Then, we used a RT-PCR assay with gene-specific primers for human ATP6V1C1 (C1) (including C1a and C1b) and ATP6V1C2 (C2) (including C2a and C2b) to test which isoform is expressed in the metastatic human breast cancer cell line MDA-MB-231 and found that only C1a (C1) was expressed in MDA-MB-231 cells, while both C1a and C2b are expressed in the human osteosarcoma cell line U2OS, and both C1a and C2a are expressed in the human osteosarcoma cell line Saos-2 (Supplementary Figure 3A-3B). The gene discussed is ATP6V1C1; the disease is osteosarcoma.