Journal: Experimental and Therapeutic Medicine

Article Title: Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2

doi: 10.3892/etm.2024.12564

Figure Lengend Snippet: PCA targets ABCG2. (A) The 2D structure of PCA is shown. The protein targets of PCA were predicted using the (B) TargetNet and (C) Super-PERD online tools. (D) The intersected proteins were predicted using both the TargetNet and Super-PERD databases. (E) The computer docking scores of PCA for ABCG2, GPR35 and TSHR are presented. (F and G) Global and local schematic presentation of PCA binding to ABCG2. PCA, purpurogallin carboxylic acid; ABCG2, ATP binding cassette subfamily G member 2; GPR35, G protein-coupled receptor 35; TSHR, thyroid stimulating hormone receptor.

Article Snippet: Purified ABCG2 proteins were obtained from Sino Biological Inc. and were then desalinated using the AKTA protein purification system (Cytiva).

Techniques: Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2

doi: 10.3892/etm.2024.12564

Figure Lengend Snippet: PCA has high affinity for ABCG2 and inhibits its activity in liver cancer cells. (A) Surface plasmon resonance assay was performed to determine the affinity of PCA for ABCG2. (B) The inhibitory effects of PCA on ABCG2 activity are demonstrated. (C) A Cell Counting Kit-8 assay was carried out to measure the non-specific toxicity of PCA on normal THLE-2 hepatocytes. Rhodamine efflux assays were performed to evaluate the drug efflux ability of (D) HepG2, (E) Huh7 and (F) Huh1 cells, following cell treatment with PCA. PCA, purpurogallin carboxylic acid; ABCG2, ATP binding cassette subfamily G member 2.

Article Snippet: Purified ABCG2 proteins were obtained from Sino Biological Inc. and were then desalinated using the AKTA protein purification system (Cytiva).

Techniques: Activity Assay, SPR Assay, Cell Counting, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2

doi: 10.3892/etm.2024.12564

Figure Lengend Snippet: PCA has no synergistic effect with 5-FU on inhibiting the proliferation and colony formation abilities of ABCG2-depleted liver cancer cells. (A) ABCG2-depleted liver cancer cells were established following cell transfection with small interfering RNAs targeting ABCG2. (B) A Cell Counting Kit-8 assay was performed to assess the effects of DMSO, 10 µM PCA, 10 µM 5-FU or their combination on the proliferation ability of ABCG2-depleted HepG2 and Huh7 cells. (C) A colony formation assay was used to evaluate the effects of DMSO, 10 µM PCA, 10 µM 5-FU or their combination on the colony formation ability of ABCG2-depleted HepG2 and Huh7 cells. ** P<0.01. PCA, purpurogallin carboxylic acid; 5-FU, 5-fluorouracil; ABCG2, ATP binding cassette subfamily G member 2; si-, small interfering RNA.

Article Snippet: Purified ABCG2 proteins were obtained from Sino Biological Inc. and were then desalinated using the AKTA protein purification system (Cytiva).

Techniques: Transfection, Cell Counting, Colony Assay, Binding Assay, Small Interfering RNA

Journal: Stem cell research

Article Title: IL6 and the human limbal stem cell niche: a mediator of epithelial-stromal interaction.

doi: 10.1016/j.scr.2010.07.002

Figure Lengend Snippet: Figure 4 (A and B) p63α (TRITC) and ABCG2 (TRITC) expression by HLE under 3.1SF conditions. Cytokeratin 3 is expressed by enlarging epithelial cells (B, FITC). (C) Semiquantitative RT-PCR data confirm the immunostaining observations. CFE assay of HLE cells derived by (D) GS and (E) 3.1SF cultures. Percentage CFE is significantly increased in 3.1SF cultures compared to GS (P=0.0286, m=6).

