Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades CD44 through the lysosome pathway. ( A ) Immunoblots to detect decreased CD44 and increased BAX, BID, and MARCH8 expression levels in MARCH8-overexpressing cells compared to GFP control cells. ( B ) Immunofluorescence staining with anti-CD44 antibody showing decreased expression of membrane protein CD44 (blue) in MARCH8-GFP expressing cells. ( C ) Flow cytometry histogram overlay (left panel) and dot plots (right panels) indicating MARCH8-decreased CD44 expression levels in negative association with MARCH8-GFP signals. ( D ) Immunoblots of endogenous CD44 and exogenous MARCH8-GFP after transient transfection of MARCH-GFP and treatment with MG-132 or chloroquine (CLQ) to block the proteasomal or lysosomal degradation pathways, respectively. ( E ) Immunoblots of MARCH8 and FLAG-tagged CD44 after anti-FLAG mediated immunoprecipitation (IP) of the lysates of HEK-293 cells after transfections with MARCH8-GFP and CD44-FLAG (standard isoform CD44s and full-length CD44f) and treatment with CLQ, indicating the interactions between MARCH8 and CD44 (CD44s or CD44f). ( F ) Immunoblots of BAX, BID, CD44, and MARCH8 in the MDA-MB-231 cells with stable expression of GFP or MARCH8-GFP with transient transfection of a FLAG vector control or restoration of CD44 expression via CD44s-FLAG. CD44 overexpression slightly inhibited the expression of proapoptotic BID and BAX in MARCH8-GFP-overexpressing cells.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Transfection, Blocking Assay, Immunoprecipitation, Plasmid Preparation, Over Expression

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades STAT3 through the proteasome pathway. ( A ) Immunoblots of AKT, pAKT, ERK, and pERK in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, both adherent and in suspension. ( B ) Immunoblots of STAT3, pSTAT3 (Y705), CD44, and MARCH8 in GFP- and MARCH8-GFP-expressing cells in the absence or presence of MG-132 and CLQ, both adherent and in suspension. ( C ) Immunoblots of MARCH8, STAT3, pSTAT3 (Y705), and ubiquitin in lysates of cells co-transfected with STAT3 (wildtype or Y705F) and MARCH8-GFP (or GFP control) and immunoprecipitated by anti-STAT3 and anti-pSTAT3 (Y705). ( D ) Immunoblots of CD44, STAT3, BID, and BAX in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, with transient transfection with CD44, STAT3, or mutant Y705F as indicated, both adherent and in suspension.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Mutagenesis

Journal: Materials Today Bio

Article Title: A bioactive composite scaffold enhances osteochondral repair by using thermosensitive chitosan hydrogel and endothelial lineage cell-derived chondrogenic cell

doi: 10.1016/j.mtbio.2024.101174

Figure Lengend Snippet: EndMT-derived tEPCs acquire an MSC-like phenotype. ( a ) Flow cytometry analysis of CD44, CD90, CD105 (mesenchymal stem cell markers), CD34 (hematopoietic and endothelial cell marker), and CD45 (leukocyte marker) expression in tEPCs. The empty areas show isotype control staining. The red-filled areas represent the expression of specific markers. ( b ) Representative immunofluorescence images of tEPC surface markers. tEPCs stain positive for MSC markers (CD44, CD90, and CD105) and negative for endothelial cell markers (CD31, eNOS, VE-cadherin, and vWF) (20 × magnification; scale bar: 50 μm). ( c , d ) CFU efficiency of EPCs and tEPCs, assessing self-renewal through the rate of colony formation in CFU assays. ( c ) Representative colonies of EPCs and tEPCs in 6-well plates (scale bar: 5 mm). ( d ) Columns illustrate the CFU efficiency. Values are reported as the mean ± standard deviation (SD) of six replicates. *** P < 0.001. ( e ) Multilineage differentiation potential of tEPCs induced to differentiate into ( i ) chondrogenic (10 × magnification; scale bar: 100 μm), ( ii ) osteogenic (10 × magnification; scale bar: 100 μm), or ( iii ) adipogenic (40 × magnification; scale bar: 10 μm) lineages. Abbreviations: CD, cluster of differentiation; CFU, colony-forming unit; EndMT, endothelial-to-mesenchymal transition; eNOS, endothelial nitric oxide synthase; EPCs, endothelial progenitor cells; MSCs, mesenchymal stem cells; tEPC, transdifferentiated EPCs; VE-cadherin, vascular endothelial cadherin; vWF, von Willebrand factor.

