Fig. 5: EPZ-6438 and azacytidine induce C32 and Malme-3M apoptosis via an IRAK-M-dependent mechanism. | Communications Biology

Fig. 5: EPZ-6438 and azacytidine induce C32 and Malme-3M apoptosis via an IRAK-M-dependent mechanism.

From: Induction of IRAK-M in melanoma induces caspase-3 dependent apoptosis by reducing TRAF6 and calpastatin levels

Fig. 5

a Heatmaps demonstrating melanoma cell death following treatment with 128 epigenetic compounds at the indicated concentrations for 48 h as measured using a fluorescence-based CellTox green cytotoxicity assay. All values were normalized to DMSO values, which were set to 1, and heatmaps are presented based on fold changes. Lower panel shows percentages of epigenetics inhibitor classes capable of inducing apoptosis in melanoma cells in an epigenetics compound library. b Expression levels of IRAK-M protein were evaluated by western blot 72 h after C32 and Malme-3M cells were treated with EPZ-6438 (EPZ) and azacytidine (Aza), respectively (25 or 50 µM). Changes in IRAK-M protein expression levels in DMSO and drug-treated melanoma cells are shown. All values were normalized to DMSO values, which were set to 1. Blots presented are representative of three independent experiments. c 72 h after melanoma cells were cultured with 50 µM EPZ-6438 or Azacytidine, cells were stained with PI and Annexin V and analyzed by flow cytometry (n = 3 per group). Data are shown as mean ± SEM. *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test. Results are representative of three independent experiments. d The relative mRNA levels of IRAK-M to GAPDH were determined by RT-PCR in melanoma cells 72 h after 50 µM EPZ-6438 or Azacytidine treatment (n = 3 per group). The relative IRAK-M mRNA values in DMSO treated C32 and Malme-3M cells were set to 1. Data are shown as mean ± SEM. **p < 0.01 by two-tailed Student’s t-test. Results are representative of two independent experiments. e, f Stable IRAK-M knockdown C32 and Malme-3M cells were treated with 75 µM EPZ-6438 and 50 µM azacytidine, respectively, for 48 h, followed by e western blot and f flow cytometric analysis of apoptosis (n = 3 per group). Results are presented as mean ± SEM. *p < 0.05 and **p < 0.01 by two-tailed Student’s t-test. Data shown are representative of at least two independent experiments.

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