Journal: Frontiers in Veterinary Science

Article Title: PDGFD maintains ovine tail ADSCs in a proliferative state by suppressing CXCL8 and activating PI3K/MAPK signaling

doi: 10.3389/fvets.2026.1777426

Figure Lengend Snippet: Immunofluorescence identification of CD44 in ovine ADSCs. (a) CD44 immunofluorescence staining (red) shows strong positive expression localized to the cell membrane and cytoplasm. (b) DAPI staining (blue) marks the cell nuclei. (c) Merged image illustrates the subcellular localization of CD44.

Article Snippet: To reduce non-specific binding, cells were blocked with 1% bovine serum albumin (BSA, Bioss, Beijing, China) at room temperature for 30 min. After blocking, cells were incubated overnight at 4 °C with a primary antibody against CD44 (rabbit polyclonal antibody, Bioss, Cat No. bs-55039R, dilution 1:1,000).

Techniques: Immunofluorescence, Staining, Expressing, Membrane

Journal: International Journal of Nanomedicine

Article Title: Long Non-Coding RNAs Within Macrophage-Derived Exosomes Promote BMSC Osteogenesis in a Bone Fracture Rat Model

doi: 10.2147/IJN.S398446

Figure Lengend Snippet: The characteristics of macrophages and BMSCs. ( a ) Primary macrophages observed under the microscope; cells were spherical and uniform in size. ( b ) Flow cytometry results of CD68 expression on the cell surface. The expression of CD68 on 10,000 cells was recorded. 90.7% of the cells expressed CD68. ( c ) The fifth generation of cultured BMSCs was observed under the microscope; the cells were long, fusiform, and translucent. ( d – f ) Alizarin red, Alcian blue, and Oil red O staining showed that the cells had osteogenic, chondrogenic, and adipogenic differentiation ability. ( g – j ). The expression of CD29, CD90, CD44, and CD34 on 10,000 cells was recorded; red indicates cell marker expression and blue indicates the isotype control. Microscopy: 100× magnification and 200 μm scale.

Article Snippet: Cells were incubated with the following specific antibodies in the dark at 4 °C for 30 min: rabbit anti-CD29/Alexa Fluor 488 (1:100 dilution), rabbit anti-CD90/Alexa Fluor 488 (1:100 dilution), rabbit anti-CD44/Alexa Fluor 488 (1:100 dilution), rabbit anti-CD34/Alexa Fluor 488 (1:100 dilution), and rabbit anti-CD68/Alexa Fluor 488 (1:100 dilution) (all purchased from Bioss, China).

Techniques: Microscopy, Flow Cytometry, Expressing, Cell Culture, Staining, Marker

Journal: Journal of Neuroinflammation

Article Title: Cytokine and chemokine alterations in tissue, CSF, and plasma in early presymptomatic phase of experimental allergic encephalomyelitis (EAE), in a rat model of multiple sclerosis

doi: 10.1186/s12974-016-0757-6

Figure Lengend Snippet: Immunohistochemistry analysis. Double immunostaining of OX42/CD163, CNPase/CD44 and CD86/NG2 in the lumbar spinal cord of control and EAE rats . a – d Double labeling for microglial cells marker (OX42) and M2 macrophages marker (CD163) in the white matter of control ( a ) and EAE experimental groups: 8, 11, and 18 DPI ( b – d ). Plus in b indicates the activated microglia expressing CD163. e – h Double labeling for oligodendrocyte marker (CNPase) and T lymphocyte marker (CD44) (see also the included high-power magnifications). CD44 is expressed by oligodendrocytes in control. Arrows in e indicate co-localization, although this expression changed at 8 DPI in f. i – l Double labeling for oligodendrocyte precursor cells (NG2) and M1 macrophages marker (CD86). The immunoreactive area of CD163 ( m ), CNPase ( n ), CD44 ( o ), and CD86 ( p ) in EAE rats compared to controls. Statistical analysis: one-way ANOVA and Dunnett’s multiple comparison test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: The primary antibodies and dilutions used were as follows: rabbit anti-CD44 (1:100, Cluster of differentiation 44, Acris Antibodies, Inc, San Diego, USA), rabbit anti-CD163 (1:100, Cluster of differentiation 163, Bioss Inc, Woburn, USA), mouse anti-CD86 (1:250, Cluster of differentiation 86, Novus Biological Europe, Cambridge, UK), mouse anti-NF200 (1:200, Neurofilament, Sigma, Saint Louse, USA), rabbit anti CSF1 (1:100, colony-stimulating factor 1, Novus Biological), mouse anti-OX42 (1:250, AbD Serotec Inc., Raleigh, USA), mouse anti-NG2 (1:100, membrane-spanning chondroitin sulfate proteoglycan, Millipore, Merck S.p.a., Milan, Italy), mouse anti-CNPase (1:250, 2′, 3′-cyclic nucleotide 3′-phosphodiesterase, Millipore, Merck S.p.a., Milan, Italy), rabbit anti-GFAP (1:500, glial fibrillary acidic protein, Dako, Milan, Italy), and mouse anti-GFAP (1:500, Sigma, Saint Louse, USA).