Article Snippet: Next, 1 × 10 6 tEPCs were suspended in 500 μL of PBS containing 20 μg/mL of fluorescein isothiocyanate (FITC)-conjugated antibodies against CD44 (Novus, Centennial, CO, USA), CD90 (Bioworld, Louis Park, MN, USA), CD105 (Bioss, Woburn, MA, USA), CD34 (Bioss), and CD45 (Bioss).

Techniques: Derivative Assay, Flow Cytometry, Marker, Expressing, Control, Staining, Immunofluorescence, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: Translocation of CD44 from cytoplasm to nuclear in the reprogramming process of C3A cells . A. Real-time PCR analysis of CD44 in indicated time points of reprogramming process. Relative gene expression of CD44 to C3A cells was calculated for C3A-D5, C3A-D15, C3A-D25 C3A-D35, C3A-iCSCs P5 and C3A-iCSCs P45. Data are presented as the means ± SD from three independent. B. Immunofluorescence staining of CD44 in indicated time points of reprogramming process. Scale bar, 25μm. C. Western blot analysis of total protein (left) and cytoplasmic/nuclear protein (right) of CD44 in C3A and C3A-iCSCs. D. Immunohistochemical staining of CD44 in clinical liver cancer samples. Arrows indicated nuclear CD44-positive staining. Scale bar, 30μm.

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques: Translocation Assay, Real-time Polymerase Chain Reaction, Gene Expression, Immunofluorescence, Staining, Western Blot, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: CD44 transport was mediated by importin β and affected by importin α. A. The interaction between CD44 and importin β / transportin 1 was demonstrated by co-immunoprecipitation assay in C3A-iCSCs. B. Western blot analysis of flag after CD44-NLS-del / CD44-WT was transduced into C3A-iCSCs. C. Immunofluorescence staining analysis of flag after CD44-NLS-del / CD44-WT was transduced into C3A-iCSCs. Scale bar, 25μm. D. Immunofluorescence staining analysis of CD44 (red) and importin β (green) after Importazole was treated in C3A-iCSCs. DMSO indicated negative control, WGA indicated positive control. Scale bar, 10μm. E. Immunofluorescence staining analysis of CD44 (red) and importin β (green) after importin α was knocked down by RNA interfere assay. Scale bar, 10μm. C-E. Percentages of colocalization in ROI were qualified.

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Negative Control, Positive Control

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: Nuclear CD44 participated in naïve genes transcriptional regulation. A. Real-time PCR analysis of the naïve pluripotent genes KLF2 , KLF5, ZFP42, ESRRB , DNMT3L, GBX2, DPPA4 and LIFR in C3A cells and C3A-iCSCs. B. Real-time PCR analysis of CD44 and naive pluripotent genes after CD44 was silenced. C. ChIP-qPCR performed using CD44-specific antibodies in C3A cells and C3A-iCSCs. KLF2 , KLF5 , ESRRB represented their promoters respectively. Samples were analyzed by real-time PCR. Error bars showed the standard deviation of three independent ChIP-qPCR assays. D. Luciferase activity assay performed in C3A-iCSCs. Luciferase reporter plasmid pGL3-KLF2 / pGL3-KLF5 / pGL3-ESRRB containing their own promoter sequences was co-transfected with Renilla control vector and Ctrl / si-CD44 RNA sequences in C3A-iCSCs. Luciferase activities were measured after 72 hrs and presented as relative to the activity of Renilla luciferase. (* p <0.05, ** p <0.01, *** p <0.001, ns indicated no significant differences)

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques: Real-time Polymerase Chain Reaction, ChIP-qPCR, Standard Deviation, Luciferase, Activity Assay, Plasmid Preparation, Transfection, Control

Journal: International Journal of Biological Sciences

Article Title: Nuclear CD44 Mediated by Importin β Participated in Naïve Genes Transcriptional Regulation in C3A-iCSCs

doi: 10.7150/ijbs.28235

Figure Lengend Snippet: Proposed model of CD44 transport from cytoplasm to nuclear . CD44 transport was mediated by importin β. Nuclear CD44 participated in transcriptional regulation of KLF2 , KLF5 , and ESRRB genes in C3A-iCSCs.

