Journal: Experimental and Therapeutic Medicine

Article Title: CCR3-shRNA promotes apoptosis and inhibits chemotaxis and degranulation of mouse mast cells

doi: 10.3892/etm.2020.8737

Figure Lengend Snippet: CCR3 interference promotes MC apoptosis. After 96 h of transfection, flow cytometry was performed following AnnexinV-FITC/PI staining. (A) Flow cytometry analysis of MCs transfected with CCR3-shRNA2. (B) Quantification of apoptosis in each group. Data are expressed as the mean ± standard deviation (n=3). * P<0.05 vs. control group. CCR3, C-C chemokine receptor type 3; FITC, fluorescein isothiocyanate; MC, mast cell; PI, propidium iodide; shRNA, short hairpin RNA.

Article Snippet: TRIzol reagent (cat. no. CW0580S), Ultrapure RNA extraction kit (cat. no. CW0581M), HiFiScript cDNA synthesis kit (cat. no. CW2569M) and UltraSYBR Mixture (cat. no. CW0957M) were all purchased from Beijing CoWin Biotech Co., Ltd. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis kit (cat. no. AP101-100-kit) was purchased from Hanzhou Multi Sciences (Lianke) Biotech Co., Ltd. RIPA Lysis Buffer (cat. no. C1053) was purchased from Applygen Technologies, Inc. SuperSignal ® West Pico chemiluminescent substrate (cat. no. RJ239676) was obtained from Thermo Fisher Scientific, Inc. Polyvinylidene difluoride (PVDF) membranes (cat. no. IPVH0001) were purchased from Merck KGaA.

Techniques: Transfection, Flow Cytometry, Staining, Standard Deviation, Control, shRNA

Journal: Experimental and Therapeutic Medicine

Article Title: CCR3-shRNA promotes apoptosis and inhibits chemotaxis and degranulation of mouse mast cells

doi: 10.3892/etm.2020.8737

Figure Lengend Snippet: CCR3 interference suppresses MC chemotaxis. (A) Representative images of flow cytometry results for MC chemotaxis from each group. (B) Quantification of the percentage of migrated c-kit cells in each group. Data are expressed as the mean ± standard deviation (n=3). * P<0.05 vs. control group. CCR3, C-C chemokine receptor type 3; FITC, fluorescein isothiocyanate; MC, mast cell; PE, phycoerythrin; shRNA, short hairpin RNA.

Article Snippet: TRIzol reagent (cat. no. CW0580S), Ultrapure RNA extraction kit (cat. no. CW0581M), HiFiScript cDNA synthesis kit (cat. no. CW2569M) and UltraSYBR Mixture (cat. no. CW0957M) were all purchased from Beijing CoWin Biotech Co., Ltd. Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis kit (cat. no. AP101-100-kit) was purchased from Hanzhou Multi Sciences (Lianke) Biotech Co., Ltd. RIPA Lysis Buffer (cat. no. C1053) was purchased from Applygen Technologies, Inc. SuperSignal ® West Pico chemiluminescent substrate (cat. no. RJ239676) was obtained from Thermo Fisher Scientific, Inc. Polyvinylidene difluoride (PVDF) membranes (cat. no. IPVH0001) were purchased from Merck KGaA.

Techniques: Chemotaxis Assay, Flow Cytometry, Standard Deviation, Control, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: The production of IL-6 is higher in human breast cancer cell line MDA-MB-231 than in MCF-7. MDA-MB-231 and MCF-7 cells were seeded in six-well plates at a density of 5 × 10 5 cells/well and incubated overnight. After incubation, the supernatants were harvested for Cytokine array assay or ELISA. ( a ) Production of cytokines from MDA-MB-231 and MCF-7 cells. ( b ) Density of spots as compared with positive control (POS). ( c ) Production of IL-6 from MDA-MB-231 and MCF-7 cells. Significant difference is shown: * p < 0.05. NEG, negative control; GCSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; GRO a/b/g, growth-regulated oncogene-a/b/g.

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: Blockade of IL-6 expression decreases the levels of pSTAT3, PI3K, and pAkt in MDA-MB-231 cells. MDA-MB-231 cells were treated with anti-IL-6 antibody or IL-6 shRNA. After 24 h of incubation, cell lysates were harvested and analyzed by western blotting. ( a ) Expression of PI3K, STAT3 (and pSTAT3), and ERK (and pERK) proteins. ( b ) Expression of IL-6, STAT3 (and pSTAT3), PI3K, and pAkt proteins. ( c ) Morphology of MDA-MB-231 cells (scale value is 100px). Significant difference is shown: * p < 0.05.

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Expressing, shRNA, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: Blockage of IL-6 expression decreases cyclin-dependent kinases (CDK) and cyclin protein expression and induces p21 expression in MDA-MB-231 cells. MDA-MB-231 cells were seeded in six-well plates at a density of 5 × 10 5 cells/well and treated with IL-6 shRNA or control shRNA. Cell lysates were harvested and analyzed by western blotting. ( a ) Expression of p53 and p21 proteins. ( b ) Expression of CDKs (CDK2, CDK4, and CDK1) and cyclins (cyclin D1 and cyclin B1). Significant difference is shown: * p < 0.05.

