Journal: Stem Cell Research & Therapy

Article Title: Hyaluronan supports the limbal stem cell phenotype during ex vivo culture

doi: 10.1186/s13287-022-03084-8

Figure Lengend Snippet: Effects of HA on TKE2 cells cultured on differently coated dishes or in the presence of HA as a supplement to the media. TKE2 cells were cultured on differently coated dishes ( A ) or on uncoated dishes with HA, HA + anti-CD44 or 4MU + Hyalase supplemented to the media ( B ). The number of ∆Np63 positive cells and the total number of cells (DAPI) were quantified using QuPath and the percentage of ∆Np63 + cells calculated ( C ). *Represents p ≤ 0.05, scale bar represents 50 μm

Article Snippet: In order to verify the mechanism by which HA supports LESCs, anti-CD44 neutralizing antibody (R&D systems, Minneapolis, USA#AF6127) was added to the culture media in order to block the binding of HA to CD44.

Techniques: Cell Culture

Journal: Stem Cell Research & Therapy

Article Title: Hyaluronan supports the limbal stem cell phenotype during ex vivo culture

doi: 10.1186/s13287-022-03084-8

Figure Lengend Snippet: Effects of HA on the differentiation of TKE2 cells cultured on differently coated dishes or in the presence of HA as a supplement to the media. TKE2 cells were cultured on differently coated dishes ( A ) or on uncoated dishes with HA of different molecular weights, HA + anti-CD44 or 4MU + Hyalase supplemented to the media ( B ) under differentiating conditions. The number of ∆Np63 positive cells and the total number of cells (DAPI) were quantified using Qupath and the percentage of ∆Np63 + cells calculated for TKE cells maintained on differently coated dishes ( C ) and with HA, HA + anti-CD44 and 4MU + Hyalase supplemented to the media ( D ). *Represents p ≤ 0.05, scale bar represents 50 μm

Article Snippet: In order to verify the mechanism by which HA supports LESCs, anti-CD44 neutralizing antibody (R&D systems, Minneapolis, USA#AF6127) was added to the culture media in order to block the binding of HA to CD44.

Techniques: Cell Culture

Journal: Cell transplantation

Article Title: Characterization and Functionality of Proliferative Human Sertoli Cells

doi: 10.3727/096368910X536563

Figure Lengend Snippet: Antibodies Used in the Study

Article Snippet: Host Type * Application Dilution GATA-4 Santa Cruz Biotechnology Sc-9053 rabbit pAb IF, FACS 1:50 (IF) Sox9 Santa Cruz Biotechnology Sc-17341 goat pAb IF, FACS 1:50 (IF) FSHr ABD Serotec 4561-8019 sheep pAb IF 1:3000 (IF) CD13 Invitrogen MHCD1301 mouse mAb FACS 1:20 CD14 Invitrogen MHCD1401 mouse mAb FACS 1:20 CD29 Invitrogen CD2901 mouse mAb FACS 1:20 CD31 BD Biosciences 555446 mouse mAb FACS 1:5 CD34 BD Biosciences 348057 mouse mAb FACS 1:5 CD44 Invitrogen MHCD4404 mouse mAb FACS 1:20 CD45 Invitrogen MHCD4501 mouse mAb FACS 1:20 CD73 BD Biosciences 550257 mouse mAb FACS 1:5 CD90 BD Biosciences 555595 mouse mAb FACS 1:50 CD105 Invitrogen MHCD10504 mouse mAb FACS 1:20 CD166 BD Biosciences 559263 mouse mAb FACS 1:5 Gal-1 Rabinovich N/A rabbit pAb IB, IHC 1:3000 Open in a separate window * pAb, polyclonal; mAb, monoclonal. caption a8 Antibodies Used in the Study

Techniques:

Journal: Cell transplantation

Article Title: Characterization and Functionality of Proliferative Human Sertoli Cells

doi: 10.3727/096368910X536563

Figure Lengend Snippet: Flow cytometry shows more than 90% of cells at passage 3–4 express the Sertoli cell markers GATA-4 and Sox9, and less than 7% express CD34, a marker for hematopoietic and endothelial progenitor cells. Gray histograms showing antibodies staining of MM-HSE-3608 (A) and MM-HSE-2305 (B) cells for CD13, CD29, CD34, CD44, Sox9, CD73, CD90, CD105, CD166, and GATA-4 compared to that of isotype control antibodies (white histograms). The percentage of cells positively stained for each antigen is shown.

