Fig 2: Effect of decreased ATAD2 levels on mtROS production in lung cancer cells. A ATAD2 mRNA expression in lung cancer cells transfected with the ATAD2 overexpression plasmid. B Cellular mtROS accumulation in A549 and SPC-A1 cells assessed using flow cytometry analysis. C, D Nuclei are stained with Hoechst (blue), and mitochondria ROS are stained using MitoSOX-red in A549 and SPCA1 cells. Merged panels demonstrate the number of MitoSOX-red positive cells among the total cells. Scale bar = 100 µm. Error bars indicate mean ± SD. All experiments performed in triplicate. *p < 0.05

Fig 3: ATAD2 expression is upregulated in LUAD tissues and cells. A Overexpression of ATAD2 mRNA is observed in LUAD tissues compared with normal samples [gene expression profiling interactive analysis (GEPIA)]. B ATAD2 mRNA expression is significantly related to the cancer stage of the patient (GEPIA). C Prognostic feature of mRNA expression of ATAD2 in LUAD patients (Kaplan–Meier plot). Overall survival (OS) curve comparing patients with high (red) and low (black) ATAD2 expression in lung cancer plotted using the Kaplan–Meier method with a threshold of p-value < 0.05. D mRNA expression of ATAD2 is increased in lung cancer cells compared with normal lung epithelial HBE cells. E, F Protein expression of ATAD2 is increased in the tissues of lung cancer patients. *p < 0.05

Fig 4: Effects of ATAD2 knockdown on lung cancer cell migration, invasion, and stemness. A, B Transwell migration assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. C, D Transwell invasion assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. E, F Tumor sphere formation assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Scale bar = 100 µm. G, H CD133 and CD44 mRNA levels in siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Error bars indicate mean ± SD. All experiments performed in triplicate. *p < 0.05

Fig 5: Effects of ATAD2 on lung cancer cell viability and proliferation. A Validation of siRNA knockdown efficiency in A549 and SPC-A1 cells as determined using RT-qPCR. B, C Cell counting kit (CCK)-8 proliferation assay in siRNA-negative control (NC) or siRNA-ATAD2-transfected A549 and SPC-A1 cells. D 5-Ethynyl-2'-deoxyuridine (EdU) proliferation assay using siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Scale bar = 100 µm. E Colony-formation proliferation assay in siRNA-NC- or siRNA-ATAD2-transfected A549 and SPC-A1 cells. Error bars indicate mean ± standard deviation (SD). All experiments performed in triplicate. *p < 0.05