Figure 12

The effect of HUVEC co-culture and augmentation of CLL glutathione levels on fludarabine-induced cell death and ID2/ID3 protein expression. CLL12 (A) and CLL18 (B) cells were pre-cultured alone or on a monolayer of HUVEC for 24 hrs. The cells were then cultured under the same conditions for a further 48 hrs in the absence or presence of fludarabine (20 μM). PEITC (5 μM for A, 20 μM for B) was added during the last 5 hrs of culture. Cell viability was assessed by MTT assay and values were normalized to the uncultured control (T0). The data show the mean ± SEM of three independent samples. Protein levels of ID2, ID3 and GAPDH were analyzed by western blotting. The original western blot images are shown in Additional file 11: Figure S6. CLL cells from patient CLL12 (C) and CLL18 (D) were pre-cultured in the absence or presence of GSH (2 mM for C, 4 mM for D) or L-cysteine (50 μM for C, 100 μM for D) for 24 hrs. The cells were then cultured for a further 48 hrs in the absence or presence of fludarabine (20 μM). L-cysteine was added to the culture medium daily. Cell viability was assessed by MTT assay and values were normalized to the uncultured control (T0). The data show the mean ± SEM of three independent samples. Protein levels of ID2, ID3 and GAPDH were analyzed by western blotting. The original western blot images are shown in Additional file 11: Figure S6. Quantification of western data by densitometric scanning is shown in Additional file 12: Figure S7.