Fig. 7

Effects of ATRA on the number of mitochondria. a and c Biological triplicates of retinoid-sensitive SK-BR-3 and retinoid-resistant HCC-1419 cells were treated with vehicle (DMSO) or ATRA (10^− 6 M) for the indicated amount of time. The left pictures show representative fluorescence images of the indicated SK-BR-3 and HCC-1419 cells treated for 24 h before staining with Hoechst for the cell nuclei (blue fluorescence) and Mitotracker to highlight the mitochondria (red fluorescence). The column diagrams on the right indicate the time-course of the effect exerted by ATRA on the number of mitochondria. The results were obtained from the quantitative image-analysis of Mitotracker fluorescence in at least 4 fields/experimental triplicate. b and d The left diagrams indicate the effect exerted by ATRA on the number of mitochondria in SK-BR-3 (b) and HCC-1419 (d) cells, as assessed by FACS analysis of Mitotracker fluorescence (Mean ± SD, N = 3), following 48 h of treatment. The right diagrams illustrate the total amounts of proteins determined in mitochondria isolated from SK-BR-3 (b) and HCC-1419 (d) cells. Mitochondria were isolated from the same number of cells and the amount of mitochondrial proteins was determined. The results are expressed as the ratio of mitochondrial protein over the total amount of cellular proteins. e Three independent cultures of SK-BR-3 cells per experimental point were treated with the indicated concentrations of ATRA for 48 h. At the end of the treatment, cells were stained with Hoechst for the cell nuclei (blue fluorescence) and Mitotracker to highlight the mitochondria (red fluorescence) as in (a) and (c). The column diagram shows the effect exerted by ATRA on the number of mitochondria. The results were obtained from the quantitative image-analysis of Mitotracker fluorescence in at least 4 fields/experimental triplicate. f and g Biological triplicates of retinoid-sensitive MD-MB-361 and the retinoid-resistant HCC-202 cells were treated with vehicle (DMSO) or ATRA (10^− 6 M) for 48 h. The upper pictures show representative fluorescence images of the indicated MD-MB-361 and HCC-202 cells treated for 24 h before staining with Hoechst and Mitotracker. The lower left diagrams show the effects exerted by ATRA on the number of mitochondria, as in (a), (b) and (e). The results were obtained from the quantitative image-analysis of Mitotracker fluorescence in at least 4 fields/experimental triplicate. The lower right diagrams indicate the effect exerted by ATRA on the number of mitochondria in MD-MB-361 (f) and HCC-202 (g) cells, as assessed by FACS analysis of Mitotracker fluorescence (Mean ± SD, N = 3), following 48 h of treatment. Each value is expressed as the Mean + SD (N = 3). *Significantly different (p < 0.05, Student’s t-test); **Significantly different (p < 0.01, Student’s t-test)