cd44v6 pe  (R&D Systems)

Journal: Stem Cell Reports

Article Title: Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy

doi: 10.1016/j.stemcr.2022.02.005

Figure Lengend Snippet: Overexpression of miR-148a represses the CSC phenotype in vitro (A) Quantification of the CD44v6-PE-positive population in CPP1 (n = 6) and HT29 (n = 4) cells transfected with miR-148a-3p ( miR-148a ) or mirVana microRNA Mimic Negative Control #1 (miCTRL) mimics. Percentage of CPP1 CD44v6-PE-positive cells is indicated in inset boxes for a representative experiment (left panel). (B) Representative pictures of tumorspheres (left panels) and percentage (right panels) of sphere-forming cells of patient-derived CPP1 and HT29 colon cancer cells transfected with miR-148a-3p or miCTRL mimics. Individual replicate values (n = 10 per experiment) are plotted, as well as the mean ± SEM (n = 3). (C and D) Percentage of ALDH-positive cells (% of ALDHred, data are expressed as mean ± SEM, n = 6) (C) and representative pictures of tumorspheres (upper panels) and percentage (lower panel; data are expressed as mean ± SEM, n = 3 with 12 individual replicate values per experiment) (D) of sphere-forming cells in HT29 cells stably overexpressing miR-148a ( miR-148a /copGFP) or empty sequence (CTRL/copGFP) transfected either with a miR-148a-insensitive PXR coding sequence ( PXR ) or the pcDNA3 empty vector (Mock). (E) RT-qPCR analyses of PXR target gene ( FGF19 , ALDH1A1 , CYP3A4 ) mRNA expression in HT29 cells stably overexpressing miR-148a /copGFP or CTRL/copGFP transfected either with a miR-148a -insensitive PXR coding sequence ( PXR ) or the pcDNA3 empty vector (Mock). Data are expressed as mean ± SEM (n = 3). ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001.

Article Snippet: CD44-allophycocyanin (APC) (559,942; BD Pharmingen), CD44v6-APC (clone REA706, 130-111-238; Macs Miltenyi), or CD44v6-phycoerythrin (PE) (FAB3660P; R&D) antibodies (1/100) were incubated with 100,000 cells in PBS containing 5% FBS for 20 min at 4°C.

Techniques: Over Expression, In Vitro, Transfection, Negative Control, Derivative Assay, Stable Transfection, Sequencing, Plasmid Preparation, Quantitative RT-PCR, Expressing

Journal: Stem Cell Reports

Article Title: Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy

doi: 10.1016/j.stemcr.2022.02.005

Figure Lengend Snippet: Niclosamide (Niclo) inhibits PXR expression and colon cancer stemness (A–C) DNMT1 and PXR mRNA expression in LS174T (A) and CPP1 colon cancer cells (B) treated for 72 h with vehicle (0.1% DMSO = CTRL) or Niclo at the indicated final concentrations. Data are expressed as mean ± SEM of three independent experiments. (C, left panel) ALDH1A1 mRNA expression (n = 3) and (right panel) percentage of Aldefluor-positive cells (% of ALDH + cells, n = 5) in CPP1 colon cancer cells treated for 72 h with vehicle (0.1% DMSO = CTRL) or Niclo at the indicated final concentrations. Data are expressed as mean ± SEM. (D) Quantification of CD44v6-APC-positive cells in CPP1 or CTC44 cells treated for 72 h with vehicle (0.1% DMSO = CTRL) or 5 μM Niclo. Data are expressed as mean ± SEM (n = 5 for CPP1, n = 6 for CTC44). (E) Percentage of sphere-forming cells of sorted live (Sytox Blue negative) CPP1 cells, pretreated for 72 h with vehicle (0.1% DMSO = CTRL) or Niclo at the indicated final concentrations. Data are expressed as mean ± SEM, n = 3 (10 individual replicate values per experiment). (F) Percentage of sphere-forming cells of sorted live (Sytox Blue negative) patient-derived cells (CPP14, CPP36, or CTC44), pretreated for 72 h with vehicle (0.1% DMSO = CTRL) or 5 μM Niclo. Data are expressed as mean ± SEM, n = 3 (10 individual replicate values per experiment). ∗ p < 0.05, ∗∗ p < 0.005, ∗∗∗ p < 0.001.

Article Snippet: CD44-allophycocyanin (APC) (559,942; BD Pharmingen), CD44v6-APC (clone REA706, 130-111-238; Macs Miltenyi), or CD44v6-phycoerythrin (PE) (FAB3660P; R&D) antibodies (1/100) were incubated with 100,000 cells in PBS containing 5% FBS for 20 min at 4°C.

