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. 2016 Dec 21;8(6):9303–9322. doi: 10.18632/oncotarget.14066

Figure 7. A single pulse of BAF A1 transiently impairs ALDH activity and H33342 exclusion in senescent HCT116 cells.

Figure 7

(A) Evaluation of percentages of HCT116 cells treated with the AFTER CHEMO protocol or the AFTER CHEMO + BAF A1 protocol showing ALDH activity at various time points. Cells co-incubated with DEAB, an ALDH inhibitor, served as a negative control. (B) Evaluation of percentage of HCT116 cells showing ALDH activity on the 17th day. Cells stained with H33342, as a ploidy discriminator, were stained for an ALDH activity. Percentage of cells with: 2C&4C, 8C and 16C DNA content displaying ALDH activity were determined by flow cytometry. (C) Quantification of cells excluding H33342 by flow cytometry. (D) Quantification of side population (SP). SP = a percentage of cells excluding H33342 in the absence of Verapamil minus a percentage of cells excluding H33342 in the presence of Verapamil. Cells stained with H33342 were co-incubated with Verapamil and analyzed by flow cytometry. (E) Visualization of spheroids in matrigel formed by untreated and doxo-treated cells co-incubated with BAF A1. Doxo-treated cells (1 000 cells) were seeded on the 14th day just after BAF A1 removal onto 96-well plate. Media were replaced every week. Representative photos were taken three weeks after seeding. Scale bar–100 μm. (F) Evaluation of the number of spheroids formed by untreated or doxo-treated cells co-incubated with BAF A1. (G) Evaluation of size of spheroids formed by untreated or doxo-treated cells co-incubated with BAF A1. Spheroids in the middle of well (hot spot) were calculated. Each bar represents mean ± SEM, N ≥ 3.*p < 0.05, **p < 0.01, ***p < 0.001–AFTER CHEMO vs. AFTER CHEMO + BAF A1.