Article Snippet: Primary antibodies included antibodies against CD44 (1:400; Proteintech), OCT4 (1:500; Santa Cruz), SOX2 (1:500; Chemicon), TRA-1-80 (1:200; Abcam), Importin β (1:1000; Abcam), Transportin 1 (1:200; Abcam).

Techniques:

Journal: Diabetology & metabolic syndrome

Article Title: Adipose stem cells-derived small extracellular vesicles transport Thrombospondin 1 cargo to promote insulin resistance in gestational diabetes mellitus.

doi: 10.1186/s13098-024-01276-1

Figure Lengend Snippet: Fig. 1 Isolation, culture and purification of GDM mice-derived ADSCs and their sEVs. A Plasma glucose values were quantified in eight normal gestation and GDM mice. B The expression of insulin, leptin, adiponectin and hs-CRP in the plasma of mice was detected by ELISA assays. C ADSCs was obtained by collagenase digestion of abdominal and inguinal adipose tissue from gestational mice and further in vitro culture. D Microscopic images of ADSCs isolated and cultured from normal gestational and GDM mice. Scale bar, 25 μm. E The expression of Cd44 marker (red) was identified by immunofluorescence. DAPI staining (blue) indicated the nucleus. Scale bar, 25 μm. F The sEVA and sEVAG were identified by transmission electron microscope. Scale bar, 100 nm. G The sEVA and sEVAG solution was diluted 500 times and particle size distribution and concentration were determined by nanoparticle tracking analysis. H The sEV markers and stem cell markers were detected by western blotting in sEVA and sEV.AG samples. Data were presented with mean ± standard deviation (SD), *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Subsequently, the cells were sealed in a PBS solution containing 0.5% Triton X-100 and 10% fetal bovine serum (FBS) for 2 h. The glass slides were then incubated with primary Cd44 antibody and Alexa Fluor 594-labeled secondary antibody (SA00006-4, Proteintech).

Techniques: Isolation, Purification, Derivative Assay, Clinical Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Cell Culture, Marker, Immunofluorescence, Staining, Transmission Assay, Microscopy, Concentration Assay, Western Blot, Standard Deviation

Journal: Biology of reproduction

Article Title: Expression of two isoforms of CD44 in human endometrium.

doi: 10.1095/biolreprod51.4.739

Figure Lengend Snippet: FIG. 1. Immunofluorescence of CD44 in human endometrium. a) Proliferative phase tissue (mAb P3H9) showing immunoreactivity in epithelial cells in a gland as well as in surrounding stroma. b) Decidua of first trimester (mAb P3H9) with strong reactivity in decidual stromal cells. c) Late secretory phase tissue (mAb PIG12) showing lateral staining in gland cells. Gland lumen is to right of the field. d) Nuclear staining in same sections as c. e) Example of tissue (late secretory phase) in which stromal reactivity is present but many glands are unstained (mAb PIG12). Note that one gland at bottom right is immunopositive. f) Nuclear staining, same field as (e). Arrowheads in a, c, and e indicate the apical cell surface of glandular epithelium.

Article Snippet: MATERIALS AND METHODS Antibodies Mouse pan anti-CD44 monoclonal antibodies (mAbs) were as follows: P1G12 and P3H9 (14,15; Chemicon, Temecula, CA); F10-44-2 (Serotec, Oxford, UK); BU52 (The Binding Site, Birmingham, UK); A1G3 (Seralab, High Wycombe, UK); and E1.2, a generous gift from Dr. C. Isacke, Imperial College, London [38].

Techniques: Immunofluorescence, Staining

Journal: Biology of reproduction

Article Title: Expression of two isoforms of CD44 in human endometrium.

doi: 10.1095/biolreprod51.4.739

Figure Lengend Snippet: FIG. 2. Immunofluorescence of CD44 in endometrial epithelial cells (mAb P3H9). al) Freshly isolated gland fragment in confocal microscopy. b) Pri- mary culture at 4 days showing mainly lateral immunoreactivity. c) Ishi- kawa endometrial adenocarcinoma cells with lateral as well as punctate surface staining.

