Figure 6.

A small molecule inhibitor of ME1²⁶ suppressed colon cancer cell numbers in vitro. (A,B) Number of viable HCT116 and HT29 cells after treatment with 50 uM of ME1 inhibitor (ME1*), 15 uM of Wnt pathway inhibitor (JW74), 50 uM ME1* plus 15 uM JW74, or vehicle (DMSO). Twenty thousand cells/well were plated and 24 h later received inhibitor(s). Cells were evaluated after 72 h of treatment (n = 3 wells/treatment group, data are representative of 2 independent experiments). (C,D) Results of MTS cell proliferation/cytotoxicity assay. Cells were plated at a density of 1000 cells per/well and 24 h later received treatments (50 uM ME1*, 15 uM JW74, 50 uM ME1* plus 15 uM JW74, or vehicle (DMSO)). Absorbance (490 nm) was measured at 48 h after treatment addition; n = 8 wells/treatment group, data are representative of 2 independent experiments. (E,F) Results of clonogenic assay. HCT116 or HT29 cells were plated at a density of 1000 cells/well and after 24 h were treated with 50 uM ME1*, 15 uM JW74, 50 uM ME1* plus 15 uM JW74, or vehicle (DMSO). After incubation for six days, cells were stained with crystal violet. (G,H) Quantification of colony forming units (CFU) from (E,F) after treatment of cells (n = 6 wells/treatment group). (I,J) Results of modified clonogenic assay. HCT116 and IEC6 cells were plated at very high density (100,000 cells/well) and after 24 h were treated with 50 uM ME1*, 15 uM JW74, 50 uM ME1* plus 15 uM JW74, or vehicle (DMSO). After 3 days, cells were stained with crystal violet. (K,L) Quantification of remaining cells from (I,J) expressed as % area of stained cells per well. Boxes show the inter-quartile range of 25–75% with mean (thick line) and median (thin line); whiskers: 10^th and 90^th percentiles. One way ANOVA was used to examine for differences between treatment groups. Different lowercase letters (a-d) designate groups that differ (P < 0.05); bars sharing the same letter are not significantly different.