1 × 10 5 cells were seeded on Matrigel (1:5 dilution) coated FluoroBlok chambers. After 6 h. incubation the cells were counted. n = 4, ** P < 0.01.

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Migration, Cell Adhesion Assay, MTT Assay, Proliferation Assay, Incubation, Staining, Invasion Assay

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Mesenchymal stem cell defects in CD13 KO mice are cell intrinsic. (A) phalloidin-stained MSC cells isolated from injured muscles of CD13 KO mice showed remarkable cytoskeletal disruption compared to cells from WT mice; Objective 40X (Bar = 100 μm). (B,C) Immunofluorescent detection of FAK phosphorylation at tyrosine residues 397 (B) and 925 (C) . Protein lysates of MSC were probed for phospho-FAK (Y397) and phospho-FAK (Y925) with β-actin as the loading control. CD13 KO MSCs expressed lower levels of phospho-FAK protein (Bar = 20 μm).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Staining, Isolation, Muscles, Disruption, Phospho-proteomics, Control

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: CD13 activation increases monolayer adhesion and FAK phosphorylation in human MSCs. (A) Human mesenchymal stem cells also express CD13 by immunofluorescence (green); Objective 63X (Bar = 20 μm) and immunoblot of human cell lysates. (B) Colorimetric quantification of adhesion of human MSCs treated with the CD13 activating mAb 452 to HUVEC monolayers. Data represents the mean ± s.e.m. n = 3 from two independent experiments ( ** P < 0.01). (C) CD13 crosslinking with activating mAb 452 temporally induces FAK tyrosine phosphorylation in human MSCs.

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Activation Assay, Phospho-proteomics, Immunofluorescence, Western Blot

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Lack of CD13 on exogenously administered MSCs impairs perfusion recovery in an in vivo hindlimb ischemia model. (A) Laser Doppler flow imaging of perfusion in mice at the indicated time points. Representative color-coded images of three groups (PBS, WT-MSC, and KO-MSC) of mice on day 0, 3, 7, 14, and 21 after surgery and cell transplantation assessed by laser Doppler imaging. Red indicates highest perfusion velocity, green intermediate, and blue, lowest velocity. (B) Cumulative results for PBS ( n = 7), WT-MSC ( n = 6), and KO-MSC ( n = 6) injected mice are shown graphically as ratios of blood flow in ischemic limb (I) to that in the non-ischemic limb (NI) at each time point. Functional assessment of ischemic muscles. Cumulative results are shown graphically as (C) the ambulatory impairment score; (D) ischemic tissue damage score as described in Methods. (E) Images of Non-ischemic and PBS, WT-MSC, and KO-MSC injected ischemic paw. Black nail indicates necrosis. # P < 0.05 compare to PBS and * P < 0.05 compare to KO-MSC; (score assessment criteria in Methods).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: In Vivo, Imaging, Transplantation Assay, Injection, Functional Assay, Muscles

Journal: Frontiers in Physiology

Article Title: CD13 promotes mesenchymal stem cell-mediated regeneration of ischemic muscle

doi: 10.3389/fphys.2013.00402

Figure Lengend Snippet: Muscle regeneration and capillary formation are impaired in mice injected with CD13 KO MSC following ischemic injury. (A) Hematoxylin and eosin (H&E) staining of gastrocnemius muscle regeneration was confirmed by the presence of multiple, centrally located myocyte nuclei, 20X objective; Bar = 100 μm. (B) Capillaries were visualized by immunofluorescent staining with CD31 (red) and nuclei with DAPI (blue); Objective, 40X objective; Bar = 50 μm. (C) The area of the injured tissue was compared among PBS, WT-MSC, and KO-MSC groups. A significant increase in muscle regeneration (average) was observed in WT-MSC group compared with PBS group at day 21. (D) The ratio of capillary density per fiber was measured in ischemic gastrocnemius muscles. Capillary density was significantly increased in WT-MSC compared with other groups, PBS and KO-MSC. All data were quantified by ImagePro Plus. Values are shown as mean ± s.e.m. ( * P < 0.05) (PBS n = 7; WT-MSC n = 6; KO-MSC n = 6).

Article Snippet: Equal amount of protein from each group were separated by SDS-PAGE and transferred on to PVDF membrane and incubated with respective primary antibodies; CD13 monoclonal antibody for mouse CD13 (SL-13, custom made by ProMab Biotechnologies, Inc. Richmond, CA); 452 for human CD13 (Dr. Meenhard Herlyn, Philadelphia, PA); pFAK 397, pFAK925, and tFAK (cell signaling); β-Actin (Sigma); followed by incubation with horseradish peroxidase-conjugated secondary antibodies.

