Figure 6. Behavior of Cpd-1 signal in an incubation chase study.

RAM2808 cells were grown in the absence or presence of 0.4% maltose and induced with 0.2% arabinose (Ara, induction of LamB expression; and Malt Ara, induction of the maltose operon and LamB expression). The bacterial suspension was then incubated with Cpd-1 in the absence or presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP, which collapses the energy driving force of efflux pumps). (A) After incubation of the RAM2808 Malt Ara cells, samples were centrifuged, and cell pellets were resuspended in the same buffer without any Cpd-1. At 0, 15, 30, 45, 60, and 90 min, samples were recovered and analyzed. The columns with bars (standard deviations) corresponded to measurements carried out in triplicate. (B) To compare the degradation of Cpd-1 in cells according to the regulation of the maltose operon (induced or not, Malt Ara or Ara conditions, respectively), the % of signal was standardized from 0 time and the degradation level (in percent) was reported at 30 and 60 min in RAM2808 cells grown with (induction of the maltose operon) or without maltose (repression of the maltose operon) (means of three independent assays performed in triplicates). Calibration curves were used to obtain the number of molecules per cell (Fig S9).