Journal: Oncogenesis

Article Title: ΔNp63 regulates the expression of hyaluronic acid-related genes in breast cancer cells

doi: 10.1038/s41389-018-0073-3

Figure Lengend Snippet: a The indicated basal-type breast tumor cells transfected with scrambled (SCR) or p63 siRNA oligos (sip63) were analyzed for the expression of CD44 by qRT-PCR. We utilized the following CD44 oligos. CD44 total: forward 5′-CAACTCCATCTGTGCAGCAAA-3′; rev 5′-GTAACCTCCTGAAGTGCTGCTC-3′. CD44v6 forward 5′-AGTACAACGGAAGAAACAGCTA-3′; rev 5′-TGTCCCTGTTGTCGAATGG-3′. Bars represent the mean of four independent experiments ( n = 4 biological replicates) ± SD. No data were excluded from the analysis. * p -value < 0.05. b The indicated basal-type breast tumor cells treated as in a were analyzed by immunoblotting using the antibodies for the indicated proteins. Mouse monoclonal antibody anti-CD44 (8E2) (Cell Signaling Technology) or mouse monoclonal anti-HCAM (CD44) (DF1485) (Santa Cruz Biotechnology) were utilized. c HCC1937 and HCC1954 cells were transfected as in a and total protein lysates were immunoblotted utilizing antibodies for the indicated proteins. Rabbit polyclonal anti-EGF Receptor (Cell Signaling Technology) and rabbit monoclonal anti-Phospho-EGF Receptor (Tyr1068) (D7A5) (Cell Signaling Technology) were utilized; d HCC1954 cells were transfected with scrambled (SCR), or p63 siRNA (sip63) oligos for 48 h and then transfected cells were plated for the sphere-forming assay. Briefly, breast cancer cells were plated in low-attachment 24-well culture plates at a density of 1000 cells per milliliter, in DMEM supplemented with 5 μg/mL insulin, 0.5 μg/mL hydrocortisone, 2% (vol/vol) B27 (Invitrogen), 20 ng/mL EGF and bFGF (BD Bio-sciences), and 4 μg/mL heparin (Sigma). The medium was made semisolid by the addition of 1% (vol/vol) methylcellulose to prevent cell aggregation. Total protein lysates were immunoblotted, utilizing antibodies for the indicated proteins. Rabbit polyclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling Technology) and rabbit monoclonal p44/42 MAPK (Erk1/2) (Cell Signaling Technology, clone 137F5) were utilized. e Representative image of clonogenic assay (left panel) performed by treating HCC1954 cells (500 cell per well) with the indicated concentration of 4-methylumbelliferone (4-MU, Sigma-Aldrich) for 1 week. Growth curve (middle panel) was performed by treating 1 × 10 5 cells with the indicated concentration of 4-MU for the indicated days. Cells were counted at the indicated time points in quadruplicates ( n = 4 technical replicates). The mean ± SD at days 2 and 5 was calculated and plotted. The graph is representative of two independent experiments. * p -value < 0.05. In parallel, protein lysates were immunoblotted for the indicated proteins (right panel)

Article Snippet: Mouse monoclonal antibody anti-CD44 (8E2) (Cell Signaling Technology) or mouse monoclonal anti-HCAM (CD44) (DF1485) (Santa Cruz Biotechnology) were utilized. c HCC1937 and HCC1954 cells were transfected as in a and total protein lysates were immunoblotted utilizing antibodies for the indicated proteins.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Clonogenic Assay, Concentration Assay

Journal: Oncogenesis

Article Title: ΔNp63 regulates the expression of hyaluronic acid-related genes in breast cancer cells

doi: 10.1038/s41389-018-0073-3

Figure Lengend Snippet: a HCC1954 cells were transfected with scrambled (SCR), HAS3 (siHAS3), or CD44 siRNA (siCD44) oligos for 48 h and then the efficiency of HAS3 or CD44 silencing has been measured by qRT-PCR. siRNA oligos against CD44 mRNA was purchased by Qiagen (Flexitube siRNA SI03098123). Bars represent the mean of three independent experiments ( n = 3, biological replicates) ± SD. No data were excluded from the analysis. * p -value < 0.05. ** p -value < 0.01. b Representative images of the mammospheres derived from HCC1954 cells transfected as in a . c HCC1954 cells transfected as in a were plated for the sphere-forming assay. The sphere-forming efficiency (SFE) was calculated as the percentage of the number of spheres per plated cells. Values represent the mean of the number of spheres counted in 12 ( n = 12) different fields. The values are representative of three independent experiments ( n = 3 biological replicates). * p -value < 0.05. d HCC1954 cells were plated for the sphere-forming assay in the presence of the indicated concentration of 4-MU. SFE was calculated as in c . Values represent the mean of the number of spheres counted in 10 ( n = 10) different fields. The values are representative of three independent experiments ( n = 3 biological replicates). ** p -value < 0.01.

