Fig 2: 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 3: 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

Fig 4: CIH-induced HIF-1α activated ATAD2 during lung cancer progression. A Gene correlation between ATAD2 and HIF-1α in LUAD determined by consulting http://timer.comp-genomics.org/. B, C Analysis of ATAD2 promoter activity. Cells were transfected with the HIF-1α-FLAG or control plasmid for 24 h. A549 and SPC-A1 cells were transfected with an ATAD2 promoter plasmid or a plasmid containing a mutated ATAD2 promoter sequence for another 24 h. D HIF-1α, ATAD2, CD133, and CD44 mRNA expression levels in A549 cells incubated under normoxia, CIH, or CIH combined with HIF-1α. E, F Flow cytometry analysis of A549 cells transfected with siRNA-NC or siRNA-ATAD2 under normoxia or CIH. Error bars indicate mean ± SD. All experiments performed in triplicate. *p < 0.05

Fig 5: 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