Fig. 10

Effects of ATRA on the growth, CL levels, the amounts of mitochondria and the mitochondrial membrane microviscosity in RARα over expressing MDA-MB-453 and RARα knock-down SK-BR-3 cells. RARA-C5 are MDA-MB-453 cells stably transfected with a plasmid construct containing the RARα cDNA, while Vect-C1 are control MDA-MB-453 cells stably transfected with the void vector. RARA-sh18 are SK-BR-3 cells stably infected with a retrovirus allowing the expression of a shRNA targeting RARα, while Vect-C6 are control SK-BR-3 cells stably infected with the void retroviral vector. a and b Left: Triplicate cultures of the indicated cells were treated with vehicle and the indicated concentrations of ATRA for 6 days. At the end of the treatment cell growth was determined with the use of a sulforhodamine assay. Right: The amounts of RARα protein were determined by Western Blot analysis in the indicated cells following treatment with vehicle (DMSO or ATRA (10^− 6 M) for 24 h. Tubulin was used as a loading control. c and e Biological triplicates of the indicated breast-cancer cells were treated with vehicle (DMSO) or ATRA (10^− 6 M) for 48 h. The box plots show the median ± SD levels of cardiolipins (CLs). The number of different CL molecules identified by mass-spectrometry is indicated in parenthesis. d and f Biological triplicates of the indicated breast-cancer cells were treated with vehicle (DMSO) or ATRA (10^− 6 M) for 48 h. At the end of the treatment, cells were stained with Mitotracker, fixed in 2% formalin and subjected to FACS analysis. The colored left diagram show representative FACS diagrams, while the colored right bar graphs show the quantitative results obtained in biological triplicates. As for the results presented in the black and white column bargraphs of panels d and f, three replicate cultures of the indicated cells were treated with vehicle (DMSO) or ATRA (10^− 6 M). In particular, Vect-C1 and RARA-C5 cells were treated for 5 days, while Vect-C6 and RARA-sh18 cells were treated for 2 days. At the end of the treatment, mitochondria were isolated and incubated with 1,6-diphenyl-1,3,5-hexatriene to assess the microviscosity of mitochondrial membranes. The values are expressed as the Mean ± SD of the membrane microviscosity values, following measurement of fluorescence anisotropy (N = 3). Panels (a-f): **Significantly different relative the vehicle treated controls (p < 0.01, Student’s t-test). *Significantly different relative the vehicle treated controls (p < 0.05, Student’s t-test)