Drug sensitivity (MTT) assay

Cell density measurements were performed using a hemacytometer to count intact cells resistant to staining with trypan blue. Approximately 20,000 cells were plated into each well of 96-well flat-bottomed micro-titer plates. After 12 h incubation at 37°C, medium containing either pre-immune IgG or anti-RLIP76 IgG (ranging 0–60 μg/mL final conc.) were added to cells followed by incubation for 24 h, then 2HF (ranging 0–200 μM) were added to cells. After 48 h incubation, 20 μL of 5 mg/mL MTT were introduced to each well and incubated for 2 h of exposure. The plates were centrifuged and the medium was decanted. Cells were dissolved in 100 μL DMSO with gentle shaking for 2 h at room temperature, followed by measurement of OD₅₇₀ [41]. Eight replicate wells in each of three separate measurements. For GI₅₀ determination of CDNB and 2HF ±CDNB in H358 cells, approximately 5,000 cells were plated as above in 96 well plate. After 12 h cells were treated with 0–15 μM CDNB or 0–150 μM 2HF and 1 μM CDNB+0–150 μM 2HF. After 48 h MTT assay was performed as described above.