Fig 2: KCNK9-US1 DMR methylation in breast epithelial cells aspirated from women at high risk for developing breast cancer. (A) Box plot of Masood Cytology Index Score versus KCNK9-US1 DMR methylation status. (B) Box plot of R/G ratio versus KCNK9-US1 DMR methylation status: methylation, partial hypomethylation, hypomethylation.

Fig 3: TASK3 expression and mitochondrial membrane potential as a function of KCNK9-US1 DMR demethylation in MCF10A cells and HMEC-15 normal cell strain. The optimal treatment conditions are 48 h for either 1.0 (red star) or 2.5 µM (green star) 5-aza-2'-deoxycytidine (5AzC) (A) Effect of 1.0 and 2.5 µM 5AzC on the DNA methylation of CpG sites 1–5 (boxes) in the KCNK9-US1 DMR in MCF10A. (B) Cell proliferation (viability) was tested using the MTT assay at 0–72 h; MCF10A cells and HMEC-15 normal cell strain were treated with 0 (blue line), 1.0 (red line), and 2.5 µM (green line) 5-AzC. Experiments were performed in triplicate. (C) Expression of TASK3 protein analyzed by western blotting at 0 and 48 h in MCF10A after treatment with 2.5 µM 5AzC and HMEC-15 normal cell strain after treatment with 1.0 and 2.5 µM 5AzC. GAPDH is used as the loading control. (D) Relative TASK3 expression in MCF10A after treatment with 2.5 µM 5AzC for 0 and 48 h and HMEC-15 normal breast cell strain after treatment with 1.0 and 2.5 µM 5AzC for 0 and 48 h. TASK3 expression was normalized to 0 h treatment (Control). Expression is for the western blots performed in triplicate for panel Figure 4C. Significant levels relative to 0 h control: *** p < 0.001. (E) JC-1 R/G ratio in MCF10A cells treated with 0, 1.0, and 2.5 µM 5AzC for 0, 24, and 48 h normalized to 0 h treatment (Control). JC-1 R/G ratio in HMEC-15 normal cell strain was treated with 0, 1.0, and 2.5 µM 5AzC for 0 and 48 h normalized to 0 h treatment (Control). Significant levels relative to 0 h control: * p < 0.05, ** p < 0.01, *** p < 0.001. Experiments were performed in triplicate. The uncropped blots and molecular weight markers are shown in Supplementary Materials.

Fig 4: Representative DNA methylation of KCNK9-US1 DMR in non-cancerous breast tissue. Commercially obtained breast genomic DNA (Biochain, Newark, CA, USA) was obtained for analysis. The results are shown from six women, ranging in age from 21 to 83 years old.

Fig 5: TASK3 expression and localization in breast cancer cell lines and mammary epithelial cell lines and cell strains. (A) Endogenous total TASK3 protein expression in human breast cell lines. TASK3 exhibited relatively increased expression in MDA231, DKAT, SUM149, SUM190, and HEK293 cells, but had lower expression in SUM225, HMEC15, and MCF10A cells. GAPDH serves as a loading control (n = 3 experiments). (B) Transient overexpression of TASK3-FLAG tag in the breast cancer cell line MCF10A results in expression of the exogenous construct in the mitochondria, as shown by western blotting. VDAC was used as a loading control for the mitochondrial fraction. Control untreated cells (Control), cells treated with transfection reagent alone (Txf), cells transfected with empty FLAG tag plasmid (Vec), and cells transfected with TASK3-FLAG tag plasmid. (C) Expression of the TASK3-FLAG construct by immunofluorescence. Control untreated cells (Control), cells transfected with empty FLAG tag plasmid (Vector Control), and cells transfected with TASK3-FLAG tag plasmid (TASK3-FLAG). TASK expression was detected by immunofluorescence using anti-FLAG tag antibody (Sigma-Aldrich, St. Louis, MO, USA). TASK3 transfected cells (TASK3-FLAG) were compared with those transfected with an empty vector control. (D) JC-1 Red/Green Ratio (R/G Ratio) in untreated MCF10A cells (Control), cells treated with transfection reagent alone (Txf), cells transfected with empty FLAG tag plasmid (Vec), and cells transfected with TASK3-FLAG tag plasmid (TK3). The R/G Ratio is a measure of mitochondrial membrane potential (??m), (n = 3 experiments). Significant levels relative to control: * p < 0.05, ** p < 0.01. (E) Caspase-3 activity, a measure of apoptosis initiation, normalized to DNA content in untreated MCF10A cells (Control); cells treated with transfection reagent alone (Txf); cells transfected with empty FLAG tag plasmid (Vec); and cells transfected with TASK3-FLAG tag plasmid (TK3). Cells were exposed for 4 h to either 0.0 µg/mL (grey bar) or 0.50 µg/mL (black bar) of staurosporine. Significant levels relative to cells not exposed to staurosporine: * p < 0.05, ** p < 0.01. The uncropped blots and molecular weight markers are shown in Supplementary Materials.