Figure 6. Supplementation with NAC and nucleosides is sufficient for oncogenic transformation of immortalized cells.

(A and B) Soft agar colonies formed by PHMLEB (A) and NIH3T3 (B) cells treated with vehicle (Ctrl), NAC (0.25 mM), nucleosides (NUC, 20 μg/mL each, 160 μg/mL total), or both NAC and NUC. (B) is replotted from Figures S5D, S5F, and S5G of same conditions.

(C and D) Athymic nude mice were injected with four million of PHMLEB (C) or NIH3T3 (D) cells and supplemented with vehicle, NAC, and/or nucleosides. Shown are frequency of tumor formation (left) and tumor images (middle) and weights (right) at the end of the experiment (see also Figures S6A–S6G).

(E) Frequency of tumor formation (left) and tumor images (middle) and weights (right) at the end of the experiment for 2^nd inoculation of four million cells from tumors that were generated with the supplementation of NAC or nucleosides in (D), in presence or absence of the initial treatment (Figures S6L and S6M).

(F–K) PHMLEB (F–H) and NIH3T3 (I–K) cells were cultured under matrix-attached (0 hr) or - detached (6 and 12 hr) conditions in presence of vehicle, NAC (0.25 mM), NUC (160 μg/mL), or both NAC and NUC. Cells were assayed for the NADPH/NADP⁺ ratio (F and I), ROS levels (G and J), and cell death (H and K).

Data are means ± SD of representative result (n = 3, or as indicated). *P < 0.05, **P < 0.01, ***P < 0.001.