Journal: PLOS ONE

Article Title: Shenqi Fuzheng injection reverses M2 macrophage-mediated cisplatin resistance through the PI3K pathway in breast cancer

doi: 10.1371/journal.pone.0279752

Figure Lengend Snippet: Tumour lysates were analysed for the expressions of p-PI3K, PI3K, P-gp, and ABCG2 with the respective antibodies. The density ratio of detected proteins to GAPDH is shown as relative expression. Values are shown as the means ± SD from three independent experiments. *p < 0.05 as compared to Cisplatin alone.

Article Snippet: Monoclonal antibodies against CD206, CD86, PI3K, P-gp, ABCG2, Bcl-2, Bax, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing

Journal: PLOS ONE

Article Title: Shenqi Fuzheng injection reverses M2 macrophage-mediated cisplatin resistance through the PI3K pathway in breast cancer

doi: 10.1371/journal.pone.0279752

Figure Lengend Snippet: Cell lysates were analysed for the expressions of P-gp, ABCG2, Bcl-2, and Bax with the respective antibodies. The density ratio of detected proteins to GAPDH is shown as relative expression. Values are shown as the means ± SD from three independent experiments. *p < 0.05 as compared to the Control. #p < 0.05 as compared to IGF-1 alone.

Article Snippet: Monoclonal antibodies against CD206, CD86, PI3K, P-gp, ABCG2, Bcl-2, Bax, and GAPDH were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: (A) The chemical structure of GS-9973. (B) The effect of the incubation of vehicle (Control), GS-9973 (1 or 3 µM) or Ko143 (3 µM) for 30, 60 or 120 min on the intracellular level of the ABCG2 transporter substrate, [ 3 H]-mitoxantrone from NCI-H460/MX20 cancer cells overexpressing the ABCG2 transporter. (C) The effect of the incubation of vehicle (Control), GS-9973 (1 or 3 µM) or Ko143 (3 µM) for 30, 60 or 120 min on the intracellular level of the ABCG2 transporter substrate, [ 3 H]-mitoxantrone from NCI-H460 parental cancer cells. (D) The effect of vehicle (Control), GS-9973 (1 or 3 µM) or Ko143 (3 µM) on the intracellular accumulation of [ 3 H]-mitoxantrone in NCI-H460 and NCI-H460/MX20 cancer cells. The columns are the mean of triplicate determinations; the error bars represent the SD. ** p ≤ 0.01 and *** p < 0.001 compared with control group.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Incubation

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: The effect of GS-9973 in parental NCI-H460 and ABCG2-overepxressing NCI-H460/MX20 cancer cell lines. (A) Th e survival fraction (%) was determined following incubation with 0.1-100 µM of GS-9973 for 72 h in NCI-H460 (blue) and NCI-H460/MX20 (red) cell lines and IC 50 values of (B) mitoxantrone, (C) doxorubicin and (D) cisplatin in parental NCI-H460 and drug-selected ABCG2 overexpressing NCI-H460/MX20 cancer cells with or without GS-9973. All data are shown as mean ± SD and represents three independent experiments. * p ≤ 0.05, ** p ≤ 0.01 and **** p < 0.0001 compared to the control group.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Incubation

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: The effect of GS-9973 in HEK293 cells transfected with the gene coding for the ABCG2 transporter. (A) The survival fraction (%) for HEK293/pcDNA3.1 (empty DNA vector control), HEK293/ABCG2-482-R2, HEK293/ABCG2-482-G2 and HEK293/ABCG2-482-T7 cell lines was determined following incubation with 0.03-30 µM of GS-9973 for 72 h. The IC 50 values of (B) mitoxantrone, (C) doxorubicin, and (D) cisplatin in HEK293/pcDNA3.1 (empty DNA vector control), HEK293/ABCG2-482-R2, HEK293/ABCG2-482-G2 and HEK293/ABCG2-482-T7 cell lines. Data are exhibited as mean ± SD and acquired from at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p < 0.001 and **** p < 0.0001 compared to the control group.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Transfection, Plasmid Preparation, Incubation

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: The effect of GS-9973 on the expression of the ABCG2 protein. Analysis of ABCG2 expression in (A) NCI-H460 and (B) NCI-H460/MX20 cancer cells after the cells were incubated with 6 µM of GS-9973 for 24, 48 and 72 h. Relative quantification of the effect of GS-9973 on ABCG2 in (C) NCI-H460 and (D) NCI-H460/MX20 cells. ABCG2 expression levels were normalized to GAPDH. Equal amounts of total cell lysates were employed for each sample and Western blot analysis was performed. * p ≤ 0.05 compared with control group. (E) The effect of GS-9973 on the expression and localization of ABCG2 using immunofluorescence. The effect of incubation of NCI-H460 and NCI-H460/MX20 cancer cells with 3 µM of GS-9973 for 0, 24, 48 and 72 h. The green color represents the presence of the ABCG2 transporter, and the blue color represents the nucleus.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Expressing, Incubation, Western Blot, Immunofluorescence

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: The effect of GS-9973 on ABCG2 mRNA expression. After incubation of NCI-H460 and NCI-H460/MX20 cancer cells with 3 µM of GS-9973 for 0, 24, 48 and 72 h, RT-PCR was conducted to determine the expression of ABCG2 mRNA.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: GS-9973 stimulated the ATPase activity of ABCG2. The graph illustrates the effect of 0-40 µM of GS-9973 on the ATPase activity of ABCG2.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Activity Assay

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: Molecular interaction of GS-9973 with the human ABCG2 model. (A) Whole ABCG2 protein with the docking site (highlighted with black box). (B) Docking pose of GS-9973 within the binding pocket of ABCG2. The protein is represented as sky blue colored ribbons. Amino acid residues are represented as follows: carbon in gray, hydrogen in white, nitrogen in blue and oxygen in red. The ligand is represented by the ball and stick model with carbon atoms are represented in green, oxygen in red nitrogen in blue and hydrogen in white. Blue dashes represent π-π stacking interaction, yellow dashes represent the hydrogen bonding. (C) 2-D ligand interaction between GS-9973 and ABCG2. Green indicates π-π interaction with amino acid residues within 5 Å of the ligand and the magenta arrow represents hydrogen bonding.

