Fig 1: Effects of M109S on cellular metabolism(A–H) M109S decreased the maximum oxygen consumption rate (OCR) and increased the extracellular acidification rate (ECAR) both in Wt and bax−/−bak−/− MEFs. OCR and ECAR represent the activities of mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis, respectively. The effect of M109S on metabolism was analyzed using the Seahorse mitochondria stress kit as described in STAR methods section. MEFs (Wt and bax−/−bak−/−) were incubated with or without M109s for 4 h before Seahorse analysis. M109S was also added to the culture medium during the Seahorse analysis procedure (approximately 3 h). (A–F) The levels of OCR of the basal (A and B), ATP production (C and D), and maximum oxygen consumption (E and F), are shown. (G and H) The basal ECAR is shown. M109S decreased maximal O2 consumption and increased glycolysis in Wt and bax−/−bak−/− MEFs. The result of the statistical analysis (one-way ANOVA) is shown in each graph. “ns”: no statistically significant difference was detected (p > 0.05). Data are presented as mean ± SD.(I and J) M109S decreases ROS levels in cell culture. ROS levels were measured by ROS-Glo-H2O2 assay kit (Promega). N-acetyl cysteine (NAC) or M109S was added to the culture medium for a total of 8 h (4 h before ROS substrate addition and 4 h during the incubation with ROS substrate). M109S addition decreased ROS levels both in Wt and bax−/−bak−/− MEFs. The result of the statistical analysis (one-way ANOVA) is shown in each graph. “ns”: no statistically significant difference was detected (p > 0.05). Data are presented as mean ± SD.(K and L) A high dose of M109S (1 μM) slowed the cell division speed both in Wt and bax−/−bak−/− MEFs. Forty thousand cells were plated in each well of 12 well plate (1 mL/well) on Day 0. The next day (24 h later), M109S was added to the medium, and cells were cultured for an additional 24 h, and the numbers of cells were counted. The cell number at Day 0 is designated 100%, and the ratio of cell number is shown. The result of the statistical analysis (one-way ANOVA) is shown in each graph. “ns”: no statistically significant difference was detected (p > 0.05).(M) Schematic explanation of the mechanism of action of CSMs. Data are presented as mean ± SD.