Techniques: Expressing, Derivative Assay

Journal: Oncotarget

Article Title: Baseline CD44v6-positive circulating tumor cells to predict first-line treatment failure in patients with metastatic colorectal cancer

doi: 10.18632/oncotarget.27794

Figure Lengend Snippet: Patient characteristics at baseline

Article Snippet: A volume of 7.5 mL of peripheral blood from each participant was collected into CellSave Preservative tubes (Menarini Silicon Biosystems), kept at room temperature and processed through CellSearch ® system within 72 h. To this end, the CellSearch ® CXC kit (Menarini Silicon Biosystems) was employed and anti-human CD44v6 Phycoerythrin (PE)-conjugated antibody (clone FAB3660P; R&D Systems, Minneapolis, USA) was added into user-defined marker channel at a concentration of 0.02 μg/mL.

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: Baseline CD44v6-positive circulating tumor cells to predict first-line treatment failure in patients with metastatic colorectal cancer

doi: 10.18632/oncotarget.27794

Figure Lengend Snippet: Clinical response and CTC number or CD44v6 status at baseline

Article Snippet: A volume of 7.5 mL of peripheral blood from each participant was collected into CellSave Preservative tubes (Menarini Silicon Biosystems), kept at room temperature and processed through CellSearch ® system within 72 h. To this end, the CellSearch ® CXC kit (Menarini Silicon Biosystems) was employed and anti-human CD44v6 Phycoerythrin (PE)-conjugated antibody (clone FAB3660P; R&D Systems, Minneapolis, USA) was added into user-defined marker channel at a concentration of 0.02 μg/mL.

Techniques:

Journal: Translational Cancer Research

Article Title: Clinicopathological significance and prognostic implication of CD44 and its splice variants (v3 and v6) in colorectal cancer

doi: 10.21037/tcr.2020.02.12

Figure Lengend Snippet: Correlation of CD44, CD44v3 and CD44v6 expression status among varied clinicopathological parameters

Article Snippet: The primary antibodies used were: Anti-human-CD44 (dilution 1:100; cat. no. ab157107; Abcam), anti-CD44v3 (dilution 1:50; cat. no. ab34229; Abcam), anti-CD44v6-monoclonal antibody (dilution 1:70; cat. no. FAB3660P; R&D Systems, Inc.) and anti-c-Kit (dilution 1:100; cat. no. ab21539; Abcam).

Techniques: Expressing, Cell Differentiation

Journal: Translational Cancer Research

Article Title: Clinicopathological significance and prognostic implication of CD44 and its splice variants (v3 and v6) in colorectal cancer

doi: 10.21037/tcr.2020.02.12

Figure Lengend Snippet: Expression CD44, CD44v3, CD44v6 and c-Kit with different intensity and extent. Most of the positive staining were located on the membrane, but some positive expression could found in the cytoplasm as the white arrows indicated. All magnifications, ×200. Well, well differentiated; Moderate, moderate differentiated; Poor, poor differentiated; c-Kit, mast/stem cell growth factor receptor Kit; GIST, gastrointestinal stromal tumor.

Article Snippet: The primary antibodies used were: Anti-human-CD44 (dilution 1:100; cat. no. ab157107; Abcam), anti-CD44v3 (dilution 1:50; cat. no. ab34229; Abcam), anti-CD44v6-monoclonal antibody (dilution 1:70; cat. no. FAB3660P; R&D Systems, Inc.) and anti-c-Kit (dilution 1:100; cat. no. ab21539; Abcam).

Techniques: Expressing, Staining, Membrane

Journal: Translational Cancer Research

Article Title: Clinicopathological significance and prognostic implication of CD44 and its splice variants (v3 and v6) in colorectal cancer

doi: 10.21037/tcr.2020.02.12

Figure Lengend Snippet: Univariable and multivariable analysis of different parameters for PFS in the patients

Article Snippet: The primary antibodies used were: Anti-human-CD44 (dilution 1:100; cat. no. ab157107; Abcam), anti-CD44v3 (dilution 1:50; cat. no. ab34229; Abcam), anti-CD44v6-monoclonal antibody (dilution 1:70; cat. no. FAB3660P; R&D Systems, Inc.) and anti-c-Kit (dilution 1:100; cat. no. ab21539; Abcam).

Techniques: Cell Differentiation

Journal: Translational Cancer Research

Article Title: Clinicopathological significance and prognostic implication of CD44 and its splice variants (v3 and v6) in colorectal cancer

doi: 10.21037/tcr.2020.02.12

Figure Lengend Snippet: Prognostic role of CD44, CD44v3 and CD44v6 regarding PFS. The cumulative survival indicates the proportion of cases surviving from the date of surgery to the end of the interval. PFS, progression-free survival; Neg, negative; Pos, positive; v3, variant 3; v6, variant 6.

Article Snippet: The primary antibodies used were: Anti-human-CD44 (dilution 1:100; cat. no. ab157107; Abcam), anti-CD44v3 (dilution 1:50; cat. no. ab34229; Abcam), anti-CD44v6-monoclonal antibody (dilution 1:70; cat. no. FAB3660P; R&D Systems, Inc.) and anti-c-Kit (dilution 1:100; cat. no. ab21539; Abcam).

Techniques: Variant Assay