Article Figures & Data
Figures
- Figure 1.
Effects of HDAC inhibitors and calpeptin on the growth and cell survival of MCF-7 and MDA-MB-231 breast cancer cells. A: MCF-7 cells were exposed to either sodium butyrate SB (0.25 mM), SAHA (7.5 μM), calpeptin (Calp, 10 μg/ml) or combination of SB and calpeptin (p value = 0.0001) or SAHA and calpeptin (p value = 0.0001) at the same concentrations. B: MDA-MB-231 cells were exposed to either SB (0.5 mM), SAHA (7.5 μM), calpeptin (7.5 μg/ml) or combination of SB and calpeptin (p-value = 0.0001) or SAHA and calpeptin (p-value = 0.0001) at the same concentrations. After 96 h, cells were washed, trypsinized, and the viable cells were counted. Results are expressed relative to the numbers of vehicle-treated (control) cells, arbitrarily assigned a value of 100%. The dark columns represent the control cells and the striped columns represent the cells exposed to inhibitors. C: MCF-7 cells were treated with inhibitors as described in A. D: MDA-MB-231 cells were treated as described in B. After 96 h, the supernatants were collected and a lactate dehydrogenase cytotoxic assay was performed. The results are expressed as the percent cell death compared to the amount of total lactate dehydrogenase present in total untreated cells.
- Figure 2.
Effects of HDAC inhibitors and calpeptin on cell cycle profile and apoptosis of MCF-7 and MDA-MB-231 breast cancer cells. A: MCF-7 cells were treated SB (0.25 mM) or SAHA (7.5μM). B: MDA-MB-231 cells were treated SB (0.5 mM) or SAHA (7.5 μM). After 48 h cells were stained with propidium iodide (PI) and cell cycle profiles were analyzed by flow cytometry. Vehicle-treated cells served as a control. Results are expressed as the percentage of cells in a particular phase of the cell cycle. C: MCF-7 cells were treated with 10μg/ml calpeptin. D: MDA-MB-231 cells were treated with 7.5 μg/ml calpeptin. After 48 h cells were stained with FITC-conjugated annexin antibody (FL1 channel) and propidium iodide (PI, FL2 channel). Apoptosis profiles were analyzed by flow cytometry. Vehicle exposed cells served as controls. E: Graphical depiction of apoptotic fractions. The data from panel C and D was expressed as percent of cells undergoing apoptosis (annexin-positive, annexin/PI-positive, PI positive fractions).
- Figure 3.
DNA methylation analysis ARHI promoter and transcript level. (A) MCF-7 and (B) MDA-MB-231 cells were treated with SB (1mM) or SAHA (10μM). Genomic DNA was isolated and treated with bisulfite. After purification, methylation-specific PCR (MS-PCR) was performed for each gene. The lower panel shows the total actin in each sample, as a loading control. (C) MDA-MB-231 cells were treated with SB (0.5 mM) or SAHA (7.5 μM), calpeptin (7.5 μg/ml) or combination of SB and calpeptin and combination of SAHA and calpeptin at the same concentrations for 48 h. Total RNA was isolated and cDNA was prepared. Real-time PCR (qPCR) for ARHI transcripts was performed. Results for each gene in each sample were normalized to the level of β-actin transcripts. The results are expressed as fold increase over the untreated control. The results are the average of 3 sets of independent experiments. Bars indicate the standard deviation.
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