Fig. 2

Loss of ATAD3A impairs mitochondrial function in HNSCC cells and inhibits tumor development. A ATAD3A protein levels in various HNSCC cell lines and normal oral keratinocytes (hTERT). B Subcellular localization of ATAD3A in HN12 cells determined by confocal microscopy after IF staining. C ATAD3A KO clones derived from the sgATAD3A-targeting HN12 cells screened by Western blotting. D Mitochondrial structure of ATAD3A KO (#1) and parental HN12 cells assessed by TEM. Mitochondrial cristae density, minor axes and aspect ratio (major axis/minor axis) were quantified from 20 randomly selected fields. Arrow heads indicate mitochondria. E OCR and ECAR profiles of ATAD3A KO (#1) and parental HN12 cells measured by a Seahorse XFe96 analyzer. In this assay, 1 μM Oligo that inhibits ATP synthesis and 1 μM FCCP that uncouples OXPHOS were injected at the indicated point (arrow). F OCR:ECAR ratio at basal and maximal respiration in ATAD3A KO and parental HN12 cells. G The effect of ATAD3A KO (#1 and #5) on cell proliferation on Day 3. H The effect of ATAD3A KO on cell colony formation within 3 weeks. Quantitative data from colony formation are shown in the lower panel (n = 3). I The effect of ATAD3A KO on soft agar cell growth on Day 14. Quantitative data from soft agar assays are shown in the lower panel (n = 3). J The effect of ATAD3A KO on HN12 cell growth on Day 14 in 3D SeedEZ scaffold. Quantitative data from 3D assays are shown in the right lower panel (n = 3). K Representative bioluminescence images showing the effect of ATAD3A KO on HN12-derived tongue tumor growth in NSG mice. Tumor progression was monitored on Day 15 by examining bioluminescence in Xenogen IVIS-200 In Vivo Imaging System. L Quantitative data of bioluminescence intensity and tumor volume in the indicated groups (n = 5 mice/group). hTERT: Human telomerase-immortalized tonsillar keratinocytes hTERT HAK Clone 41. *p < 0.05; **p < 0.01