Article Snippet: Primary antibodies were supplied as follows: mouse monoclonal antibody for cytokeratin 15, Santa Cruz, mouse monoclonal antibody for ABCG2, mouse monoclonal antibody for cytokeratin 3 and mouse monoclonal antibody for Ki67, Millipore; rabbit polyclonal antibody for p63α, Cell Signalling, and rabbit polyclonal antibody for IL6, Abcam.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Immunostaining, Derivative Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2

doi: 10.3892/etm.2024.12564

Figure Lengend Snippet: PCA targets ABCG2. (A) The 2D structure of PCA is shown. The protein targets of PCA were predicted using the (B) TargetNet and (C) Super-PERD online tools. (D) The intersected proteins were predicted using both the TargetNet and Super-PERD databases. (E) The computer docking scores of PCA for ABCG2, GPR35 and TSHR are presented. (F and G) Global and local schematic presentation of PCA binding to ABCG2. PCA, purpurogallin carboxylic acid; ABCG2, ATP binding cassette subfamily G member 2; GPR35, G protein-coupled receptor 35; TSHR, thyroid stimulating hormone receptor.

Article Snippet: Following blocking with 8% skim milk powder in TBS-Tween-20 (0.1%) (TBST) for 2 h at 28˚C, the membranes were incubated with primary antibodies against cyclin-dependent kinase (CDK) 4 (1:2,000; cat no. 11026-1-AP), CDK6 (1:1,000; cat no. 14052-1-AP), ABCG2 (1:1,000; cat no. 27286-1-AP) and GAPDH (1:50,000; cat no. 60004-1-Ig) all from Proteintech Group, Inc. for 16 h at 4˚C.

Techniques: Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2

doi: 10.3892/etm.2024.12564

Figure Lengend Snippet: PCA has high affinity for ABCG2 and inhibits its activity in liver cancer cells. (A) Surface plasmon resonance assay was performed to determine the affinity of PCA for ABCG2. (B) The inhibitory effects of PCA on ABCG2 activity are demonstrated. (C) A Cell Counting Kit-8 assay was carried out to measure the non-specific toxicity of PCA on normal THLE-2 hepatocytes. Rhodamine efflux assays were performed to evaluate the drug efflux ability of (D) HepG2, (E) Huh7 and (F) Huh1 cells, following cell treatment with PCA. PCA, purpurogallin carboxylic acid; ABCG2, ATP binding cassette subfamily G member 2.

Article Snippet: Following blocking with 8% skim milk powder in TBS-Tween-20 (0.1%) (TBST) for 2 h at 28˚C, the membranes were incubated with primary antibodies against cyclin-dependent kinase (CDK) 4 (1:2,000; cat no. 11026-1-AP), CDK6 (1:1,000; cat no. 14052-1-AP), ABCG2 (1:1,000; cat no. 27286-1-AP) and GAPDH (1:50,000; cat no. 60004-1-Ig) all from Proteintech Group, Inc. for 16 h at 4˚C.

Techniques: Activity Assay, SPR Assay, Cell Counting, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Purpurogallin carboxylic acid exhibits synergistic effects with 5‑fluorouracil on liver cancer cells in vitro by targeting ABCG2

doi: 10.3892/etm.2024.12564

Figure Lengend Snippet: PCA has no synergistic effect with 5-FU on inhibiting the proliferation and colony formation abilities of ABCG2-depleted liver cancer cells. (A) ABCG2-depleted liver cancer cells were established following cell transfection with small interfering RNAs targeting ABCG2. (B) A Cell Counting Kit-8 assay was performed to assess the effects of DMSO, 10 µM PCA, 10 µM 5-FU or their combination on the proliferation ability of ABCG2-depleted HepG2 and Huh7 cells. (C) A colony formation assay was used to evaluate the effects of DMSO, 10 µM PCA, 10 µM 5-FU or their combination on the colony formation ability of ABCG2-depleted HepG2 and Huh7 cells. ** P<0.01. PCA, purpurogallin carboxylic acid; 5-FU, 5-fluorouracil; ABCG2, ATP binding cassette subfamily G member 2; si-, small interfering RNA.

Article Snippet: Following blocking with 8% skim milk powder in TBS-Tween-20 (0.1%) (TBST) for 2 h at 28˚C, the membranes were incubated with primary antibodies against cyclin-dependent kinase (CDK) 4 (1:2,000; cat no. 11026-1-AP), CDK6 (1:1,000; cat no. 14052-1-AP), ABCG2 (1:1,000; cat no. 27286-1-AP) and GAPDH (1:50,000; cat no. 60004-1-Ig) all from Proteintech Group, Inc. for 16 h at 4˚C.

