To validate the hypothesis that calcium overload triggered by P2X4 activation induces mitochondrial ROS production and membrane damage, leading to the cytosolic release of mtDNA, we applied the ROS scavenger N-acetyl-cysteine (NAC) or substitution with calcium-free CRC cell conditioned medium during TAM induction, Both interventions effectively suppressed mitochondrial ROS levels and mtDNA leakage (Figure 3F). The gene discussed is P2RX4; the disease is colorectal carcinoma.