Cell Cycle Arrest Flow Cytometry

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Ailanthone is a major quassinoid derived from the Chinese herbal medicine Ailanthus altissima, which is believed to have antiproliferative effects on various cancer cells. The present study aims to investigate the antitumor effect of ailanthone on SGC-7901 cells and analyze the underlying molecular structure. After ailanthone treatment, an 8-cell counting kit was used to detect the cytotoxic effects of ailanthone on SGC-7901 cells in vitro. Typical apoptotic morphology of SGC-7901 cells was observed by Hoechst 33258 staining. Cell cycle progression and apoptosis were measured by flow cytometry, and protein and mRNA levels in Bcl-2 and Bax were measured. were analyzed by Western blot analysis and quantitative polymerase chain by transcription. reaction (RT-qPCR) respectively, in SGC-7901 cells. . The results of the present study indicated that ailanthone inhibited the proliferation of adjoining and time-dependent SGC-7901 cells in vitro, and also showed that ailanthone induced G2/M cell cycle arrest and apoptosis of SGC-7901 cells. Furthermore, basic molecular mechanics analysis revealed that ailanthone reduced Bcl-2 expression levels, while Bax levels were elevated by protein and mRNA levels. In conclusion, ailanthone can inhibit the proliferation of SGC-7901 cells by inducing G2/M cell cycle uptake and apoptosis by altering protein and mRNA levels of Bcl-2 and Bax in SGC-7901 cells. 7901.

Gastric cancer (GC) is one of the deadliest tumors in the world. According to GLOBOCAN estimates, GC is the fourth most common cancer, with the third highest cancer mortality rate among men worldwide (1). Additionally, GC is more prevalent in East Asian countries (2). It is currently accepted that surgery is the only effective treatment for GC; However, chemotherapy is the most important treatment for patients with advanced GC, which can improve quality of life and increase patient survival (3). However, chemotherapy can cause a number of problems, including drug resistance and side effects; therefore, it is not considered appropriate for the treatment of advanced GCs. Therefore, there is a need to develop effective antitumor drugs, which have high efficacy and low toxicity.

Cell Cycle Arrest Flow Cytometry

Cell Cycle Arrest Flow Cytometry

Today, most antitumor drugs are natural products derived from plants, which have been used in the treatment of cancer, including vincristine, paclitaxel and docetaxel (4, 5). The Chinese medicinal herb Ailanthus altissima has long been used to treat colds and stomach ailments in traditional Chinese medicine (6). Ailanthone, derived from Ailanthus altissima (7), is a type of quassinoid that has been reported to have a pronounced antitumor effect. Previous studies have reported that ailanthone has many biological activities, including antimalarial, antibacterial, and anti-inflammatory activities (8, 9). Previous research has indicated that ailanthone affects the growth of HeLa, Jurkat, HepG2, Hep3B, R-HepG2, MCF-7, MDA-MB-231 and A549 tumor cells in vitro (10-13). ). Additionally, ailanthone promotes apoptosis in Huh-7 cells and inhibits the growth of Huh-7 tumors in nude mouse xenograft models, with no adverse side effects (14). Apoptosis is a type of regulated cell death, which is controlled by genetic levels and results in the destruction of damaged cells (15). Anti-apoptotic mechanisms play an important role in cancer growth and progression, and are considered cancer markers and may be a factor in adverse effects on cancer treatment (16, 17). Promoting apoptosis is known to be an effective method of treating cancer; Therefore, ailanthone can be used to treat tumors in the future.

Cell Cycle Arrest Induced By Aphidicolin (a) And Cell Cycle…

The effect of ailanthone has not been reported in GC cells. The present study aims to investigate the effect of ailanthone on SGC-7901 human GC cells and elucidate the molecular mechanics potential in vitro.

Clean ailanthones (Fig. 1A) were extracted and isolated from Ailanthus altissima. Ailanthone samples (purity ≥98%) were provided by the Institute of Traditional Chinese Medicine and Natural Products, University of Jinan (Guangzhou, China). Taxol is obtained from Beijing SL Pharmaceutical Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Cell Count-8 (CCK-8) Kit (Cat. No. KGA317) was obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanking, China). RPMI-1640 (cats # 11875-093) and penicillin-streptomycin (PS; cats # 15140-122) were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Fetal bovine serum (FBS; cat no. 100-700) is obtained from Gemini Bio Products (West Sacramento, CA, USA). Antibodies against monoclonal mouse β-actin (cat. no. BM0626), monoclonal mouse B-cell lymphoma 2 (Bcl-2; cat. no. BM0200) and polyclonal rabbit Bcl-2-related protein X ( Bax; catalog no. BA0315-2) was purchased from Wuhan Boster Biological Technology Ltd. (Wuhan, China). Horseradish Peroxidase (HRP) Conjugated Goat Anti-Mouse (Cat. No. 62-6520) and Anti-Rabbit (Cat. No. G-21234) Immunoglobulin (Ig) G is derived from Invitrogen; ThermoFisher Scientific, Inc.

