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

Figure 2. Senescent HCT116 cells exhibit certain features of cancer stem cells and re-populate in vitro culture after doxorubicin removal.

Figure 2

(A) Quantification of cell number at various time points of the AFTER CHEMO protocol. Cells were counted in Bürker's chamber. (B) Senescent cells produce progeny. HCT116 cells were seeded in 6-well plate and their behaviour was recorded starting from the 17th day of the AFTER CHEMO protocol. Representative photos were acquired by time-lapse imaging. Progeny originating from senescent, hypertrophic cells is indicated by black arrows. Original magnification 160×. (C) Evaluation of percentages of CD24+ cells on the 13th day (CHEMO). Cells were incubated with an anti-CD24-FITC antibody and percentages of positive cells were determined by flow cytometry. Cells labeled with an isotypic IgG were used as a negative control. (D) Quantification of H33342 excluding cells by flow cytometry. (E) Quantification of side population (SP) on the 13th day (CHEMO). Cells stained with H33342 were co-incubated with Verapamil and analyzed by flow cytometry. SP = a percentage of cells excluding H33342 in the absence of Verapamil minus a percentage of cells excluding H33342 when Verapamil is present. (F) Representative blot shows the expression of NANOG. GAPDH served as a loading control. (G) HCT116 cells persist in non-proliferating state during doxorubicin administration. Cells were stained with lipophilic, red-fluorescent dye Dil on the 1st day and the percentage of cells retaining Dil+ was determined with flow cytometry, at each time point. Unstained cells were used as a negative control. (H) Representative photos show Ki67 staining in untreated and treated cultures on the 13th day (CHEMO); immunofluorescence for Ki67-visualized as green (AlexaFluor 488), nuclei-as grey (H33342). Original magnification 400×. Data acquired by fluorescent confocal microscopy. (I) Automatic evaluation of a percentage of Ki67 positive cells. Each bar represents mean ± SEM, N ≥ 3; statistical significance #p < 0.05, ## p < 0.01, ###p < 0.001-untreated vs. CHEMO (day 13th); $p < 0.05, $$p < 0.01, $$$p < 0.001 – AFTER CHEMO vs. day 13th (CHEMO).