Techniques: Immunohistochemistry, Double Immunostaining, Labeling, Marker, Expressing

Journal: Molecular Cancer

Article Title: NAC1 promotes stemness and regulates myeloid-derived cell status in triple-negative breast cancer

doi: 10.1186/s12943-024-02102-y

Figure Lengend Snippet: Effect of NAC1 on stemness of TNBC cells. A-C Western blot analysis of the stemness-associated markers in HCC1806 and BT549 TNBC cells with or without knockdown of NAC1. D Flow cytometry analysis of CD44 protein surface expression in MDA-MB-231 cells with or without knockdown of NAC1. E Flow cytometry analysis of CD24 protein surface expression in MDA-MB-231 cells with or without knockdown of NAC1. F Right : Mammosphere formation of TNBC cells with or without depletion of NAC1; Left : quantification of the number of spheres larger than 45 µM. G Tumor initiation and growth of MDA-MB-231 cells with or without depletion of NAC1 in nu/nu mice. Number of tumors(n) = 2, number of tumors per mouse (Tn) = 4. Luminescence intensity signifies a relative number of detectable live cells

Article Snippet: The antibodies used were: GAPDH Mouse mAb (Cell Signaling Technology, #97,166), β-actin antibody (Cell Signaling Technology, #4967), NAC1 (Biolegend, cat#849,302), ADAM17 (Thermo, cat#H00006868-M01A), MMP9 antibody (Santa cruz biotechnology, cat#sc-21733), MMP1 antibody (Santa cruz biotechnology, cat#sc-21731), CD44 (E7K2Y) XP® Rabbit mAb (Cell signaling technology, cat#37,259), CD44-ECD (extracellular domain), anti-human CD44-ICD (intracellular domain) polyclonal antibody (Diagnocine, cat#FNK-KO601), CD24 (Santa Cruz Biotechnology, cat#-sc-19585), Sox2 Rabbit mAb (cell signaling technology, cat#14,962), Nanog (D73G4) XP® Rabbit mAb (Cell Signaling Technology, cat#4903), Cyclin D1 (E3P5S) XP® Rabbit mAb (Cell Signaling Technology, cat#55,506), ALDH1A1 (Cell Signaling Technology, cat#12,035), STAT3 mouse mAb (Cell Signaling Technology, cat#9139), Phospho-STAT3 (Tyr705) Rabbit mAb (Cell Signaling Technology, cat#9145), JAK1 antibody (Santa Cruz Biotechnology, cat#sc-376996), CD130/gp130 antibody (Santa Cruz Biotechnology, cat#sc-376280), Vimentin Rabbit mAb (Cell signaling technology, cat#5741), mouse anti-E-Cadherin antibody (BD Transduction Laboratories™, cat#610,181).

Techniques: Western Blot, Knockdown, Flow Cytometry, Expressing

Journal: Molecular Cancer

Article Title: NAC1 promotes stemness and regulates myeloid-derived cell status in triple-negative breast cancer

