FIGURE 2.

CAIX protects cells against irradiation-induced cell death in an acidic environment. (A) Cell cycle distribution of NHE-1-disrupted fibroblasts PS120 cells expressing (pca9) or not (pev) CAIX, in normoxia in a ${\text{HCO}}_3^{-}$/CO₂-free environment at pH_o 7.0 or 7.5 for 24 h. (B–D) PS120-pev and PS120-pca9 cells (1 × 10⁴) were plated in 60 mm dishes. Once attached cells were incubated in 30 mM HEPES-buffered ${\text{HCO}}_3^{-}$-free medium adjusted to pH_o 7.0 in the absence (B) or in the presence of 10 mM ${\text{HCO}}_3^{-}$ (D) or to pH_o 7.5 (C) for 24 h in a CO₂-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a CO₂-containing incubator with fresh regular ${\text{HCO}}_3^{-}$-containing (44 mM) medium for 4 days. Cell death was determined by the trypan blue exclusion assay. Data represent the average of three independent experiments. (E) The clonogenic capacity of PS120-pev and PS120-pca9 cells exposed to a medium adjusted to pH_o 7.0 or 7.5 was measured 10 days after irradiation (0,1, 2, 4, 6, and 8 Gy). Dishes were stained with Giemsa (Fluka). The colonies were counted with ImageJ software according to the following parameters: particles size = 0.15–5 mm² and circularity = 0.1–1.