Unlike HDAC inhibitors, chaetocin-induced reductions in A549 cell-colony formation are attenuated by cotreatment with glutathione or NAC. (A) Glutathione or NAC, but not inhibitors of DNA (aphidicolin), RNA (DRB), or protein (cycloheximide) synthesis, attenuate chaetocin-induced reductions in colony formation in A549 cells. (B-D) In contrast, apicidin-, LAQ 824–, and trichostatin A–induced reductions in A549 cell-colony formation are not altered by cotreatment with NAC (B, C, and D, respectively). (E) The ability of glutathione to attenuate chaetocin-induced inhibition of colony formation is highly time dependent and is maximal when glutathione is added before initiation of chaetocin exposure. (F-G) The effects of glutathione pretreatment on intracellular (F) and extracellular (media; G) chaetocin concentration in response to treatment with 10 μM chaetocin for 5 minutes, without or with 100 μM glutathione added 5 minutes before chaetocin addition. (H) Double reciprocal plot indicating the effects of 5-minute pretreatment with 100 μM glutathione or diluent on intracellular chaetocin concentrations resulting from treatment with varying concentrations of chaetocin for 5 minutes. (F-H) Intracellular and extracellular chaetocin concentrations were assessed in A549 cells using HPLC as described in “Patients, materials, and methods,” under “Measurement of intracellular and extracellular chaetocin levels.” Results shown are representative of 3 independent experiments. Error bars indicate 1 standard deviation.