Mitochondrial Oxidative DNA Damage Assessment

To assess oxidative DNA damage, AC16 Cells were incubated with an anti-DNA/RNA Damage (8-oxoG) antibody (Abcam, ab62623), as described previously [31 (link)], and images were acquired using ZEN 2.1 software (Carl Zeiss, Oberkochen, Germany). Co-localization of DNA oxidation and mitochondria was assessed by immunofluorescent analysis of Translocase Of Outer Mitochondrial Membrane (TOM20, proteintech, 11802-1-AP) and 8-oxoG (Abcam, ab62623) in AC16 cells. To explore whether the place where CAV3 interacts with NDUFA10 is located in mitochondria, AC16 cells were labelled with the MitoTracker Red CMXRos probe (Thermo Fisher Scientific, Waltham, MA, USA), an anti-CAV3 antibody (Abcam, ab289544), an anti-NDUFA10 antibody (Santa Cruz, sc-376357), and DAPI (Servicebio, Wuhan, China). The cells were also incubated with an anti-Lamp1 antibody (Abcam, ab208943). Immunofluorescence co-localization images were captured using a laser scanning confocal microscope (Leica, SP8, Germany) and processed using LAS X 3.7.4 software (Leica, SP8, Germany).