CAIX activity assay

In-Cell CAIX catalytic activity assays were carried out as described (33). Assays were performed using SUM159PT cells following a 72-hour incubation in 1% O₂. Cells were harvested using 0.5 mmol/L EDTA in PBS. For each sample, 5 × 10⁵ cells were suspended in 100 μL of CO₂-free isotonic buffer [20 mmol/L HEPES, 130 mmol/L NaCl, and 5 mmol/L KCl (pH 8.0)] in a 2-mL flat bottom vial equipped with an 8-mm magnetic stir bar. SLC-0111 was added to the cell suspension to a final concentration of 50 μmol/L or an equivalent volume of DMSO vehicle was added, and the sample was incubated at room temperature for 30 minutes. An additional 700 μL of ice-cold buffer was then added to the cell-inhibitor mixture, and a narrow pH electrode (Accumet) was immersed in the sample and equilibrated for 3 minutes. A total of 200 μL of CO₂-saturated water was added to initiate the assay, and pH readings were recorded at 5-second intervals for up to 150 seconds. The change in pH was plotted as a function of time. To determine the rate of spontaneous CO₂ hydration, measurements were performed on cell-free (“buffer”) samples. The increase in hydration rate above the spontaneous rate is a measure of CA activity. Three replicates were assayed for each condition evaluated in the assay.