Techniques: Injection, Staining, Muscles

Journal: Scientific Reports

Article Title: CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

doi: 10.1038/s41598-021-90271-x

Figure Lengend Snippet: Immunofluorescence analysis of persistent expression of fusion-regulatory proteins in CD13-deficient multinucleated OC. Expression of the fusion regulatory proteins dynamin ( a , d ), DCST1 ( b , e ) and CD9 ( c , f ) is maintained in CD13 KO multinucleated osteoclasts but not in WT cells (**). High levels of dynamin co-localize with actin and DCST1 in OCPs (*), imaged using Zeiss LSM 880 confocal fluorescence microscope and analyzed by Zeiss Zen 2.0 Pro blue edition software ( https://www.zeiss.com/content/dam/Microscopy/Downloads/Pdf/FAQs/zen2-blue-edition_installation-guide.pdf ). Scale bar; 10 µm. Data represents average of three independent experiments. N = 3/genotype. Dynamin, CD9, DCST1; green. Phalloidin; red. Magnification ×63 oil.

Article Snippet: Antibodies to DC-STAMP1 (DCST1; Biorbyt, orb2242, rabbit polyclonal Ab), Dynamin (Abcam, ab3457, rabbit polyclonal Ab), CD9 (Abcam, ab223052, rabbit polyclonal Ab), Phalloidin-TRITC (Sigma-Aldrich, P1951), CD13 (SL13, Millipore, MABC950, rat monoclonal Ab).

Techniques: Immunofluorescence, Expressing, Fluorescence, Microscopy, Software

Journal: Scientific Reports

Article Title: CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

doi: 10.1038/s41598-021-90271-x

Figure Lengend Snippet: Immunoblot analysis of sustained expression of fusion-regulatory proteins in CD13-deficient OC over time. Expression of the fusion-promoting proteins dynamin, DC-STAMP (DCST1) and CD9 is aberrantly sustained in CD13 KO but not in WT multinucleated osteoclasts ( a , b ) in presence of M-CSF and RANKL. A cropped image is presented, see Supplementary Fig. for full-length blots and Supplementary Fig. for cropped replicates. ( c , d ). CD13 expression is unaltered in BM-derived OCs in response to M-CSF and RANKL stimulation over time. Blots were imaged by ChemiDoc Imaging system version 3.0.1 ( https://www.bio-rad.com/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15 ) (Biorad). A cropped image is presented, see Supplementary Fig. for full-length blots and cropped replicates. Data represents average of two isolates. N = 3/genotype. **p < 0.01, *p < 0.05.

Article Snippet: Antibodies to DC-STAMP1 (DCST1; Biorbyt, orb2242, rabbit polyclonal Ab), Dynamin (Abcam, ab3457, rabbit polyclonal Ab), CD9 (Abcam, ab223052, rabbit polyclonal Ab), Phalloidin-TRITC (Sigma-Aldrich, P1951), CD13 (SL13, Millipore, MABC950, rat monoclonal Ab).

Techniques: Western Blot, Expressing, Derivative Assay, Imaging

Journal: Scientific Reports

Article Title: CD13 is a critical regulator of cell–cell fusion in osteoclastogenesis

doi: 10.1038/s41598-021-90271-x

Figure Lengend Snippet: Fusion-regulatory proteins, dynamin and DCST1 are regulated by a CD13-dependent post transcriptional mechanism. ( a ) Quantitative RT-PCR analysis of fusion regulatory transcripts normalized to GAPDH in flow sorted mouse BM cells stimulated with M-CSF and RANKL over indicated time. Expression of the genes regulating osteoclast fusion -dynamin 2 (DNM2), DCST1, CD9 and CD81 are highly induced upon M-CSF and RANKL over time but was equivalent between genotypes. All data was analyzed using CFX Manager version 3.1 (( https://www.bio-rad.com/en-us/sku/1845000-cfx-manager-software?ID=1845000 ) (Biorad). Data represents average of two independent experiments. N = 3/genotype. ( b – c ) Dynamin and DCST1 protein stability are enhanced in absence of CD13. Immunoblot analysis of dynamin and DCST1 of WT and CD13 KO BM-derived OC treated with cycloheximide (CHX) for indicated time. Blots were imaged by ChemiDoc Imaging system version 3.0.1 ( https://www.bio-rad.com/en-us/category/chemidoc-imaging-systems?ID=NINJ0Z15 ) (Biorad). A cropped image is presented, see Supplementary Fig. for full-length blots and cropped replicates. Data represents average of two isolates. N = 3/genotype. **p < 0.01, *p < 0.05.

Article Snippet: Antibodies to DC-STAMP1 (DCST1; Biorbyt, orb2242, rabbit polyclonal Ab), Dynamin (Abcam, ab3457, rabbit polyclonal Ab), CD9 (Abcam, ab223052, rabbit polyclonal Ab), Phalloidin-TRITC (Sigma-Aldrich, P1951), CD13 (SL13, Millipore, MABC950, rat monoclonal Ab).

Techniques: Quantitative RT-PCR, Expressing, Software, Western Blot, Derivative Assay, Imaging