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Fig. 4 Compound 2 binds to CAIX of CT26 colon cancer cells. (A) Flow cytometric analysis of CT-26 cells treated with 1 and 2 (10 μM), respectively; (B) median fluorescence intensity of (A); (C) flow cytometric analysis of wild type and CAIX knock-down CT-26 cells treated with 2 (10 μM) and western blotting analysis of the CAIX knock-down cells by siRNA; (D) median fluorescence intensity of (C); (E) flow cytometric analysis of CT26 cells treated with acetazolamide (10 μM) and 2 (10 μM), respectively, as well as the acetazolamide-preincubated CT26 cells treated with 2; (F) median fluorescence intensity of (E). (G) CT26 cells were cultured under normoxia and hypoxia (0.5% O2), and 24 h later the cells were incubated with or without 2 (10 μM) and subjected to flow cytometry analysis, besides the expression of CAIX was determined by western blotting at different time points; (H) median fluorescence intensity of (G). Results are expressed as mean ± SEM from two independent experiments (*p < 0.05, **p < 0.01, t-test).