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Expressing, shRNA, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: Blockade of IL-6 expression inhibits invasion and metastasis factors in MDA-MB-231 cells. MDA-MB-231 cells were seeded at a density of 2 × 10 5 cells/mL in a transwell and treated with anti-IL-6 antibody or IL-6 shRNA. After 12 h of incubation, cells were stained with crystal violet dye for invasion assay and cell lysates were harvested for western blotting. ( a ) Invasive capability of MDA-MB-231 cells (scale value is 100px). ( b ) Expression of E-cadherin and N-cadherin proteins. ( c ) Meta-analysis-based biomarker assessment with Kaplan–Meier Plotter showed a negative correlation between the blood level of IL-6 and relapse-free survival in patients with Triple-negative breast cancer (TNBC).

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Expressing, shRNA, Incubation, Staining, Invasion Assay, Western Blot, Biomarker Assay

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: Blockade of IL-6 expression inhibits MDA-MB-231-derived tumor growth and metastasis. MDA-MB-231 cells were transfected with IL-6 shRNA or control shRNA lentivirus plasmid. BALB/c nude mice were subcutaneously injected with 5 × 10 6 MDA-MB-231 cells transfected with IL-6 shRNA or control shRNA into the dorsum next to right hind leg. After 14 days, tumor volume was measured using caliper every other day. ( a ) Tumor growth curve. ( b ) Tumor photograph. Expression of proteins in tumor tissues analyzed with western blotting and immunohistochemistry (IHC). Expression of STAT3 (and pSTAT3), ERK (pERK), PI3K, pAkt, snail, vimentin, and N-cadherin ( c ). Black arrows indicate the dots of pSTAT3, pAkt, and N-cadherin ( d ) (scale value is 100px). Significant differences are shown: * p < 0.05, ** p < 0.001.

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Expressing, Derivative Assay, Transfection, shRNA, Plasmid Preparation, Injection, Western Blot, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: Apigenin treatment decreases the expression of snail and N-cadherin via IL-6 inhibition in MDA-MB-231 cells. MDA-MB-231 cells were plated at a density of 5 × 10 5 cells/well in a six-well plate and incubated with different concentrations of apigenin (10 to 50 μM) for 24 h. The cells were harvested for western blot analysis and qPCR, and the supernatants were used for ELISA. ( a ) The structure of apigenin. ( b ) Production of IL-6 by ELISA. ( c ) Relative mRNA expression of IL-6 by qPCR. ( d ) Expression of IL-6, snail, and N-cadherin proteins by western blot analysis. Migration and invasiveness of MDA-MB-231 cells were assessed using scratch motility assay and invasion assay, respectively. ( e ) Migratory capability of MDA-MB-231 cells. For scratch motility assay, cells were plated at a density of 2 × 10 5 cells/well in a 24-well plate and treated with different concentrations (0, 10, 20, and 40 μM) of apigenin for different time points (0, 6, and 12 h). ( f ) Invasive capability of MDA-MB-231 cells. For invasion assay, a transwell insert was coated with Matrigel. Cells were seeded in the upper chamber of transwell at a density of 2–5 × 10 5 cells/mL and treated with different concentrations of apigenin (0, 10, 20, and 40 μM). After 24 h of incubation, cells were stained with crystal violet (scale value is 100px). Significant differences are shown: * p < 0.05, ** p < 0.001, *** p < 0.002.

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Expressing, Inhibition, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Motility Assay, Invasion Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Antitumor and Anti-Invasive Effect of Apigenin on Human Breast Carcinoma through Suppression of IL-6 Expression

doi: 10.3390/ijms20133143

Figure Lengend Snippet: The inhibitory effect of apigenin on the growth and metastasis of a MDA-MB-231 cell-derived tumor is mediated through the reduced expression of pSTAT3, pERK, IL-6, and pAkt. BALB/c nude mice were subcutaneously injected with 5 × 10 6 MDA-MB-231 cells into the dorsum next to right hind leg. After 14 days, the mice were orally administrated drinking water or apigenin (25 or 50 mg/kg) every day for another 2 weeks. Tumor size was measured by caliper every other day. ( a ) Tumor growth curve. ( b ) Tumor photograph. ( c,d ) Expression of proteins in tumor tissues analyzed by western blotting. ( e ) Expression of proteins in tumor tissues analyzed by immunohistochemistry (IHC). Expression of STAT3 (and pSTAT3), ERK (pERK), PI3K, and pAkt proteins (c,d). Black arrows indicate the dots of pSTAT3, pAkt, and N-cadherin (e) (scale value is 100px). Significant differences are shown: * p < 0.05, ** p < 0.001.

Article Snippet: Human IL-6 shRNA lentivirus plasmid with pGFP vector kit (#TL312162, Origene, Rockville, MD, USA) was used for blocking IL-6 expression in MDA-MB-231 cells.

Techniques: Derivative Assay, Expressing, Injection, Western Blot, Immunohistochemistry