Article Snippet: Host Type * Application Dilution GATA-4 Santa Cruz Biotechnology Sc-9053 rabbit pAb IF, FACS 1:50 (IF) Sox9 Santa Cruz Biotechnology Sc-17341 goat pAb IF, FACS 1:50 (IF) FSHr ABD Serotec 4561-8019 sheep pAb IF 1:3000 (IF) CD13 Invitrogen MHCD1301 mouse mAb FACS 1:20 CD14 Invitrogen MHCD1401 mouse mAb FACS 1:20 CD29 Invitrogen CD2901 mouse mAb FACS 1:20 CD31 BD Biosciences 555446 mouse mAb FACS 1:5 CD34 BD Biosciences 348057 mouse mAb FACS 1:5 CD44 Invitrogen MHCD4404 mouse mAb FACS 1:20 CD45 Invitrogen MHCD4501 mouse mAb FACS 1:20 CD73 BD Biosciences 550257 mouse mAb FACS 1:5 CD90 BD Biosciences 555595 mouse mAb FACS 1:50 CD105 Invitrogen MHCD10504 mouse mAb FACS 1:20 CD166 BD Biosciences 559263 mouse mAb FACS 1:5 Gal-1 Rabinovich N/A rabbit pAb IB, IHC 1:3000 Open in a separate window * pAb, polyclonal; mAb, monoclonal. caption a8 Antibodies Used in the Study

Techniques: Flow Cytometry, Marker, Staining

Journal: The Journal of Reproduction and Development

Article Title: Hyaluronan improves neither the long-term storage nor the cryosurvival of liquid-stored CD44-bearing AI boar spermatozoa

doi: 10.1262/jrd.2017-141

Figure Lengend Snippet: Detection of the CD44 receptor by Western blotting (WB) in human and pig spermatozoa. In A, using a porcine-specific anti-CD44 monoclonal antibody (60224-1-Ig, Nordic BioSite, Proteintech), the CD-44 receptor was detected in pig and human spermatozoa at 85 kDa. Gel B depicts co-incubation of this monoclonal primary antibody with its specific blocking peptide (Ag7633, Nordic BioSite, Proteintech Europe, Manchester, UK), which eliminated the 85 kDa band of the full-sized CD-44 receptor in all cells/tissues. L1: ladder, L2: human, L3: non-capacitated boar spermatozoa, L4: capacitated boar spermatozoa, L5: boar seminal vesicle (somatic positive control).

Article Snippet: The insets in the top-right corner in each figure (marked A´–C´) depict ICC negative control images (where the antibody was neutralized by treatment with the specific blocking peptide CD44 fusion protein Ag7633 (Nordic BioSite, Proteintech Europe, Manchester, UK)).

Techniques: Western Blot, Incubation, Blocking Assay, Positive Control

Journal: The Journal of Reproduction and Development

Article Title: Hyaluronan improves neither the long-term storage nor the cryosurvival of liquid-stored CD44-bearing AI boar spermatozoa

doi: 10.1262/jrd.2017-141

Figure Lengend Snippet: Localization of the CD44 receptor on immunocytochemistry (ICC) analysis in human (A) and pig (B: uncapacitated, C: capacitated) spermatozoa using a monoclonal antibody (60224-1-Ig, Nordic BioSite, Proteintech Europe, Manchester, UK). The insets in the top-right corner in each figure (marked A´–C´) depict ICC negative control images (where the antibody was neutralized by treatment with the specific blocking peptide CD44 fusion protein Ag7633 (Nordic BioSite, Proteintech Europe, Manchester, UK)). Human spermatozoa (positive control) had clear CD44-immunostaining over the post-acrosome area of the sperm head (Fig. 2A). In pig spermatozoa (Fig. 2B; 2C), the CD44 receptor appeared consistently localized on the plasma membrane over the post-acrosomal region, neck, and midpiece. Confocal laser scanning microscopy, scale bar: 10 μm.

Article Snippet: The insets in the top-right corner in each figure (marked A´–C´) depict ICC negative control images (where the antibody was neutralized by treatment with the specific blocking peptide CD44 fusion protein Ag7633 (Nordic BioSite, Proteintech Europe, Manchester, UK)).

Techniques: Immunocytochemistry, Negative Control, Blocking Assay, Positive Control, Immunostaining, Confocal Laser Scanning Microscopy

Journal:

Article Title: A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells

doi:

Figure Lengend Snippet: Western blot analysis of KG1a CD44 with HECA-452 or anti-CD44 mAbs. SDS/PAGE (6% reducing gel) resolution of the 98-kDa gel fragment from the third round of SDS/PAGE purification immunoblotted with either HECA-452 (lane 1) or anti-CD44 mAbs A3D8 (lane 2) and Hermes-1 (lane 3).

Article Snippet: Anti-CD44 mouse mAb A3D8 was from Sigma.

