Fig 1: TriPcides cause increased ROS and ATP levels in MRSA-1369.(A) Transposon insertion mutants represented in the library following treatment relative to the vehicle control. Log10 ratio of insertions in the experimental condition relative to the control (x axis) and corrected P values (y axis) of transposon insertions in indicated genes in the PS1962 (1.04 μg/ml, 2 μM)–treated group relative to DMSO control. Genes that significantly differ in insertions relative to the control are indicated in blue (ratio < 0.5, corrected (corr.) P value < 0.05) and red (ratio > 2, corr. P value < 0.05). (B) Changes in ATP levels in exponential-phase MRSA-1369. The MRSA-1369 cells were treated with DMSO (vehicle control) or SS1045B (0.5 to 4 μg/ml; 0.9 to 7 μM) for 30 min, and then the ATP levels were determined using a BacTiter-Glo Microbial Cell Viability Assay kit from Promega. Bar graphs are represented as the mean and SD derived from 10 independent biological replicates. (C) Changes in ROS levels in exponential-phase MRSA-1369 cells incubated in BHI medium supplemented with 10 μM H2DCFDA fluorescence probe in the presence of DMSO (vehicle control), H2O2 (68 to 272 μg/ml, 2 to 8 mM), or SS1045B (1 to 4 μg/ml; 1.8 to 7 μM) at 37°C for 1 hour. ROS levels were determined by H2DCFDA fluorescence intensity normalized with corresponding OD600 values. (D) OD600 values of the MRSA-1369 samples shown in (C). Bar graphs in (C) and (D) are represented as means and SD from five biological replicates. Statistical analysis by ordinary one-way ANOVA test with Tukey’s comparison. Samples marked with (*) showed a statistically significant difference compared to the DMSO control (P ≤ 0.0001), whereas samples marked with (n.s.) were not significantly different from the DMSO control (P = 0.9996). RFU, relative fluorescence unit.