Article Snippet: MATERIALS AND METHODS Antibodies Mouse pan anti-CD44 monoclonal antibodies (mAbs) were as follows: P1G12 and P3H9 (14,15; Chemicon, Temecula, CA); F10-44-2 (Serotec, Oxford, UK); BU52 (The Binding Site, Birmingham, UK); A1G3 (Seralab, High Wycombe, UK); and E1.2, a generous gift from Dr. C. Isacke, Imperial College, London [38].

Techniques: Immunofluorescence, Isolation, Confocal Microscopy, Staining

Journal: Biology of reproduction

Article Title: Expression of two isoforms of CD44 in human endometrium.

doi: 10.1095/biolreprod51.4.739

Figure Lengend Snippet: FIG. 3. Immunoprecipitation of CD44 from surface iodinated gland cells with mAb PIG12 gives a band at approximately 130 kDa. Right lane: con- trol, irrelevant mAb. Dots at right indicate positions of molecular size mark- ers (from the top down) of 200, 116, 67, and 45 kDa.

Article Snippet: MATERIALS AND METHODS Antibodies Mouse pan anti-CD44 monoclonal antibodies (mAbs) were as follows: P1G12 and P3H9 (14,15; Chemicon, Temecula, CA); F10-44-2 (Serotec, Oxford, UK); BU52 (The Binding Site, Birmingham, UK); A1G3 (Seralab, High Wycombe, UK); and E1.2, a generous gift from Dr. C. Isacke, Imperial College, London [38].

Techniques: Immunoprecipitation

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades CD44 through the lysosome pathway. ( A ) Immunoblots to detect decreased CD44 and increased BAX, BID, and MARCH8 expression levels in MARCH8-overexpressing cells compared to GFP control cells. ( B ) Immunofluorescence staining with anti-CD44 antibody showing decreased expression of membrane protein CD44 (blue) in MARCH8-GFP expressing cells. ( C ) Flow cytometry histogram overlay (left panel) and dot plots (right panels) indicating MARCH8-decreased CD44 expression levels in negative association with MARCH8-GFP signals. ( D ) Immunoblots of endogenous CD44 and exogenous MARCH8-GFP after transient transfection of MARCH-GFP and treatment with MG-132 or chloroquine (CLQ) to block the proteasomal or lysosomal degradation pathways, respectively. ( E ) Immunoblots of MARCH8 and FLAG-tagged CD44 after anti-FLAG mediated immunoprecipitation (IP) of the lysates of HEK-293 cells after transfections with MARCH8-GFP and CD44-FLAG (standard isoform CD44s and full-length CD44f) and treatment with CLQ, indicating the interactions between MARCH8 and CD44 (CD44s or CD44f). ( F ) Immunoblots of BAX, BID, CD44, and MARCH8 in the MDA-MB-231 cells with stable expression of GFP or MARCH8-GFP with transient transfection of a FLAG vector control or restoration of CD44 expression via CD44s-FLAG. CD44 overexpression slightly inhibited the expression of proapoptotic BID and BAX in MARCH8-GFP-overexpressing cells.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Flow Cytometry, Transfection, Blocking Assay, Immunoprecipitation, Plasmid Preparation, Over Expression

Journal: Cancers

Article Title: MARCH8 Suppresses Tumor Metastasis and Mediates Degradation of STAT3 and CD44 in Breast Cancer Cells

doi: 10.3390/cancers13112550

Figure Lengend Snippet: MARCH8 interacts with and degrades STAT3 through the proteasome pathway. ( A ) Immunoblots of AKT, pAKT, ERK, and pERK in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, both adherent and in suspension. ( B ) Immunoblots of STAT3, pSTAT3 (Y705), CD44, and MARCH8 in GFP- and MARCH8-GFP-expressing cells in the absence or presence of MG-132 and CLQ, both adherent and in suspension. ( C ) Immunoblots of MARCH8, STAT3, pSTAT3 (Y705), and ubiquitin in lysates of cells co-transfected with STAT3 (wildtype or Y705F) and MARCH8-GFP (or GFP control) and immunoprecipitated by anti-STAT3 and anti-pSTAT3 (Y705). ( D ) Immunoblots of CD44, STAT3, BID, and BAX in MDA-MB-231 cells with stable expression of GFP and MARCH8-GFP, with transient transfection with CD44, STAT3, or mutant Y705F as indicated, both adherent and in suspension.