Article Snippet: Mouse monoclonal antibody anti-CD44 (8E2) (Cell Signaling Technology) or mouse monoclonal anti-HCAM (CD44) (DF1485) (Santa Cruz Biotechnology) were utilized. c HCC1937 and HCC1954 cells were transfected as in a and total protein lysates were immunoblotted utilizing antibodies for the indicated proteins.

Techniques: Transfection, Quantitative RT-PCR, Derivative Assay, Concentration Assay

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Distribution of CD44 + cells within intestinal epithelium. a , b Immunohistochemical (IHC) staining for Lgr5 + ISCs ( black dotted lines ) in vivo. c , d IHC staining for CD44 + cells ( black dotted lines ) in vivo. a , c Magnification ×400. Bars 50 μm. b , d Magnification ×1000. Bars 20 μm. e–n Immunocytochemical (ICC) staining for CD44 + cells in vitro. e , j Differential interference contrast (DIC) imaging. f , k Propidium iodide (PI) staining for nuclei. g , l Fluorescein isothiocyanate (FITC) for CD44 + cells ( white arrowheads crypt cells strongly positive for CD44). h , m Overlay of PI image and FITC image. i , n Overlay of FITC image and DIC image. e–i Magnification ×200. Bars 200 μm. j–n Magnification ×630. Bars 100 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Immunohistochemical staining, Immunohistochemistry, In Vivo, Staining, In Vitro, Imaging

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Identification of CD44 + cells. a–a’’’’’ Fluorescence-activated cell sorting (FACS) analysis for cellular phenotype. a Isotype control, IgG2a-phycoerythrin (PE). a’ CD31-PE. a’’ CD34-PE. a’’’ Isotype control, IgG2b-allophycocyanin (APC). a’’’’ CD44-APC ( lo low-positive for CD44, hi high-positive for CD44). a’’’’’ CD45-APC. b Development of CD44 + ISC in 3D-culture system. Numbers represent days. Top Magnification ×400. Bar 50 μm. Bottom Magnification ×200. Bars 100 μm. c–c’’ Colony-forming efficacies of CD44 - cells and CD44 + cells. c CD44 - cells in 3D-culture system for 14 days. c’ CD44 + cells in 3D-culture system for 14 days. c , c’ Magnification ×40. Bars 500 μm. c’’ Comparision of colony-forming efficacy per 100 sorted cells seeded in one well of a 96-well plate. CD44 - group in 48 wells; CD44 + group in 48 wells. Data represent means ± SD of 48 independent measurements ( n = 48). Bars SD values. Paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 + group versus CD44 - group). All experimental procedures were repeated twice. d–d’’’’ Transmission electron microscope imaging of CD44 + ISC differentiation. CD44 + ISC were cultured in the 3D-system for 6 days and formed a cystic structure. d Cystic structure of a single CD44 + ISC-derived organoid at 6 days. Boxed areas are shown at higher magnification in d’–d’’’’ ( Lu lumen). Magnification ×400. Bar 50 μm. d’ Absorptive cell ( Ab ). d’’ Endocrine cell ( En ). d’’’ Goblet cell ( Go ). d’’’’ Paneth cell ( Pa ). Black arrowhead in d’ indicates brush border. Black arrowheads in d’’–d’’’’ indicates granules. d’–d’’’’ Magnification ×1500. Bars 5 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Fluorescence, FACS, Control, Transmission Assay, Microscopy, Imaging, Cell Culture, Derivative Assay

