CMA2 has distinct effects on β cells compared with the analogue mithramycin. (A) Ins-GLuc MIN6 cells were treated 24 h with either CMA2 or mithramycin (MTM) at the indicated doses, and then secretion was stimulated using the diazoxide (Dz; 250 µM) paradigm as previously in the presence or absence of 40 mM KCl and 20 mM glucose (Gluc). Secreted luciferase activity was measured, and the data are the mean ± SEM from three independent experiments. *, P < 0.05 versus respective DMSO by two-way ANOVA. (B) Lysates of the diazoxide-treated samples in A were subjected to Cell Titer Glo assays to measure viability. The luciferase activity is shown with respect to DMSO control, and the data are the mean ± SEM (n = 3). (C) The Ins-GLuc MIN6 cells were incubated in culture media for 24 h with indicated treatments and then assayed for glucose-stimulated secretion in KRBH in the absence of treatment (no washout; left). In parallel, cells were also incubated for 24 h with indicated treatments followed by a 24-h washout in culture media before the secretion assay (24-h washout; right). Data are the mean ± SEM (n = 3). *, P < 0.05 versus respective DMSO by two-way ANOVA. (D) The MIN6 cells were treated with CMA2 for 24 h, and RNA was isolated for RT-qPCR against INSI/II, PDX1, and UCN3. Data were normalized to 18S rRNA by the 2^^-ΔΔCt method and are the mean ± SEM (n = 3). *, P < 0.05 versus DMSO control. (E) Insulin content was measured in lysates from human islets that were treated 24 h with CMA2 (100 nM) or DMSO (0.1%). Data are the mean ± SEM (n = 3). (F) The MIN6 cells were treated with the indicated doses of CMA2 for 72 h and subjected to a Cell Titer Glo assay to measure viability. Data are the mean ± SEM normalized to the percent of DMSO (n = 3). *, P < 0.05 versus DMSO by Student’s t test. (G) The data in F were confirmed by treating MIN6 cells with CMA2 for 48 h followed by fixation and staining with DAPI. Nuclei counts per well are shown as the mean ± SEM (n = 3). *, P < 0.05 versus DMSO by Student’s t test.