Article Snippet: Plasmids that were used for overexpression include human CD44 standard form (NM_001001391), FLAG-tagged ORF Clone pCMV6-Flag-CD44s (OriGene Technologies, Rockville, MD, USA, Cat RC221820), CD44 full-length pCMV3-CD44f-HA ((Sino Biological, Beijing, China, Cat HG12211-CY), Lenti ORF clone of human MARCH8-GFP (OriGene Technologies, Rockville, MD, USA, Cat RC209891L2), GFP control (OriGene Technologies, Rockville, MD, USA, Cat PS10007), STAT3 (Addgene, Watertown, MA, USA, Cat 71450), and STAT3 Y705F (Addgene, Watertown, MA, USA, Cat 71445) mutant.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Mutagenesis

Journal: Stem Cell Research & Therapy

Article Title: Effects of intra-arterial transplantation of adipose-derived stem cells on the expression of netrin-1 and its receptor DCC in the peri-infarct cortex after experimental stroke

doi: 10.1186/s13287-017-0671-6

Figure Lengend Snippet: Characteristics and immigration of ADCSs after transplantation. a Passage 3 of cultured ADSCs. Scale bar = 50 μm. Immunofluorescence staining showed that ADSCs at passages 3–4 were positive for surface antigens CD44 ( b ), while negative for CD34 ( c ) and CD45 ( d ). Scale bar = 50 μm. Immunofluorescence staining showed that PKH-26 ( red )-labeled ADSCs were mainly located in the peri-infarct area at days 7 ( e ) and 14 ( f ) after MCAO, and the number of PKH-26 + cells decreased at day 14 when compared to day 7. The white dotted line represents the boundary line between the peri-infarct area (left side) and infarct core (right side). Scale bar = 50 μm

Article Snippet: The cells were then incubated with primary mouse antibodies against CD34, CD44, and CD45 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h. After washing with PBS three times, cells were incubated with Dylight488 affinipure donkey anti-mouse immunoglobulin G (IgG; 1:400, Jackson Immunoresearch) for 45 min.

Techniques: Transplantation Assay, Cell Culture, Immunofluorescence, Staining, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury

doi: 10.3390/ijms20194815

Figure Lengend Snippet: Modulation of macrophage functional polarity towards an anti-inflammatory phenotype by MSCs occurs via CD44 on rat macrophages. ( a ) Rat macrophages were transfected with scrambled siRNA (50 nM) or rat CD44 siRNA (50 nM) for 24 h. After CD44 siRNA transfections, LPS stimulation was performed. Rat CD44 protein expression on macrophages was analyzed by western blotting. Intensity was normalized to macrophages only. ( b , b’ – d , d’ ) CD44 knockdown in macrophages disrupted macrophage polarization. Confocal microscopy of CD44 siRNA-transfected macrophages showed an increased expression of the M1 marker, CD11b and decreased expression of the M2 marker, CD163, despite treatment with MSCs. Quantitative analysis showed the significant results of knockdown CD44 on macrophages. Nuclei were stained with Hoechst 33342. Red (CD11b) and green (CD163 and CD44) staining indicate positive cells. ( e – g ) Cytokine analysis was performed by ELISA. Supernatants from CD44 siRNA-transfected macrophage cultures also showed high levels of IL-8 and TNF-α, but low levels of IL-10. Scale bar = 20 μm. Data are presented as mean ± SD, n = 5 per group. ** p < 0.01, * p < 0.05. n/s, not significant; MΦ, macrophage; L, LPS; Con siR, scrambled siRNA-transfected control group; rCD44 siR, rat CD44 siRNA-transfected group.

Article Snippet: Blocked membranes were incubated with a primary anti-CD44 antibody (Novus Biologicals, Centennial, CO, USA), followed by horseradish peroxidase-conjugated secondary antibodies.

