Cell lines generation to study ATAD3 role in mitochondrial function and cell metabolism

ATAD3 (ATPase family AAA domain-containing protein 3) is a mitochondrial protein that regulates mitochondrial function, dynamics, and metabolism. In this study, we focused on one of the main components of this protein family ATAD3B, with the latter being specifically expressed in cancer and stem cells. It has been demonstrated that ATAD3B expression correlates with the malignancy of cancer cell lines, likely affecting tumor progression and metastasis. Here, we employed two different strategies to create a stable cell line knockdown (KD) for ATAD3B: 1) A Lentiviral expression system, and 2) A Piggybac transposon system, both of which express a short hairpin RNA (shRNA) targeting ATAD3B mRNA, which is then inserted into the genome of the host cell line. We selected MDA-436, MCF7, and H9 cell lines as the models for genetically modifying ATAD3B protein levels. After confirming the success of the strategy by analyzing protein levels through western blotting, we studied the impact of the KD on cell proliferation and mitochondrial function. Our results will significantly contribute to understanding the mitochondrial function and cellular metabolism of ATAD3B.