Fig 2: Intracellular ROS-mediated activation of autophagy in response to EHMT2 inhibitiona. NF-?B nuclear translocation was inhibited by a ROS scavenger, NAC. MCF-7 cells were treated with 10 µM BIX for 4 h in the presence of 4 mM NAC. Cells were then examined by fluorescence microscopy. NF-?B was detected using a Alexa488 (green)-conjugated antibody against the 65 kDa subunit. Nuclei were visualized with Hoechst 33258 (blue). Scale bar, 50 µm. Lower panels show the quantification of translocated NF-?B/p65. The percentage of cells with nuclear NF-?B/p65 is indicated, error bars represent the S.D. (four experiments with at least 50 cells analyzed per condition and experiment). Data are expressed as the means ± SEM of three independent experiments. *P < 0.05 compared with BIX by one-way ANOVA. b. Inhibition of ROS by NAC resulted in the reduction of Beclin-1 expression. MCF-7 cells were treated with 10 µM BIX for 4 h in the presence or absence of 4 mM NAC. Western blotting was carried out using the specified antibodies.

Fig 3: Inhibition of EHMT2 increases Beclin-1 expressiona. GFP-MCF-7 cells were treated with 10 μM BIX for 2 h. Cells were examined using fluorescence microscopy. Scale bar, 50 μm. b. Two breast cancer cell lines (MCF-7, Beclin-1+/−; HS578T, Beclin-1+/+) were treated with 10 μM BIX for the indicated amounts of time. Beclin-1 transcripts and protein expression were determined by RT-PCR (lower panel) and Western blotting (upper panel), respectively. c. RT2 Profiler PCR analysis for ATG expression. This figure includes a “heat map,” which is the part of the figure containing colors (red, green and black), in which the color represents the expression level of the gene; red and green represent high and low expression, respectively. Expression levels are continuously mapped on the color scale provided at the top of the figure. The heat map represents the differential gene expression pattern in MCF-7 cells treated without or with 10 μM BIX for 4 or 24 h. d. Graph represents the fold-change in the level of mRNA transcript in cells treated with BIX for 24 h versus untreated cells. Each circle represents an autophagy gene; those with the greatest fold-changes are indicated in red. The central line represents no changes in expression; above the central line indicates genes with increased expression; below the line are those genes with reduced levels. Grey lines indicate a 2-fold increase or decrease. e. MCF-7 cells were transfected with siCont or EHMT2 siRNA (siEHMT2) for 48 h. Western blot analysis was performed using the indicated antibodies. f. MCF-7 cells were transfected with 2 μg pcDNA3 empty vector (MOCK) or 1 or 2 μg pcDNA3-Flag-EHMT2 (FLAG-EHMT2) for 24 h; pcDNA3 empty vector was used as a negative control. Western blot analyses were performed using the indicated antibodies.

Fig 4: Coordination of DNMT1 and EHMT2 in the suppression of Beclin-1 expressiona. Western blotting after IP showed that BIX treatment reduced the physical binding between EHMT2 and DNMT1. b. Representative Western blots for the detection of Beclin-1 expression before and after MCF7 cells were treated with 10 µM 5-Aza-Cd, 10 µM BIX, or a combination of both treatments. c. Methylation-specific PCR (MSP) analysis of DNA methylation in the Beclin-1 promoter region in MCF7 cells treated with 10 µM 5-Aza-Cd for 48 h, 10 µM BIX for 1 h, or a combination of both treatments.

Fig 5: Epigenetic transcriptional activation of Beclin-1 by EHMT2 inhibitiona. A pair of breast cancer cell lines (MCF-7: Beclin-1+/- and HS578T: Beclin-1+/+) were treated with 10 µM BIX, 10 µg/ml CHX, 1 µg/ml ACD, or combinations of these compounds as indicated for 4 h. Beclin-1 transcripts and protein expression were determined by RT-PCR (upper panel) and Western blotting (lower panel), respectively. b. MCF-7 cells were treated with 10 µM BIX and/or 1 µg/ml ACD for 4 h. Cell morphology was examined under a light microscope. Scale bar: 50 µm. c, d and e. MCF-7 cells and HS578T cells were treated with 10 µM BIX for 4 h or MCF-7 cells were transfected with 1 µM siEHMT2. Treated cells were analyzed by ChIP. The ChIP analysis performed with anti-H3K9me2, anti-EHMT2, and anti-RNA pol II antibodies was compared with normal rabbit IgG as a negative control. An equal amount (input) of DNA–protein complex was applied. Real-time quantification of the Beclin-1 promoter sequences was carried out with anti-H3K9me2 ChIP, anti-EHMT2 ChIP, and anti-RNA pol II ChIP (right panel). Results are presented relative to the input and are the fold-changes over the control expressed as means ± SEM of three independent experiments. *P < 0.001 compared with control or siCont by one-way ANOVA.