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Binding Assay

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: The effect of GS-9973 on reversal of ABCG2 mediated MDR in the drug-resistant cell lines

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques:

Journal: International Journal of Biological Sciences

Article Title: The Spleen Tyrosine Kinase Inhibitor, Entospletinib (GS-9973) Restores Chemosensitivity in Lung Cancer Cells by Modulating ABCG2-mediated Multidrug Resistance

doi: 10.7150/ijbs.61229

Figure Lengend Snippet: The effect of GS-9973 on reversal of ABCG2 mediated MDR in the transfected cell lines

Article Snippet: ABCG2 (D752K) and GAPDH (D16H1) selective monoclonal antibodies and secondary anti-rabbit antibody linked with HRP (7074S) were procured from Cell Signaling Technologies (Danvers, MA).

Techniques: Transfection

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: Expression of ABCG2 in human HCC tissues. (a) ABCG2 was expressed in placenta tissues (positive control). (b) Primary antibody omitted placenta tissue slide was set as negative control. (c) Positive ABCG2 expression in HCC tissue. (d) Negative ABCG2 expression in HCC tissue.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Expressing, Positive Control, Negative Control

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: ABCG2 expression and characteristics of patients.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Expressing

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: ABCG2 was expressed in a minor population of SMMC-7721 cells. (a) The positive rate of ABCG2 in SMMC-7721 cells determined by flow cytometry was about 8.8% before cell sorting. P2 gate represents ABCG2 positive cells. Mouse IgG2b was used as isotype control. (b) Positive rate of ABCG2 in sorted ABCG2+/ABCG2− cells after 3 passages of culture. (c) Detection of ABCG2 protein level in freshly sorted cells by western blot. β -Actin was used as loading control. The optical density of specific bands was quantified and normalized to β -Actin. ** P < 0.01.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Flow Cytometry, FACS, Western Blot

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: Difference of tumorigenic ability between ABCG2+ and ABCG2− cells.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques:

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: Expression level of ABCG2 correlated with cell proliferation and chemoresistance. (a), (b) Freshly sorted ABCG2+ cells were transfected with ABCG2-specific siRNA. ABCG2− cells were transfected with ABCG2-overexpression plasmid. The proliferation of cells after transfection was compared with untransfected cells and negative control (scrambled siRNA or empty plasmid) using MTT assay after indicated time. (c), (d) The expression level of ABCG2 was manipulated as described above. Survival rates after exposure to indicated dose of doxorubicin for 24 h were determined by MTT assay. (e), (f) IC50 value to doxorubicin was calculated for different groups. ** P < 0.01.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Negative Control, MTT Assay

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: RNA interference and plasmid-mediated overexpression of ABCG2 in SMMC-7721 cells. (a) RT-PCR analysis of ABCG2 mRNA in SMCC-7721 cells after transfection with siRNA or plasmid for 48 h. Normal SMMC-7721 cells were used as blank control. Scrambled siRNA was set as negative control. For overexpression, empty plasmid was transfected as negative control. (b), (c) Western blot analysis confirmed the efficiency of downregulation of ABCG2 by siRNA and upregulation by overexpression, respectively. β -Actin served as loading control. The optical density was quantified and normalized to β -Actin. ** P < 0.01, *** P < 0.001.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Plasmid Preparation, Over Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, Negative Control, Western Blot

Journal: Gastroenterology Research and Practice

Article Title: Expression of Potential Cancer Stem Cell Marker ABCG2 is Associated with Malignant Behaviors of Hepatocellular Carcinoma

doi: 10.1155/2013/782581

Figure Lengend Snippet: Downregulation or upregulation of ABCG2 had effect on migration and invasion potential in HCC cells. (a), (b) Wound healing assay evaluated the potential of migration of HCC cells. Untreated cells served as normal control. ABCG2+ cells were transfected with ABCG2 siRNA while ABCG2− cells were transfected with ABCG2-expressing plasmid. Scrambled siRNA and empty plasmid were used as negative control. The pictures above represent the wound right after tip scratch. Pictures below showed wound-healing status after 48 h. (c), (d) Results of transwell invasion assay. After 48 h of invasion, cells invaded to the lower chamber were stained and photographed. Invaded cells were counted in three random visions and the average numbers were presented. *** P < 0.001.

Article Snippet: Sections were incubated with rabbit anti-ABCG2 polyclonal antibody (1 : 200, 4477S, Cell Signaling Technology, Danvers, MA) and rabbit anti-Ki67 monoclonal antibody (1 : 900, ab16667, Abcam, Cambridge, MA) at 4°C overnight.

Techniques: Migration, Wound Healing Assay, Transfection, Expressing, Plasmid Preparation, Negative Control, Transwell Invasion Assay, Staining