Fig. 2

HMA stimulates ROS formation and mitochondrial membrane depolarization and affects DNA integrity. Effect of scavengers. a Quantification of ROS by cytofluorimetric analysis of DCF (dichlorofluorescein) fluorescence in HCT-116 cells treated for increasing times (4 h to 24 h) with HMA (30 μM) alone, or co-incubated with HMA and antioxidants (15 mM NAC or 400 μM α-Tocopherol, Toc) for the same time periods. b Measurement of mitochondrial membrane potential by cytofluorimetric analysis of TMRM (tetramethylrodamine methylester) fluorescence in HCT-116 cells treated as in (a). In a and b, data are expressed as fold increase with respect to untreated cells (mean ± s.d. calculated on three independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001. c Detection of DNA damage by 8-oxoguanine immunostaining of untreated and HMA (20 μM, 24 h)-treated HCT-116 cells; parallel samples were incubated simultaneously with HMA and with 15 mM NAC. d Detection of γ-H2AX by immunostaining in untreated and HMA treated (20 μM, 24 h) HCT-116 cells. Scale bar: 50 μm. A representative experiment out of three is shown in panels (c) and (d), where nuclei were counterstained with DAPI (blue fluorescence)