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: CD44 + ISCs resemble CBC stem cells. a–a’’ Strategy for sorting CD44 - , CD44 low+ and CD44 hi+ subpopulations by using the FACS technique ( R1 determining the cell-zone, R2 determining viable cells, PI propidium iodide, APC allophycocyanin). b Semi-quantitative reverse transcription (RT) followed by the polymerase chain reaction (PCR) for ISC-related gene-expression in sorted cells. Fold expression values were normalized to the CD44 - group. Data represent means ± SD of six independent measurements ( n = 6). Bars indicate value of SD. The paired t -test was used for data analysis. * P ≤ 0.05 represents high significance (CD44 hi+ group versus CD44 low+ group); $ P ≤ 0.05 represents low significance (CD44 hi+ group versus CD44 low+ group); ns represents no statistic differences between the CD44 hi+ group and CD44 low+ group. P -values for Lgr5 , Bmi1 , Hopx , mTERT , Ascl2 , Smoc2 , Lrig1 , Rnf43 and Prominin-1 are respectively 0.016, 0.036, 0.010, 0.844, 0.007, 0.001, 0.041, 0.005 and 0.859. c , c’ Colony-forming efficacy of CD44 low+ and CD44 hi+ cells in 3D-culture system for 14 days. c CD44 low+ group. c’ CD44 hi+ group. Magnification ×40. Bars 500 μm

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Gene Expression, Expressing

Journal: Cell and Tissue Research

Article Title: Mensenchymal stem cells can delay radiation-induced crypt death: impact on intestinal CD44 + fragments

doi: 10.1007/s00441-015-2313-6

Figure Lengend Snippet: Epithelial homeostasis in CD44 + ISC-derived organoid. a , b TUNEL staining for apoptotic cells in normal epithelium. c , d IHC staining of Ki67 for proliferative cells within normal epithelium. a , c Magnification ×200. Bars 100 μm. b , d Magnification ×400. Bars 50 μm. e–j Epithelial homeostasis in vitro. e–g TUNEL staining for apoptotic cells in CD44 + ISC-derived organoid. e DAPI staining ( blue ) for nuclei. f dUTP-FITC ( green ) for apoptotic cells. g DAPI image merged with dUTP-FITC image. Magnification ×100. Bars 200 μm. h–j ICC staining for proliferative cells in CD44 + ISC-derived organoid. h DAPI staining for nuclei. i Ki67-FITC for proliferative cells. j DAPI image merged with Ki67-FITC image. Magnification ×200. Bar 100 μm. k Representation of an organoid

Article Snippet: During incubation for single cell releasing, rabbit anti-mouse CD44 primary antibody (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) was added to SRM at the ratio of 1:50 (w/v).

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, In Vitro

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: Colocalization of CD44HSPG and MMP-7 in cell lines. (A) (Top panel) Phorbolester-treated neuroblastoma SH-SY5Y cells display colocalization of MMP-7 and CD44HSPG on the cell surface and along neurite extensions (inset). (Bottom panel) CHO cells cotransfected with cDNAs encoding human CD44v3-10 and MMP-7 display colocalization of the two molecules in cell protrusions. (B) Recombinant purified MMP-7 binds weakly to Ltk− [L-M(TK−)] cells, but strongly to Ltk− cells transfected with CD44v3-10. Binding is abrogated by washing the transfectants with 0.2 mg/mL heparin.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Recombinant, Purification, Transfection, Binding Assay

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: Reconstitution of the CD44HSPG/MMP 7/HB-EGF/ErB4 complex in Namalwa cells. (A) Confocal microscopy of Namalwa CD44 transfectants incubated with anti-CD44 (red fluorescence) and anti-MMP-7 (green fluorescence) mAb. Namalwa transfectants expressing CD44v3-10 (top) and CD44H (bottom) are shown. (B) Coimmunoprecipitation of CD44v3 and MMP-7. Indicated Namalwa transfectants were incubated with anti-MMP-7 C-M7 antibody, washed extensively, and lysed. Lysates were incubated with protein A sepharose beads and the immunoprecipitates were subjected to SDS-PAGE, transferred onto Hybond-C nitrocellulose membranes and immunoblotted with anti-CD44 mAb. (C) Transferrin zymogram of heparin eluates from anti-CD44 antibody immunoprecipitates of Namalwa CD44v3.8-10 and CD44v3-10 transfectant lysates (left). Transferrin zymogram of chondroitin lyase (Ch)− and heparanase (Hep)-treated anti-CD44 mAb immunoprecipitates from CD44v3-10 and CD44v7-10 Namalwa transfectants (right). C1 and C2 are control lanes containing protein A beads only (C1) and heparinase, chondroitinase, and digestion buffer in the absence of substrate (C2). MMP-3 (M3) and pro- and active-recombinant MMP-7 (M7) are used as marker controls. Migration of pro- and active MMP-7 are indicated on the left along with the molecular weight markers.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Confocal Microscopy, Incubation, Fluorescence, Expressing, SDS Page, Transfection, Recombinant, Marker, Migration, Molecular Weight