doi: 10.1186/s12943-024-02102-y

Figure Lengend Snippet: CD44/JAK-STAT3 is involved in the NAC1-mediated control of TNBC stemness and progression. A Analysis of the transcription factors (TFs) associated with the differentially expressed genes in NAC1 knockdown cells demonstrates STAT3 as an important TF in NAC1-induced phenotypes. B GSEA analysis demonstrates reduction of genes associated with tumor progression phenotypes and pathways including angiogenesis, cell migration, cell motility and JAK/STAT3 cascade. C STAT3 qPCR mRNA analysis of MDA-MB-231 cells. D STAT3 mRNA expression in MDA-MB-231 cells with depletion of NAC1. E TCGA STAT3 mRNA expression analysis reveals insignificant change ( p > 0.05) in tumor samples compared to adjacent normal tissues. F STAT3 protein expression significantly increases in CPTAC dataset tumor samples compared to normal. G Correlation between NAC1, proliferation marker KI67, caspase 3, and phospho-STAT3 in TNBC patients’ tissue from the University of Kentucky retrospective tissue bank. H Depletion of NAC1 downregulates STAT3 and phospho-STAT3 protein expression in TNBC cells. I JAK1 mRNA expression in MDA-MB-231 cells with depletion of NAC1. J Western blot analysis of JAK/STAT3 pathway-associated proteins in MDA-MB-231 cells with depletion of NAC1. K CD44 mRNA expression in MDA-MB-231 cells with depletion of NAC1. L Depletion of CD44 caused downregulation of JAK1 in MDA-MB-231 cells

Article Snippet: The antibodies used were: GAPDH Mouse mAb (Cell Signaling Technology, #97,166), β-actin antibody (Cell Signaling Technology, #4967), NAC1 (Biolegend, cat#849,302), ADAM17 (Thermo, cat#H00006868-M01A), MMP9 antibody (Santa cruz biotechnology, cat#sc-21733), MMP1 antibody (Santa cruz biotechnology, cat#sc-21731), CD44 (E7K2Y) XP® Rabbit mAb (Cell signaling technology, cat#37,259), CD44-ECD (extracellular domain), anti-human CD44-ICD (intracellular domain) polyclonal antibody (Diagnocine, cat#FNK-KO601), CD24 (Santa Cruz Biotechnology, cat#-sc-19585), Sox2 Rabbit mAb (cell signaling technology, cat#14,962), Nanog (D73G4) XP® Rabbit mAb (Cell Signaling Technology, cat#4903), Cyclin D1 (E3P5S) XP® Rabbit mAb (Cell Signaling Technology, cat#55,506), ALDH1A1 (Cell Signaling Technology, cat#12,035), STAT3 mouse mAb (Cell Signaling Technology, cat#9139), Phospho-STAT3 (Tyr705) Rabbit mAb (Cell Signaling Technology, cat#9145), JAK1 antibody (Santa Cruz Biotechnology, cat#sc-376996), CD130/gp130 antibody (Santa Cruz Biotechnology, cat#sc-376280), Vimentin Rabbit mAb (Cell signaling technology, cat#5741), mouse anti-E-Cadherin antibody (BD Transduction Laboratories™, cat#610,181).

Techniques: Control, Knockdown, Migration, Expressing, Marker, Western Blot

Journal: Molecular Cancer

Article Title: NAC1 promotes stemness and regulates myeloid-derived cell status in triple-negative breast cancer

doi: 10.1186/s12943-024-02102-y

Figure Lengend Snippet: Analysis of bulky RNA sequencing data reveals the immunosuppressive TME-associated factors potentially regulated by NAC1. A Altered oncogenic-associated pathways and genes in NAC1-deficient tumor cells. B Reactome analysis shows downregulation of innate immunity-associated genes in tumor cells with NAC1 knockdown. C Enrichment of the genes associated with neutrophil degranulation in tumor cells with NAC1 knockdown. D Expression of G-CSF, CCL2, and SOD2 in MDA-MB-231 cells with or without depletion of NAC1. E Expression of CD44 in MDA-MB-231 cells with or without depletion of NAC1. F IL6 mRNA expression in MDA-MB-231 cells with or without depletion of NAC1. G Level of soluble G-CSF in MDA-MB-231 cells with or without depletion of NAC1. H Soluble IL6 concentration in EO771 cells with forced expression of NAC1

