FIGURE 1.

Intracellular carbonic anhydrase activity. A, panel i, determination of cytoplasmic CO₂ hydration rate from pH_i dynamics in intact HCT116 cells (AM-loaded with pH dye cSNARF1). Switching between CO₂/HCO₃⁻-free and 5% CO₂, 22 mm HCO₃⁻-containing superfusate triggers intracellular CO₂ hydration. The experiment was repeated with 100 μm ATZ to inhibit CA. Inset, pH_i response to CO₂ addition. Panel ii, CA_i activity in cancer and fibroblast/fibroblast-related cells (n > 20 each), normalized to measurements in the presence of ATZ (k_f = 0.18 s⁻¹). B, panel i, measuring CA activity in membrane-free RT112 lysates. Panel ii, CA_s activity normalized to spontaneous rate determined in ATZ (expressed per mg/ml of total protein). C, panel i, knockdown of CAII in HCT116 cells using one of four shRNA constructs, scrambled shRNA (sham) and non-transfected wild type (WT). *, p < 0.05, relative to WT. Panel ii, total CA_i activity in knockdown, sham, and WT HCT116 cells (n > 20 each). Panel iii, Western blot for soluble CA isoforms other than CAII in HCT116 cells treated with shRNA. D, Western blot for CAII and CAIX under control (normoxic) incubation or 48 h in 1 mm dimethyl oxalylglycine (DMOG). Error bars represent S.E.