Techniques: Functional Assay, Transfection, Expressing, Western Blot, Knockdown, Confocal Microscopy, Marker, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury

doi: 10.3390/ijms20194815

Figure Lengend Snippet: Decorin knockdown in MSCs attenuates macrophage polarization. ( a – c ) Decorin siRNA-treated MSCs were co-cultured with LPS-induced macrophages and macrophage polarization was assessed. ( a’ – c’ ) Immunofluorescent staining with CD11b, CD163, and CD44 were analyzed according to the percentage of positively stained cells. Red (CD11b) and green (CD163 and CD44) staining indicate positive cells. Nuclei were stained with Hoechst 33342. ( d ) IL-8, ( e ) IL-6, and ( f ) IL-10 levels in co-cultures of macrophages and MSCs were analyzed by ELISA. Data are presented as mean ± SD, n = 5 per group. Scale bar = 20 μm. ** p < 0.01. n/s, not significant; MΦ, macrophage; L, LPS.

Article Snippet: Blocked membranes were incubated with a primary anti-CD44 antibody (Novus Biologicals, Centennial, CO, USA), followed by horseradish peroxidase-conjugated secondary antibodies.

Techniques: Knockdown, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury

doi: 10.3390/ijms20194815

Figure Lengend Snippet: MSCs promote tissue repair in a hyperoxic lung injury rat model. The normal group was raised under normoxic conditions, whereas the experimental groups (bronchopulmonary dysplasia (BPD), BPD + MSC H, or MSC L) were raised under hyperoxia conditions (90% oxygen) from 10 h after birth to postnatal day (P14). On day 5, 1 × 10 5 MSCs were injected intratracheally. Rat pups were sacrificed and lung tissues and bronchoalveolar lavage fluid (BALF) samples were collected on P14. ( a ) MSCs were divided into two groups (MSC-H and MSC-L) according to their decorin expression levels. Decorin secretion by MSCs was quantified by secretome analysis and ELISA. Supernatants from co-cultures of macrophages and MSCs showed different levels of decorin. ( b ) MSC H lot was selected from MSC-H groups and MSC L lot was selected from MSC-L groups. Kaplan–Meier survival curve at birth and P14. ( c ) Morphometric analysis of lung tissues was performed to determine the degree of alveolarization by measuring the mean linear intercept (MLI). ( d ) Lung tissue samples were sectioned and stained with hematoxylin and eosin (H&E). ( e – g ) CD11b, CD163, and CD44 were stained in rat lung tissues. ( h ) Immunofluorescence staining of human β2 microglobulin (hβ2MG) was used to visualize the engrafted MSCs. ( e’ – h’ ) Positively stained cells were analyzed and presented as percentages. Red (CD11b) and green (CD163, CD44, and hβ2MG) staining indicate positive cells. Nuclei were stained with Hoechst 33342. ( i – k ) Lung bronchoalveolar lavage fluid (BALF) samples were collected to analyze the pro-inflammatory cytokines, IL-8 and IL-6 and the anti-inflammatory cytokine, IL-10, by ELISA. Data are presented as mean ± SD for n = 11 (normal) or 15 (BPD, BPD + MSC H, and MSC L) per group. Scale bar = 40 μm. ** p < 0.01, * p < 0.05. n/s, not significant; BPD, bronchopulmonary dysplasia.

Article Snippet: Blocked membranes were incubated with a primary anti-CD44 antibody (Novus Biologicals, Centennial, CO, USA), followed by horseradish peroxidase-conjugated secondary antibodies.

