Transient receptor potential melastatin‐4 (TRPM4) knockout (KO) reduces tumor spheroid outgrowth and increases cell death in tumor spheroids. (A) Representative microscopic images of tumor spheroid outgrowth from DU145 cells and corresponding TRPM4 KO clones (n = 4). Scale bar indicates 250 μm. (B) Spheroid outgrowth was quantified as the ratio of outgrowth area to core spheroid size at 48 and 72 h. All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. (C) Cell death was quantified as the relative intensity of CellTox Green staining in tumor spheroids from DU145 cells and corresponding TRPM4 KO clones on days 3, 5, and 10 postseeding (n = 4). All individual values are plotted as dots in the graph. The median is shown as a red line, whiskers represent the interquartile range. Significance levels for data from both clones in B and C are given relative to DU145 cells. Differences between samples were analyzed using one‐way ANOVA followed by Dunnett's post‐hoc test. (D) Representative fluorescence microscopic image of a tumor spheroid from DU145 TRPM4 KO2 cells on day 5 postseeding (n = 4). Live cells were stained with calcein‐AM (green), dead cells with propidium iodide (red), and the nuclei of all cells with Hoechst 33342 (blue). Scale bar indicates 100 μm.
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