Fig. 2: KDM1A knockdown induces apoptosis, cell cycle arrest, and defective DDR. | Cell Death Discovery

Fig. 2: KDM1A knockdown induces apoptosis, cell cycle arrest, and defective DDR.

From: Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation

Fig. 2

Western blots showing KDM1A protein silencing upon shRNA-mediated transduction of HCT116 cells and CRC-SCs with shNT and the shKDM1A (sh68 and sh71) on day 3 after transduction (protein quantification normalized on tubulin, relative to shNT) (A). Cell viability assay using CellTiter-Glo to measure ATP content in CRC cells on days 5, 7, and 9 after transduction. Data represent the mean ± SD of at least three independent experiments (B). Viability of CRC-SCs (n = 4) 11 days after transduction with shNT, sh68, and sh71 shRNA expressing lentiviruses. Data represent the mean ± SD of at least three independent experiments (C). Representative dots plots showing HCT116 cells distribution after AxV/PI staining (D). Graphs showing the analysis of apoptotic cells (AxV pos/PI neg) after KDM1A silencing in HCT116 and CRC-SC#1 cells on day 4 and day 7 after transduction. Data represent the mean ± SD of at least three independent experiments (E). Cell cycle distribution quantification (F) of transduced HCT116 cells by flow cytometry. The percentage of cells in sub G0, G0/G1, S, and G2 phases of the cell cycle was assessed 48 h after serum addition to starved transduced cells (day 2 after transduction). Data represent the mean ± SD of three independent experiments. For DDR investigation, cells (day 2 after transduction) were pretreated with oxaliplatin 10 μmol/L for 8 h to induce DSBs formation. After oxaliplatin removal cells were stained for nuclei (blue), 53BP1 (red), or pH2AX (red), and mounted on microscope slides 8- and 24 h following treatment removal. Pictures were acquired by confocal microscopy (63X magnification, scale bar 25 μm). Representative pictures (G, I). Graphs showing 53BP1 or pH2AX foci average number per cell (H, J). Data show the mean ± SEM of three independent experiments. RT-qPCR analysis of BUB1, PLK1, and AURKA in CRC-SC#1 and CRC-SC#2 KDM1A-silenced cells (K). Data represent the mean ± SD of at least three independent experiments. Student’s t-test was conducted (sh68 or sh71 versus shNT): *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

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