Fig. 4.

LLM-induced ROS partially contribute to apoptotic cell death in human breast cancer cells. A Representative confocal images for MitoSOX Red fluorescence (63× objective magnification; scale bar = 20 μm) in MitoGFP-MDA-MB-231, MitoGFP-MCF-7, and SUM159 cells treated with ethanol or 5 μM of LLM for 6 hours. B Quantitation of MitoSOX Red fluorescence enrichment relative to corresponding control in MDA-MB-231, MCF-7, and SUM159 cells after 3- or 6-hour treatment with LLM. Results shown are mean ± SD (n = 3). *Statistical significance compared with ethanol-treated control was analyzed by Student’s t test (P < 0.05). C Quantitation of histone-associated DNA fragment release into the cytosol in stably transfected MCF-7 cells after treatment with ethanol (control) or the indicated concentrations of LLM for 24 hours (Inset: Immunoblotting for MnSOD using lysates from pcDNA3.1 vector-transfected control cells or MnSOD-overexpressing MCF-7 cells). Results shown are mean ± SD (n = 3). Statistically significant (P < 0.05) ^acompared with corresponding control and ^b between pcDNA3.1 transfected and MnSOD overexpressing cells by one-way ANOVA with Newman-Keuls multiple comparisons test. D Quantitation of histone-associated DNA fragment release into the cytosol in wild-type (WT) MDA-MB-231 cells and its Rho-0 variant after 24-hour treatment with ethanol (control) or the indicated concentrations of LLM. The enrichment results shown are relative to ethanol-treated WT cells. E The effect of LLM treatment (24 hours) on viability in WT and Rho-0 MDA-MB-231 cells. The results shown are relative to ethanol-treated WT MDA-MB-231 cells (mean ± SD; n = 3). Statistically significant (P < 0.05) ^a compared with corresponding control and ^b between WT and Rho-0 cells by one-way ANOVA with Newman-Keuls multiple comparisons test (D and E). Each experiment was done at least twice and representative data from one such experiment are shown.