Fig 2: Suspension-cultured breast cancer cell lines expressed high levels of carbonic anhydrase IX. (A) Breast cancer cell lines were collected from the monolayer (D0) following 7 days of suspension culture (D7) and analyzed using immunoblotting for markers identified in the top dysregulated cell surface proteins as well as HIF1α. Carbonic Anhydrase IX (CAIX) and ENO2 were significantly upregulated following suspension culture in the cell lines studied. (B) The surface expression of CAIX was detected using MIFC with gating performed on the isotype and unlabeled controls for each cell line. (C) CAIX surface expression increased in each cell line over the 7 days of culture, with the most prominent expression seen in MB-231, MCF7, and ZR75-1 cells. CAIX+ populations were quantified at each indicated time point. (n = 3, * p < 0.05, relative to monolayer culture).

Fig 3: Validation of glycolysis pathway in HLE cells. Images represent protein levels of glycolysis-related proteins in HLECs under hypoxia and normoxia conditions. The protein levels of HK1, HK2, PGK1, ENO2, and LDHA were analysed via Western blot (A). (B) Bar graph shows quantification. Results are combined data from four experiments with different cell preparations, and each value represents mean ± SEM; *: p < 0.05, **: p < 0.01, ns: p > 0.05. (C) Light photomicrographs of HLECs cultured with or without glycolysis inhibitor 2DG under hypoxia conditions. Scale bars: 500 μm. (D) CCK8 assay was used to detect the cell viability and the optical density (OD) values of HLECs; those cultured with glycolysis inhibitor 2DG have significantly lower values under hypoxia conditions. Images represent protein levels of glycolysis-related proteins in HLECs cultured with or without glycolysis inhibitor 2DG under hypoxia conditions (***: p < 0.001). The protein levels of HK1, HK2, PGK1, ENO2, and LDHA were analysed via Western blot (E). (F) Bar graph shows quantification. Results are combined data from four experiments with different cell preparations, and each value represents mean ± SEM; *: p < 0.05, **: p < 0.01, ns: p > 0.05.