Techniques: Western Blot, SDS Page, Purification

Journal:

Article Title: A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells

doi:

Figure Lengend Snippet: L-selectin ligand activity of immunoprecipitated CD44. KG1a CD44 immunoprecipitates (Hermes-1 mAb; lanes 3 and 6) resolved on a reducing 9% SDS/PAGE gel, transferred to PVDF membrane, and immunostained with either Hermes-1 or HECA-452. Immunoblots included total cell lysate (lanes 1 and 4) and rat IgG isotype control immunoprecipitate (lanes 2 and 5) groups. L-selectin-dependent lymphocyte rolling was observed in the parallel-plate flow chamber (2.3 dynes/cm2) only over the 98-kDa protein (lymphocyte-binding data presented in parentheses as the mean number of lymphocytes rolling per field at ×200 magnification from more than five different fields).

Article Snippet: Anti-CD44 mouse mAb A3D8 was from Sigma.

Techniques: Activity Assay, Immunoprecipitation, SDS Page, Western Blot, Binding Assay

Journal:

Article Title: A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells

doi:

Figure Lengend Snippet: Effect of N-glycosidase-F on L-selectin ligand activity of immunoprecipitated CD44 from KG1a cells and from AML (M5) blasts. Immunoprecipitated CD44 from KG1a cells (A) or from AML (M5) blasts (B) were untreated (−) or treated (+) with N-glycosidase-F, resolved on reduced 9% SDS/PAGE, and immunoblotted with HECA-452. L-selectin ligand activity of the 98-kDa band in each case was observed in the parallel-plate flow chamber (2.3 dynes/cm2), and lymphocyte-binding data are presented in parentheses as the mean number of lymphocytes rolling at ×200 magnification; five fields counted.

Article Snippet: Anti-CD44 mouse mAb A3D8 was from Sigma.

Techniques: Activity Assay, Immunoprecipitation, SDS Page, Binding Assay

Journal:

Article Title: A distinct glycoform of CD44 is an L-selectin ligand on human hematopoietic cells

doi:

Figure Lengend Snippet: Effect of chlorate treatment on sulfation and L-selectin ligand activity of KG1a CD44. (A) SDS/PAGE (6%, reduced) autoradiogram of immunoprecipitated CD44 from non-chlorate- and chlorate-treated KG1a cells radiolabeled with [35S]SO4. (B) HECA-452 immunoblots of immunoprecipitated CD44 from chlorate-treated (+) and non-chlorate-treated (−) KG1a cells. L-selectin ligand activity of the 98-kDa band observed in the parallel-plate flow chamber (2.3 dynes/cm2) was equivalent in sulfated and nonsulfated (chlorate-treated) CD44 (binding data presented in parentheses as the mean number of lymphocytes at ×200 magnification per field; five fields counted).

Article Snippet: Anti-CD44 mouse mAb A3D8 was from Sigma.

Techniques: Activity Assay, SDS Page, Immunoprecipitation, Western Blot, Binding Assay

Journal: Scientific Reports

Article Title: Melanocortin-4 receptor in macrophages attenuated angiotensin II-induced abdominal aortic aneurysm in mice

doi: 10.1038/s41598-023-46831-4

Figure Lengend Snippet: An osteopontin receptor CD44 mediates vascular vulnerability of MC4R TB/TB mice. ( A ) Leptin (100 nM)-induced Spp1 gene expression in primary vascular smooth muscle cells from WT male mice pretreated with or without the PI3K inhibitor LY294002 (LY, 10 nM; n = 4). ( B ) Representative pictures and ( C ) quantification of osteopontin (OPN) immunostaining in the aorta of WD-fed MC4R + / + and MC4R TB/TB mice (n = 6, scale bar is 100 μm). ( D ) Representative pictures of DAPI, α-smooth muscle cells, and merged images in the aorta of WD-fed MC4R TB/TB mice (scale bars represent 20 μm). ( E ) Changes in the sBP of 14–18-weeks-old MC4R TB/TB mice during Ang II (500 ng/kg/min) infusion concomitantly administered with anti-CD44 or control antibodies (Ab; n = 6–12). ( F ) Representative pictures of the aorta and ( G ) AAA incidence and ( H ) diameter after Ang II infusion into WD-fed MC4R + / + and MC4R TB/TB mice administered with anti-CD44 or control Abs (n = 6–12). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: In the experiment of α-MSH (M4135, Sigma-Aldrich, 1 mg/kg/day) or anti-CD44 neutralizing antibody (BE0039, BioXCell, 200 μg/day) treatment, they were injected intraperitoneally into ApoE KO or MC4R TB/TB mice for 4 weeks starting on the day the minipumps were implanted.

