Figure 4 from Acetalax (Oxyphenisatin Acetate, NSC 59687) and Bisacodyl Cause Oncosis in Triple-Negative Breast Cancer Cell Lines by Poisoning the Ion Exchange Membrane Protein TRPM4

Role of TRPM4 in the action of Acetalax. A, Scatter plot demonstrating the correlation between Acetalax activity versus TRPM4 expression in the TNBC cell lines of the GDSC-MGH-Sanger database used in the present study (Supplementary Fig. S4 for the overall dataset with all cancer tissue types). The x-axis shows Acetalax activity. The y-axis shows the TRPM4 transcript level measured by RNA-seq (log₂). Both were visualized using CellMinerCDB. Each point is a TNBC cell line. The red line is the regression line. The Acetalax-sensitive cell lines are highlighted in red, and the resistant cell lines in blue. B, Western blots of TRPM4 levels in the five TNBC cell lines. GADPH was used as the loading control. C, Western blot of TRPM4 protein expression in MDA-MB468 and BT549 parental and TRPM4-KO cells. D, Acetalax cell viability assay in parental, TRPM4-KO, and CE cells. Cells were treated with Acetalax for 72 hours. Cell viability was determined using CellTiter-Glo 2. Experiments were performed in triplicate (n = 3). E, Western blot of TRPM4 expression in MDA-MB468 and BT549 parental and Acetalax-resistant (CE) cells. GADPH was used as the loading control. F, Representative microscopy images of parental MDA-MB468 cells and their resistant (CE) and TRPM4-KO counterparts. Cells were stained in blue with DAPI (binds DNA) and green with phalloidin (stains actin). Control, without Acetalax treatment. Scale bars, 50 μm. Cells were treated with Acetalax at 10 μmol/L for 30 minutes. Control, no drug. TRPM4 was degraded in the Acetalax-sensitive MDA-MB468 cells following Acetalax treatment for 4 hours. G, Quantification of repeated experiments as in F. Each cell and each nucleus are indicated by a dot. Numbers indicate the number of cells and nuclei analyzed.