Therefore, we first demonstrated that treatment with 50 μM of bumetanide could inhibit NKCC1 activity in HCC cell lines MHCC97H and Huh7, using fluorescence intensity measurement of enzyme activity (Supplementary Figure 5A, Supplementary Figure 5C) and detection of phosphorylated NKCC1 (Supplementary Figure 5B, Supplementary Figure 5D). Here, SLC12A2 is linked to hepatocellular carcinoma.