SGC-7901 human GC cell line (cat. No. KG026) is from Nanjing KeyGen Biotech Co., Ltd. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% PS in an incubator containing 5% CO2 and 95% air at 37°C for cell subculture and all experiences. Ailanthone stock solution was prepared in DMSO, and stored at 20°C. Before use, the stock solution was immediately purified to the required concentration of complete RPMI -1640; the terminal concentration of DMSO in the culture medium was ≤ 0.1%. Control cells were treated with DMSO (0.1%), without ailanthone or taxol.

The CCK-8 assay was used to measure cell viability. Taxol was used in a well-controlled control group. Growth-stage SGC-7901 cells (5×103 cells/length) were incubated and cultured in 96-length plates for 24 h, then treated with 0.1% DMSO (follow-up group.-maso), ailanthone ( 0.5, 1, 2). , 4 and 8 μM) or taxol (1.25, 2.5, 5, 10 and 20 μM) for 24, 48 and 72 h at 37°C, each group was examined four times. Then 10 µl of CCK-8 solution was added to each well. After 3 h at 37°C, the optical density was measured at a wavelength of 450 nm using a microplate reader (RT-6000; Rayto Life and Analytical Sciences Co., Ltd., Shenzhen , China). Right cell viability was determined using the following formula: Relative cell viability = (mean A450 in test group/mean A450 in control group) × 100%.

The Ubiquitin Peptidase Uchl1 Induces G0/g1 Cell Cycle Arrest And Apoptosis Through Stabilizing P53 And Is Frequently Silenced In Breast Cancer

Hoechst 33258 staining was used to examine the apoptotic morphology of cells. Cells from the SGC-7901 incubator were incubated on glass plates in 6-length plates (3 × 105 cells/hole) for 24 h, then treated with 0.1% DMSO or ailanthone (1 and 2 M) for 48 h at 37°. C. Cells were dried and placed in 4% paraformaldehyde for 30 min at room temperature. Cells were then washed three times with phosphate buffered saline (PBS) and stained with 10 µg/ml Hoechst 33258 (Catalogue No. KGA211-10; Nanjing KeyGen Biotech Co., Ltd.) for 10 minutes at ambient temperature. . . Cells were washed twice with PBS and placed with an antifade coater (cat no. P0126; Beyotime Institute of Biotechnology, Shanghai, China). Finally, nuclear morphology was observed by fluorescence microscopy (Olympus BX43; Olympus Corporation, Tokyo, Japan).

Growing SGC-7901 cells were selected and cultured in 6-well plates (3 × 105 cells/well) for 24 h, then treated with 0.1% DMSO or ailanthone (1-4 μM) for 48 hours at 37°C. Cells were washed twice with cold PBS and then incubated with APC/7-ADD Annexin V (Catalogue No. KGA1026; Nanjing KeyGen Biotech Co., Ltd.) for 15 min at room temperature in a dark room. according to the manufacturer. protocol. Then the cells were examined by flow cytometry. Fluorescence was measured using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The experiment was repeated three times independently and the proportion of apoptotic cells was calculated using the internal software system FACSCalibur (BD Biosciences).

SGC-7901 cells were selected and cultured in 6-well plates (3 × 105 cells/well) for 24 h, then treated with 0.1% DMSO or ailanthone (1–4 μM) for 48 hours at 37°C. The cells were harvested, washed with PBS and placed in 70% ethanol at 4°C overnight. Subsequently, the cells were incubated with 1% RNase A at 37°C for 30 min and with a solution of propidium iodide (cat No. KGA511; Nanjing KeyGen Biotech Co., Ltd.) in’ at 4 °C for 30 min in the dark. The DNA content of the cells was measured using a FACSCalibur flow cytometer (BD Biosciences). Experiments were repeated three times independently and data were analyzed using MultiCycle DNA content and cell cycle analysis software (FlowJo, version 7.6.5; FlowJo LLC, Ashland, OR). , United States).

Cell Cycle Arrest Flow Cytometry

After treatment with 0.1% DMSO or ailanthone (1-4 μM) for 48 h at 37°C, SGC-7901 cells were washed twice with cold PBS and suspended by assay of radioimmunoprecipitation with lysis buffer (cat no. KGP702; Nanjing KeyGen Biotech Co., Ltd.) on ice for 30 min. The lysate was removed by centrifugation at 12,000 × g for 15 min at 4°C. Then the bicinchoninic acid protein test kit (cat No. KGP902; Nanjing KeyGen Biotech Co., Ltd.) was used to measure the total protein content of each sample according to the manufacturer’s protocol. Protein samples (30 µg) from each group were separated by 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pall Corporation, Port Washington, NY,

Cell Cycle Analysis

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