FIGURE 3.

Cellular target engagement of rhein. A, plate killing assay to show the resistance of E. coli Gold that overexpresses AlkB to MMS in the presence of rhein. The starting density is A₆₀₀ 0.008. B, cfu count assay to show resistance of E. coli AB1157 (wild type), HK82 (AlkB mutant), and AlkB complemented HK82 to MMS by rhein. The error bars are means ± S.E. (n = 6). **, p < 0.01; ***, p < 0.001. C, rhein could not sensitize E. coli Gold to MNNG. The density of bacteria in the top line is A₆₀₀ 0.005. D, rhein could not sensitize E. coli growth to other DNA-damaging agents such as the oxidizing agent (H₂O₂). E, CETSA showing that rhein increases the thermal stability of AlkB in E. coli cell lysate. The data are presented as means ± S.E., and experiments were performed in triplicate. F, CETSA shows that rhein stabilizes AlkB in intact bacterial cells. ns, not significant.