FIGURE 2 from Pharmacologic Targeting of Histone H3K27 Acetylation/BRD4-dependent Induction of ALDH1A3 for Early-phase Drug Tolerance of Gastric Cancer

Induction of ALDH1A3-overexpressing DTP cells upon treatment with 5-FU. A, Sensitivity of 5FU-TP cells to 5-FU. Parental JSC15-3 (top) and JSC18-1 (bottom) cells and their 5FU-TP cells were treated with 5-FU at the indicated concentrations for 9 days. Data are representative of at least three independent experiments. **, P < 0.01; ****, P < 0.0001, two-tailed t test. B, ALDH1A3 induction by 5-FU. Cells were treated with 3 µmol/L 5-FU or DMSO and then subjected to qRT-PCR. Data are representative of at least three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, one-way ANOVA. C, Experimental design of gfp knock-in into the ALDH1A3 locus using CRISPR/Cas9-mediated genome editing. IRES, internal ribosome entry site. In construct schemes, only representative genetic elements are shown. D, Visualization of ALDH1A3-GFP expression. Cells were treated with 3 µmol/L 5-FU or DMSO for 5 days and then analyzed by fluorescence microscopy. E, Live imaging of ALDH1A3-GFP cells. Cells were treated with 3 µmol/L 5-FU or DMSO for 60 hours. Images were obtained at 0, 12, 36, 60 hours. Blue arrows indicate ALDH1A3 (GFP) induction. F, Schema for experimental schedule for cell sorting with flow cytometry. G, FACS of ALDH1A3-GFP expression. ALDH1A3-GFP cells were divided into ALDH1A3high and ALDH1A3low cells (day −1). Each cell fraction (day 0) was treated with 3 µmol/L 5-FU or DMSO for 9 days and then subjected to flow cytometry (day 9). Right bar graph shows a quantitative representation of triplicate data. **, P < 0.01; ns, not significant, one-way ANOVA.