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: HB-EGF association with and processing by MMP 7. (A) (Top panel) Localization of HB-EGF on the surface of CD44v3 isoform expressing Namalwa cells, as assessed by neutralizing anti-HB-EGF antibody reactivity. (Bottom panel) Colocalization of proHB-EGF (C-18 antibody) and MMP-7 (RM7-C antibody) in Namalwa cells transfected with CD44v3-10, as assessed by confocal microscopy (magnification, 63×). (B) Coimmunoprecipitation of HB-EGF and MMP-7. MMP-7 was immunoprecipitated from Namalwa cells expressing the indicated CD44 isoforms with the indicated anti-MMP-7 antibody. The immunoprecipitates were subjected to SDS-PAGE, transferred onto nylon filters, and immunoblotted with the indicated anti-HB-EGF mAb. Lanes indicated pep C-18 and Mock C, respectively denote immunoprecipitates in the presence of immunogenic peptide and immunoprecipitates derived from vector only transfectants. (C) Proteolytic cleavage of pro-HB-EGF by MMP-7. Namalwa transfectant-derived anti-CD44 mAb immunoprecipitates were washed with a buffer containing EDTA to remove the coimmunoprecipitated MMP-7 and any other possibly associated MMP activity. The immunoprecipitates were then incubated with recombinant active MMP-7 in the presence or absence of the synthetic inhibitor SC44463 and subjected to SDS-PAGE, transfer to nitrocellulose membranes, and immunoblotting with a combination of neutralizing and cytoplasmic domain anti-HB-EGF mAb. Molecular weight markers are indicated.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Expressing, Transfection, Confocal Microscopy, Immunoprecipitation, SDS Page, Derivative Assay, Plasmid Preparation, Activity Assay, Incubation, Recombinant, Western Blot, Molecular Weight

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: . ErbB4 complexes with CD44 but is phosphorylated preferentially when associated with CD44v3. (A) Confocal microscopy shows colocalization of ErbB4 (red fluorescence) and CD44 (green fluorescence) in Namalwa CD44 transfectants. (B) (Top panel) Western blot analysis of anti-CD44 mAb immunoprecipitates from Namalwa transfectant lysates using anti-phosphotyrosine mAb pY20. (Bottom panel) Western blot analysis of anti-CD44 mAb immunoprecipitates in B immunoblotted with anti-c-ErbB4 mAb HFR-1. (C) HB-EGF neutralizing Ab (100 ng/mL), proMMP-7E219Q mutant (300 ng/mL), TIMP-3 (100 ng/mL), and MMP inhibitors SC44463 and BB94 (50 ng/mL) can reduce the level of tyrosine phosphorylated ErbB-4 in CD44V3-10 transfected Namalwa cells. (Top panel) Anti-tyrosine Ab pY20 blot. (Bottom panel) Anti-C-erbB-4 Ab-4 (HFR-1) blot. (D) Pro-MMP-7E219Q binds to the surface of Namalwa CD44HSPG and promotes apoptosis in serum-free medium. Namalwa transfectants expressing CD44v3-10 were incubated in serum free medium in the presence or absence of 100 ng/mL of recombinant proMMP-7E219Q alone or proMMP-7E219Q and recombinant active HB-EGF (50 ng/mL) as indicated. The cells were then tested for cell surface-bound MMP 7E219Q, using rat N-terminal propeptide MMP-7-specific antibody RM7-N (red fluorescence), and apoptosis, as assessed by TUNEL staining (green fluorescence). The RM7-N antibody did not react with CD44v3-10 Namalwa transfectants in the absence of exogenous rat proMMP 7E219Q (left). RM7-N stained CD44v3-10 Namalwa transfectants incubated with pro-MMP 7E219Q, but not CD44 transfectants lacking HSPG (data not shown). The majority of MMP-7E219Q bound cells tested TUNEL positive. Recombinant active HB-EGF (50 ng/mL) bound to the surface of transfectants preincubated with proMMP 7E219Q, as assessed by staining with neutralizing anti-HB-EGF antibody (right) and could rescue the majority of the cells from MMP 7E219Q binding-induced apoptosis.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Confocal Microscopy, Fluorescence, Western Blot, Transfection, Mutagenesis, Expressing, Incubation, Recombinant, TUNEL Assay, Staining, Binding Assay

Journal:

Article Title: CD44 anchors the assembly of matrilysin/MMP-7 with heparin-binding epidermal growth factor precursor and ErbB4 and regulates female reproductive organ remodeling

doi: 10.1101/gad.925702

Figure Lengend Snippet: CD44 regulates postpartum uterus remodeling. (A). Gross anatomy (a) and histology (b–g) of 36 h postpartum uteri from wild-type (+/+) and CD44 deficient (−/−) DBA/l mice. Note the comparable diameter of the cervix (a). Low power (8×) magnification of uterine cross-sections shows a preserved endometrial and myometrial structure in wild-type postpartum uterus (b) that contrasts with disorganized architecture of the CD44−/− counterpart (c), characterized by the loss of myometrial mass. Higher magnification (40×) shows predominantly round nuclei in the epithelial cells of wild-type uterus (d) compared to elongated, hyperchromatic nuclei in the more columnar epithelial cells of CD44−/− uterus (e). Smooth muscle layers are preserved in wild-type uterus (f), but appear disorganized and condensed, with hyperchromatic nuclei in CD44−/− uterus (g). (B) CD44HSPG-dependent localization of MMP-7 to the apical surface of uterine epithelium. (Top panels) 36 h postpartum uteri from wild-type and CD44−/− mice were stained with anti-CD44v3 mAb (red fluorescence) and anti-MMP-7 mAb (green fluorescence). (Bottom panels) Comparison of the localization of anti-MMP-7 antibody staining to that of anti-basement membrane proteoglycan perlecan antibody staining in 36 h postpartum epithelium of wild-type and CD44−/− mice. White and red arrows indicate the apical and basal epithelial surface, respectively. (C) MMP-7 and HB-EGF localization in wild-type and CD44−/− uterine epithelium. Thirty-six h postpartum uteri from wild-type and CD44−/− mice were stained with anti-MMP-7 antibody (red fluorescence) and anti-HB-EGF neutralizing antibody that recognizes the mature form only (HB-EGF), or the C-18 antibody against a peptide in the cytoplasmic tail of pro-HB-EGF (proHB-EGF, green fluorescence). White and red arrows denote the apical and basal epithelial surface, respectively. (D) MMP-7 and HB-EGF localization in wild-type and CD44−/− postpartum uterine smooth muscle. Staining was performed as in C and shows colocalization of MMP-7 and mature HB-EGF in wild-type smooth muscle cells (top). In CD44−/− postpartum uteri, MMP-7 is redistributed to the cell periphery, MMP-7 and pro-HB-EGF show only occasional colocalization (middle), and mature HB-EGF is barely detectable (bottom). (E) Colocalization of ErbB4 and phosphotyrosine in postpartum uterine epithelium. Anti-ErbB4 antibody (red fluorescence) and anti-phosphotyrosine pY20 mAb (green fluorescence) predominantly stain the apical epithelial surface in wild-type postpartum uterus (top). In CD44−/− postpartum uterus, ErbB4 distribution is unchanged, whereas apical epithelial tyrosine phosphorylation is abrogated, and only intracellular phosphorylation remains detectable. White and red arrows indicate the apical and basal epithelial surface, respectively (F) TUNEL (green fluorescence) and anti-CD44 mAb staining (red fluorescence) in wild-type and CD44−/− 48 h postpartum uteri. Wild-type (top) and CD44−/− (second panel) epithelial, and wild-type (third panel), and CD44−/− smooth muscle (bottom) staining is shown, indicating massive apoptotic cell death in both cell types in the absence of CD44.

Article Snippet: Primary antibodies included: polyclonal rabbit anti-MMP-7 antibody RM7-C and polyclonal rabbit anti-MMP-7 antibody RM7-N for rat proMMP-7 ( Yu and Woessner 2000 ); anti-panCD44 mAb clone 241, and anti-CD44V3 mAb (R&D Systems); anti-pro-HB-EGF C-18 antibody (Santa Cruz Biotechnology) for human samples and anti-pro-HB-EGF M-18 antibody (Santa Cruz) for mouse samples; anti-HB-EGF neutralizing antibody (R&D Systems); monoclonal anti-ErbB4 Ab-4 (HFR-1), polyclonal rabbit anti-ErbB4 Ab-2 (NeoMarkers), rat anti-human and mouse perlecan antibody (Chemi-Con).

Techniques: Staining, Fluorescence, Comparison, Membrane, TUNEL Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Computer controlled expansion of equine cord blood mesenchymal stromal cells on microcarriers in 3 L vertical-wheel ® bioreactors

doi: 10.3389/fbioe.2023.1250077

Figure Lengend Snippet: Antibodies used for flow cytometry.

Article Snippet: CD44 , Horse , Bio-Rad , FITC , CVS18 , , MSC.

Techniques: Cytometry