Techniques: Injection, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury

doi: 10.3390/ijms20194815

Figure Lengend Snippet: Decorin knockdown suppressed the therapeutic effects of MSCs by down-regulating macrophage polarization. Decorin-silenced MSCs were intratracheally injected in BPD rats. ( a ) Decorin siRNA-transfected MSCs were intratracheally injected on day 5. Daily survival rates during 14 d from birth are presented as Kaplan–Meier survival curves. ( b ) Lung tissues on P14 were analyzed by histological and morphogenic comparison with the control group (normal). The mean linear intercept (MLI) values were determined by the degree of alveolarization. ( c ) Lung tissue samples were sectioned and stained with H&E. Data are presented as mean ± SD for n = 11 (normal), 15 (BPD, BPD with MSC), or 14 (co-cultured with MSCs or scrambled siRNA-transfected MSCs (Con siR-MSC), Decorin siR-MSC) per group. ** p < 0.01, * p < 0.05. n/s, not significant; BPD, bronchopulmonary dysplasia. ( d,d’ – f,f’ ) Immunofluorescence analysis was performed with CD11b, CD163, and CD44 antibodies in lung tissues obtained from rats with hyperoxic lung injury. ( g , g’ ) Transplanted MSCs were detected by hβ2MG (green) staining of rat lung tissues. Blue staining represents nuclei stained with Hoechst 33342. Red (CD11b) and green (CD163 and CD44) staining indicate positive cells. ( h – j ) The levels of cytokines IL-8, IL-6, and IL-10, were analyzed by ELISA on P14. Data are presented as mean ± SD, n = 5 per group. Scale bar = 40 μm. ** p < 0.01, * p < 0.05. n/s, not significant; MΦ, macrophage; L, LPS.

Article Snippet: Blocked membranes were incubated with a primary anti-CD44 antibody (Novus Biologicals, Centennial, CO, USA), followed by horseradish peroxidase-conjugated secondary antibodies.

Techniques: Knockdown, Injection, Transfection, Comparison, Control, Staining, Cell Culture, Immunofluorescence, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Decorin Secreted by Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Induces Macrophage Polarization via CD44 to Repair Hyperoxic Lung Injury

doi: 10.3390/ijms20194815

Figure Lengend Snippet: Sequences of primers used for indicated target genes.

Article Snippet: Blocked membranes were incubated with a primary anti-CD44 antibody (Novus Biologicals, Centennial, CO, USA), followed by horseradish peroxidase-conjugated secondary antibodies.

Techniques: Sequencing

Journal: Scientific reports

Article Title: Dynamics of CD44 + bovine nucleus pulposus cells with inflammation.

doi: 10.1038/s41598-024-59504-7

Figure Lengend Snippet: Figure 1. Gene expression profile of CD44+ versus CD44- bovine NP cells. Fresh bovine NP cells were sorted for the expression of CD44 and analyzed for the gene expression of phenotypic markers. (A) Acquired cell sorting events were filtered based on: (i) size (FSC/SSC); (ii) symmetry for the removal of duplets (FSC-H/ FSC-A); and (iii) viability (according to staining with viability dye). Filtered events were then separated into CD44+ and CD44-, according to the fluorescence intensity for the APC channel, and considering the non- specific fluorescence displayed by the unstained population. (B) Total mRNA isolated from the sorted cells was translated to cDNA and analyzed by qRT-PCR for the expression of: CD14, LGALS3, FoxF1, IL-6, IL-8, VEGFA, ADAMTS5, MMP3, TIMP1, TIMP2, COL2A and ACAN (n = 4). Results are presented as box and whiskers plots with representation of median, min and max values (statistical significance, *p < 0.05).

Article Snippet: NP cells obtained upon digestion were labelled with an FITC-conjugated CD44 antibody (1:50, AbD Serotec, clone IL-A118) as described for cell sorting and with the nuclear DRAQ5TM fluorescent probe (1:500, eBioscience) for accurate detection of the cells in stream.

Techniques: Gene Expression, Expressing, FACS, Staining, Fluorescence, Isolation, Quantitative RT-PCR

Journal: Scientific reports

Article Title: Dynamics of CD44 + bovine nucleus pulposus cells with inflammation.

doi: 10.1038/s41598-024-59504-7

Figure Lengend Snippet: Figure 2. Proteomic analysis of CD44+ vs CD44- bovine NP cells. Proteomic analysis of fresh NP cells sorted based on expression (or lack) of CD44. (A) Scheme of the protocol used to compare the proteomic profile of CD44+ vs CD44- bovine NP cells. (B) Principal component analysis (PCA) results. (C) Volcano plot with threshold lines for p-value < 0.05 (Y axis) and CD44+/CD44- fold-change >|1.5| (X axis). (D) proteins found significantly altered between CD44+ and CD44- cells (fold-change >|1.5|, p < 0.05). (E) REACTOME software analysis of the proteins significantly up-regulated in CD44+ vs CD44- bovine NP cells.

