Figure 4.

The potency of CORM-2 is influenced by certain compounds added to the growth medium. (I,II) CORM-2 stocks were prepared by dilution in 10% DMSO/90% H₂O (‘CORM-2′) or diluted in 10% DMSO/90% medium: (I) minimal salts-based media (phosphate-buffered saline, PBS, glucose-defined minimal medium, GDMM) or (II) rich nutrient broths (lysogeny broth, LB, Mueller-Hinton II, MH-II, Dulbecco’s modified Eagle medium, DMEM or Roswell Park Memorial Institute medium, RPMI) and left to incubate for 10 min. These stocks were then added at a CORM-2 concentration of 30 µM to E. coli grown on GDMM (arrow). Growth (OD₅₉₅) was monitored thereafter. Rich growth media (II) protected cells from the growth inhibitory effects of 30 µM CORM-2. In (III), cultures grown on GDMM were supplemented with 0.25% (w/v) casamino acids, and 0–500 µM CORM-2 was added to determine whether a mixture of extracellular amino acids protects against CORM-2-mediated growth inhibition. To determine which specific components of rich media were responsible for protective effects against CORM-2-induced growth inhibition, CORM-2 stocks were mixed with a 2-fold excess of (IV) non-polar amino acids, (V) polar, uncharged amino acids, (VI) aromatic amino acids, (VII) polar charged amino acids, (VIII) reduced thiol compounds (N-acetyl cysteine, NAC, reduced glutathione, GSH, sodium hydrosulphide, NaHS) or (IX) oxidised thiol compounds (cystine, OxCys, oxidised glutathione, GSSG). These stocks were then added to E. coli cells grown on GDMM and growth was monitored relative to no-CORM controls (dashed line) and 30 µM CORM-2 prepared by dilution in 10% DMSO/90% H₂O as standard (grey line). The lines in red indicate amino acids/sulphur compounds that protect against the growth inhibitory effects of 30 µM CORM-2, whereas black lines indicate that the compounds did not exert protective effects. All data are representative of 3–4 biological repeats ± SD.