Figure 3. IC₅₀ determination of VR16-09, VD11-4-2, and VD12-09 in hypoxic MDA-MB-231 cells and CA IX-dependent mode of action for inhibitors in hypoxic HeLa CA IX KO cells.

(A) Original recordings of the log enrichment of MDA-MB-231 cells pre-incubated with VD11-4-2 for up to 3 h. The beginning of each trace shows the rate of degradation of the ¹⁸O-labeled substrate in the non-catalyzed reaction. (B–D) – Relative CA activity in MDA-MB-231 cells, incubated under hypoxia (1% O₂) for 3 days. Cells were pre-incubated with VR16-09 (B), VD11-4-2 (C), or VD12-09 (D) for up to 3 h. CA activity was determined by MS gas-analysis from the increase in the rate of log enrichment after addition of cell suspension. CA activity in the presence of inhibitor was normalized to the activity in the absence of inhibitor. (E) CA activity in HeLa-WT and HeLa-CA IX-KO cells, before (white bars) and after addition of VR16-09, VD11-4-2 or VD12-09 (black bars). Average + SD of 4 independent experiments per cell line are shown. Asterisks indicate significant difference between CA activity before and after addition of inhibitor (^**p < 0.01, ^***p < 0.001, n.s.: not significant).