In contrast to the poor response to gemcitabine individually in the HyPer-low subpopulation of CCA cells, the CCK-8 assay proved that the combination of MTX and gemcitabine impaired cell viability in HyPer-low CCA cells under gemcitabine treatment (Fig. 5F) and Annexin-V/PI staining for flow cytometric analysis reassure our conclusion (Fig. 5G, H). The gene discussed is ANXA5; the disease is cholangiocarcinoma.