Article Snippet: NP cells obtained upon digestion were labelled with an FITC-conjugated CD44 antibody (1:50, AbD Serotec, clone IL-A118) as described for cell sorting and with the nuclear DRAQ5TM fluorescent probe (1:500, eBioscience) for accurate detection of the cells in stream.

Techniques: Expressing, Software

Journal: Scientific reports

Article Title: Dynamics of CD44 + bovine nucleus pulposus cells with inflammation.

doi: 10.1038/s41598-024-59504-7

Figure Lengend Snippet: Figure 3. NP CD44 expression in response to a pro-inflammatory stimulus. CD44 expression was analyzed in bovine NPs stimulated by needle puncture and IL-1β. (A) IL-6, IL-8, MMP3, ACAN, COL2A, CD44 and CD14 gene expression were analyzed in days 2, 7 and 14 (n = 3–8). (B) CD44 cell surface quantification by flow cytometry (n = 22). Results are depicted as box and whiskers plots with representation of median, min and max. Statistical significance (*p < 0.05); **p < 0.01); ***p < 0.001).

Article Snippet: NP cells obtained upon digestion were labelled with an FITC-conjugated CD44 antibody (1:50, AbD Serotec, clone IL-A118) as described for cell sorting and with the nuclear DRAQ5TM fluorescent probe (1:500, eBioscience) for accurate detection of the cells in stream.

Techniques: Expressing, Gene Expression, Flow Cytometry

Journal: Scientific reports

Article Title: Dynamics of CD44 + bovine nucleus pulposus cells with inflammation.

doi: 10.1038/s41598-024-59504-7

Figure Lengend Snippet: Figure 4. Morphological analysis of CD44 NP cells by imaging flow cytometry, conventional flow cytometry and immunofluorescence. (A) Morphological characterization of CD44+ vs CD44- bovine NP cells through: (a) flow cytometry (difference in size assessed by backgating analysis). Results are presented as box and whiskers plots with representation of median, min, and max values (n = 12); (b) imaging flow cytometry of bovine NP cells stimulated with IL-1β. FITC-CD44 antibody (green, Ch02) and DRAQ5 nuclear staining (red, Ch05) were used to complement NP cell imaging (in brightfied, Ch01 and darkfield, Ch06). Results are presented in a table indicating the number of cells quantified (n = 1). (B) CD44 immunofluorescence staining after 2 days of culture. Representative images of sagittal sections of IVD explants (scale bar: 50 μm) Quantification of the percentage of cells positive for CD44 was performed according with the scheme presented. Results are presented as box and whiskers plots with representation of median, min, and max values (n = 4). Distribution of CD44+ cells according to percentage of the cell area stained for each marker is represented as an histogram for average value of n = 4.

Article Snippet: NP cells obtained upon digestion were labelled with an FITC-conjugated CD44 antibody (1:50, AbD Serotec, clone IL-A118) as described for cell sorting and with the nuclear DRAQ5TM fluorescent probe (1:500, eBioscience) for accurate detection of the cells in stream.

Techniques: Imaging, Flow Cytometry, Immunofluorescence, Staining, Marker

Journal: Scientific reports

Article Title: Dynamics of CD44 + bovine nucleus pulposus cells with inflammation.

doi: 10.1038/s41598-024-59504-7

Figure Lengend Snippet: Figure 5. Co-expression analysis of CD44, CD45 and CD14 in NP cells in response to a pro-inflammatory/ degenerative stimulus. (A) Representative images of sagittal sections of IVD explants with immunofluorescence staining for CD44, CD45 and CD14 in bovine NPs after 2 days in culture (scale bar: 50 μm). (B) Quantification of the percentage of cells simultaneously expressing CD44, CD45 and CD14 markers was performed, as previously described (n = 4). Results are presented as box and whiskers plots with representation of median, min and max. Statistical analysis was performed and p values indicated in the plots.

Article Snippet: NP cells obtained upon digestion were labelled with an FITC-conjugated CD44 antibody (1:50, AbD Serotec, clone IL-A118) as described for cell sorting and with the nuclear DRAQ5TM fluorescent probe (1:500, eBioscience) for accurate detection of the cells in stream.

Techniques: Expressing, Immunofluorescence, Staining