Article Snippet: The antibodies used were: GAPDH Mouse mAb (Cell Signaling Technology, #97,166), β-actin antibody (Cell Signaling Technology, #4967), NAC1 (Biolegend, cat#849,302), ADAM17 (Thermo, cat#H00006868-M01A), MMP9 antibody (Santa cruz biotechnology, cat#sc-21733), MMP1 antibody (Santa cruz biotechnology, cat#sc-21731), CD44 (E7K2Y) XP® Rabbit mAb (Cell signaling technology, cat#37,259), CD44-ECD (extracellular domain), anti-human CD44-ICD (intracellular domain) polyclonal antibody (Diagnocine, cat#FNK-KO601), CD24 (Santa Cruz Biotechnology, cat#-sc-19585), Sox2 Rabbit mAb (cell signaling technology, cat#14,962), Nanog (D73G4) XP® Rabbit mAb (Cell Signaling Technology, cat#4903), Cyclin D1 (E3P5S) XP® Rabbit mAb (Cell Signaling Technology, cat#55,506), ALDH1A1 (Cell Signaling Technology, cat#12,035), STAT3 mouse mAb (Cell Signaling Technology, cat#9139), Phospho-STAT3 (Tyr705) Rabbit mAb (Cell Signaling Technology, cat#9145), JAK1 antibody (Santa Cruz Biotechnology, cat#sc-376996), CD130/gp130 antibody (Santa Cruz Biotechnology, cat#sc-376280), Vimentin Rabbit mAb (Cell signaling technology, cat#5741), mouse anti-E-Cadherin antibody (BD Transduction Laboratories™, cat#610,181).

Techniques: RNA Sequencing Assay, Knockdown, Expressing, Concentration Assay

Journal: Molecular Cancer

Article Title: NAC1 promotes stemness and regulates myeloid-derived cell status in triple-negative breast cancer

doi: 10.1186/s12943-024-02102-y

Figure Lengend Snippet: Myeloid-derived cells with expression of NAC1 supports CSCs. A Tumor growth rate for knockdown 4T1 allografted cells with or without NK cell depletion. B Expression of NAC1 in MDSCs from 4T1 tumor-bearing or tumor-free BALB/C mice. C Gr1 + /CD11b + cells were isolated from the NACC1 +/+ or NACC1 −/− mice bearing EO771 tumors through negative selection to obtain MDSCs and were further purified using CD45 positive selection assay kit (Stemcell technologies) to eliminate contaminating tumor cells. D EO771 cells co-cultured with NACC1 −/− Gr1 + /CD11b + cells showed reduced CD44 expression compared to the co-culture with NACC1 +/+ Gr1 + /CD11b + cells. E Aldolase activity of EO771 cells co-cultured with NACC1 +/+ or NACC1 −/− mice Gr1 + /CD11b + cells. F Tumor initiation ability of EO771 cells orthotopically inoculated in NACC1 +/+ or NACC1 −/− mice ( n = 5, Tn = 2). G-I EO771 cells were co-cultured with NACC1 +/+ or NACC1 −/− Gr1 + /CD11b + cells, and cell viability was determined using the CellTrace™ CFSE Cell Proliferation Kit ( G ) or luciferase assay ( H )

Article Snippet: The antibodies used were: GAPDH Mouse mAb (Cell Signaling Technology, #97,166), β-actin antibody (Cell Signaling Technology, #4967), NAC1 (Biolegend, cat#849,302), ADAM17 (Thermo, cat#H00006868-M01A), MMP9 antibody (Santa cruz biotechnology, cat#sc-21733), MMP1 antibody (Santa cruz biotechnology, cat#sc-21731), CD44 (E7K2Y) XP® Rabbit mAb (Cell signaling technology, cat#37,259), CD44-ECD (extracellular domain), anti-human CD44-ICD (intracellular domain) polyclonal antibody (Diagnocine, cat#FNK-KO601), CD24 (Santa Cruz Biotechnology, cat#-sc-19585), Sox2 Rabbit mAb (cell signaling technology, cat#14,962), Nanog (D73G4) XP® Rabbit mAb (Cell Signaling Technology, cat#4903), Cyclin D1 (E3P5S) XP® Rabbit mAb (Cell Signaling Technology, cat#55,506), ALDH1A1 (Cell Signaling Technology, cat#12,035), STAT3 mouse mAb (Cell Signaling Technology, cat#9139), Phospho-STAT3 (Tyr705) Rabbit mAb (Cell Signaling Technology, cat#9145), JAK1 antibody (Santa Cruz Biotechnology, cat#sc-376996), CD130/gp130 antibody (Santa Cruz Biotechnology, cat#sc-376280), Vimentin Rabbit mAb (Cell signaling technology, cat#5741), mouse anti-E-Cadherin antibody (BD Transduction Laboratories™, cat#610,181).

Techniques: Derivative Assay, Expressing, Knockdown, Isolation, Selection, Purification, Cell Culture, Co-Culture Assay, Activity Assay, Luciferase