Techniques: Expressing, Immunostaining

Journal: PLoS ONE

Article Title: Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

doi: 10.1371/journal.pone.0058760

Figure Lengend Snippet: (A) Total RNA was extracted from the injured arteries at 21 days after vascular injury and analyzed for mRNA expression of CD44 by real-time RT-PCR. Data are expressed as mean ± SEM (n = 10 for each, * p <0.001). (B) After VSMCs were transfected with either CD44-siRNA or NC-siRNA, total RNA was extracted from the cells and analyzed for CD44 mRNA expression by real-time RT-PCR. Data are expressed as mean ± SEM (n = 8 for each, * p <0.001). (C–F) VSMCs were transfected with either NC- or CD44-targeted siRNA for 48 h, and their migration in response to LMW-HA or HMW-HA (1 mg/mL) was measured using a modified Boyden chamber transwell migration assay (6 h, C and D) and scratch-wound migration assay (48 h, E and F) Data are mean ± SEM (n = 10 for each, * p <0.001). (G) VSMCs were transfected with either NC- or CD44-targeted siRNA for 48 h and then stimulated with LMW-HA or HMW-HA for 60 min. Cell lysates were analyzed by Western blotting with antibodies against activated RhoA. The results are representative of 3 independent experiments.

Article Snippet: HMW-HA (>950 kDa), LMW-HA (15–40 kDa), and neutralizing anti-CD44 antibody were obtained from R&D Systems.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Migration, Modification, Transwell Migration Assay, Western Blot

Journal: PLoS ONE

Article Title: Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

doi: 10.1371/journal.pone.0058760

Figure Lengend Snippet: VSMCs were pretreated with either control IgG or neutralizing anti-CD44 antibody (5 μg/mL) for 6 h and then VSMC migration in response to LMW-HA or HMW-HA (1 mg/mL) was then measured using a modified Boyden chamber transwell migration assay (6 h, A and B) and scratch-wound migration assay (48 h, C and D). Data are mean ± SEM (n = 8 for each, * p <0.001). (E) VSMCs were pretreated with control IgG or neutralizing anti-CD44 antibody (5 μg/mL) for 6 h and then stimulated with LMW-HA or HMW-HA for 60 min. Cell lysates were analyzed by western blotting with antibodies against activated RhoA. The results are representative of 3 independent experiments.

Article Snippet: HMW-HA (>950 kDa), LMW-HA (15–40 kDa), and neutralizing anti-CD44 antibody were obtained from R&D Systems.

Techniques: Migration, Modification, Transwell Migration Assay, Western Blot

Journal: PLoS ONE

Article Title: Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

doi: 10.1371/journal.pone.0058760

Figure Lengend Snippet: (A) Primary VSMCs were pretreated with vehicle, U0126 (ERK inhibitor), SP600125 (JNK inhibitor), or SB203580 (p38 inhibitor) for 1 h, and the proliferation of VSMCs in response to LMW-HA or HMW-HA (1 mg/mL) was measured using a BrdU incorporation assay. Data are expressed as mean ± SEM (n = 10 for each, * p <0.001). (B) VSMCs were transfected with either NC- or CD44-targeted siRNA for 48 h and then stimulated with LMW-HA or HMW-HA for 60 min. Cell lysates were analyzed by Western blotting with antibodies against p-ERK1/2, p-JNK, p-p38, and β-actin. The results are representative of 3 independent experiments. (C) Effect of anti-CD44 antibody on ERK1/2 activation. VSMCs were pretreated with control IgG or neutralizing anti-CD44 antibody (5 μg/mL) for 6 h and then stimulated with LMW-HA or HMW-HA for 20 min. Cell lysates were analyzed by Western blotting with antibodies against p-ERK1/2 and β-actin. The results are representative of 3 independent experiments.

Article Snippet: HMW-HA (>950 kDa), LMW-HA (15–40 kDa), and neutralizing anti-CD44 antibody were obtained from R&D Systems.

Techniques: BrdU Incorporation Assay, Transfection, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

doi: 10.1371/journal.pone.0058760

Figure Lengend Snippet: (A and B) Primary VSMCs were stimulated with LMW-HA or HMW-HA for 24 h. The levels of IL-6 (A) and MCP-1 (B) in the supernatants were assessed. Data are expressed as mean ± SEM (n = 10 for each, * p <0.001). (C) VSMCs were stimulated with LMW-HA or HMW-HA for 24 h and stained with DHE. The results are representative of 3 independent experiments. (D) VSMCs were stimulated with LMW-HA or HMW-HA for 24 h. The levels of d-ROM in the supernatants were assessed. Data are expressed as mean ± SEM (n = 10 for each, * p <0.001). (E–G) VSMCs were transfected with either NC- or CD44-targeted siRNA for 48 h and then stimulated with LMW-HA or HMW-HA for 24 h. The levels of IL-6 (E), MCP-1 (F), and d-ROMs (G) in the supernatants were assessed. Data are expressed as mean ± SEM (n = 10 for each, * p <0.001).

Article Snippet: HMW-HA (>950 kDa), LMW-HA (15–40 kDa), and neutralizing anti-CD44 antibody were obtained from R&D Systems.

Techniques: Staining, Transfection