Techniques: Transfection, Cell Counting, Colony Assay, Binding Assay, Small Interfering RNA

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: Cell Surface Protein Markers.

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques:

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: Freshly isolated aortic pVICs were stained with antibodies for CD31, OB–CDH, ABCG2, NG2, SSEA-4, and the corresponding control antibodies. Staining was quantified by flow cytometry. In the figure, the y-axis is fluorescence intensity and the x-axis is forward scattering. Percentage in the rectangular gates represents the fraction of positively stained cells. After subtracting the background, about 7.70% of these aortic pVICs stained positive for CD31, 4.71% stained positive for OB–CDH, 5.60% stained positive for ABCG2, 5.56% stained positive for NG2, and 6.59% stained positive for SSEA-4.

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques: Isolation, Staining, Control, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: Unique cell subpopulations identified based on cell surface markers are present in both aortic and pulmonary valves.

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques: Marker

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: To examine co-expression of these different markers, freshly isolated pVICs were co-stained with pairwise combinations of the markers OB–CDH, NG2 and SSEA-4, or with ABCG2 and NG2. Both the x-axes and the y-axes are fluorescence intensity of the antibody staining. (A) OB–CDH was not co-expressed with SSEA-4 in the same cell population. (B) OB–CDH was not co-expressed with NG2 in the same cell population. (C) SSEA-4 was not co-expressed with NG2 in the same cell population. These data support that these markers (OB–CDH, NG2 and SSEA-4) label distinct subpopulations relative to each other in pVICs. (D) However, NG2 and ABCG2 were co-expression by the same cell subpopulation in aortic valves.

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques: Expressing, Isolation, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: To examine the origin of these cell subpopulations, aortic pVICs were double stained with CD31 and one of the following markers: SSEA-4, OB–CDH or ABCG2. CD31 is expressed by porcine valvular endothelial cells but not by pVICs. From (A), SSEA4 and CD31 labeled distinct cell subpopulations. However, OB–CDH + or ABCG2 + cells showed heterogeneous CD31 staining. About 52% of OB–CDH + cells were CD31 - and ~61% of ABCG2 + cells were CD31 - , indicating that a majority of these cells are from pVICs rather than endothelium.

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques: Staining, Labeling

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: Sorted ABCG2 + and ABCG2 - valvular cells were grown to confluence and re-plated in osteogenic media for 8 days. (A) Brightfield images of cells cultured in control conditions or osteogenic conditions were taken at day 8. ABCG2 + progeny secreted more dark mineralization spots than ABCG2 - progeny on plastic plates. Scale bar: 100 µm. (B) Calcium deposition by the cells was quantified as described in the Materials and Methods. Data was represented as mean + standard error. Compared with ABCG2 - progeny, ABCG2 + progeny produced higher amounts of calcium composites at day 8 (* indicates p<0.05 in between the groups).

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques: Cell Culture, Control, Produced

Journal: PLoS ONE

Article Title: Characterization of Cell Subpopulations Expressing Progenitor Cell Markers in Porcine Cardiac Valves

doi: 10.1371/journal.pone.0069667

Figure Lengend Snippet: Based on previous literature and our flow cytometry data, porcine aortic valves are comprised of layers of endothelial cells and VICs. pVICs are further divided into multiple different subpopulations, including ABCG2 + and NG2 + pVICs, OB–CDH + pVICs and SSEA-4 + pVICs. The functions of these different subpopulations in the diseased valves are speculated based on literature and our experimental data, and these serve as hypotheses to be tested in future.

Article Snippet: The cells were subsequently stained with selected primary antibodies raised against ABCG2 (R&D Systems, Cat # FAB995P), CD31 (AbD Serotec, Cat# MCA1746APC), OB–CDH (clone 15F7 [ ], a generous gift from Dr. Micheal Brenner’s group at the Brigham and Women’s Hospital) and SSEA4 (R&D Systems, Cat# FAB1435P) for 1 hour at 4°C.

Techniques: Flow Cytometry