To normalize readings, we used Ct values from 18?s as internal controls for each run, obtaining a delta Ct value for each gene. Small-interfering RNA The specific small-interfering RNA of GADD45 (Stealth RNAi siRNA Duplex Oligoribonucleotides) and Lipofectamine RNAiMAX gene transfection system were purchased from Invitrogen (Thermo Fisher Scientific, USA). malignant tumor common in Southeast Asia and Taiwan. The age of NPC onset tends to be younger than that of other tumors, affecting most patients at approximately 30C50 years of age1. Infections with Epstein-Barr virus, genetic predisposition, as well as various dietary and environmental factors are believed to play important roles in the development of carcinogenesis2. Radiotherapy is the mainstay of treatment, for which the five-year survival rate is approximately 25%3. Cucurbitacins are a group of tetracyclic triterpenes with medicinal properties derived from the climbing stem of 0.05 versus the control group. Non-CuE-induced apoptosis/necrosis of Detroit 562 and HONE-1 cells To identify the role played by CuE in the apoptosis/necrosis of Detroit 562 and HONE-1 cells, we employed Annexin V-FITC and propidium iodide staining to reveal the formation of apoptotic cells following 4?hours of exposure to CuE. The percentage of apoptotic cells was assessed by flow cytometric analysis (Supplemental Figure S1A and Figure S1B). A dot-plot of Annexin V-FITC fluorescence versus PI fluorescence indicates a nonsignificant increase in the percentage of apoptotic cells treated with CuE, compared with untreated cells. No significant increase was observed in the percentage of cells undergoing necrosis, apoptosis (Supplemental Figure S1C) or caspase 3 activation at CuE concentrations of 0.625 to 2.5?M (Supplemental Figure S2A, Figure S2B and Figure S2C). However, the results summarized in Supplemental Figure S1 and Figure S2 indicate that CuE may mediate the survival of Detroit 562 and HONE-1 cells. Thus, we hypothesize that the proliferation of these cells was inhibited by pathways other than apoptosis/necrosis. CuE-induced accumulation of G2/M phase in CuE-treated cells The cell-cycle distribution of CuE-treated cells Syringic acid was analyzed by flow cytometry. Cells were exposed to CuE for 24?hours Syringic acid prior to processing and analysis. As shown in Figure 2(A), exposure to CuE resulted in an increase in the number of G2/M phase, cells, which may imply that the Detroit 562 and HONE-1 cells underwent cell cycle arrest. Our results indicate that treatment with CuE increased the cell populations in G2/M phase, while simultaneously reducing the number of cells in the S and G1 phases (* p 0.05 vs CuE 0?M) (Figure 2B). Open in a Syringic acid separate window Figure 2 Influence of CuE on cell cycle progression/distribution in Detroit 562 and Hone-1 cells: (A) Cell cycle analysis of Detroit 562 and Hone-1 cells after being cultured with CuE for 24?h. (B) CuE induced an increase in G2/M phase cells (%). (C) MPM-2 (anti-phospho-Ser/Thr-Pro) expression in untreated and treated cancer cells. MPM-2 is an antibody that recognizes proteins which are only phosphorylated in mitosis. Cells were dually stained using propidium iodide to analyze DNA content and protein expression was quantified by flow Tnfsf10 cytometry. As a positive control, separate groups of cells were treated for 24?h with nocodazole (15?g/mL), an anti-fungal agent known to induce metaphase arrest. Cell-cycle analysis and quantification of MPM-2 expression (gated cells) were performed by flow cytometry following treatment with CuE for 24?h. (D) CuE enhanced the level of MPM-2 in Detroit 562 and Hone-1 cells. Symbol (*) in each group of bars indicates that the difference resulting from treatment with CuE 0?M is statistically significant at P 0.05. Effects of CuE on the mitotic Syringic acid index To distinguish G2 arrest from mitotic arrest, we employed an additional marker, MPM-2 (anti-phospho-Ser/Thr-Pro). Syringic acid This antibody is capable of recognizing proteins whose epitopes are exclusively phosphorylated during mitosis, specifically from early prophase to metaphase13. MPM-2 is commonly used as an indicator of mitotic disturbance. To provide a positive control, we treated separate groups of Detroit 562 and HONE-1 cells with nocodazole (15?g/mL), an inducer of metaphase arrest14. Treating the two types of cells with nocodazole for 24?hours resulted in synchronization of entire cell populations in the G2/M phase as well as an increase in MPM-2 labeling (Figure 2C and 2D). Among all cells treated with CuE, the MPM-2 level was elevated compared with control group (19% and 31% for Detroit 562 and Hone-1 cells treated with CuE, respectively) (Figure 2D). However, MPM-2 staining was not as strong as that achieved with nocodazole. This is likely because MPM-2 stained cells were in various stages of mitosis, some of which could not be identified using this early prophase marker. Specifically, the accumulated.