F and HN cooperate synergistically to enhance mitochondrial biogenesis. A Detection of TOM 20 and TIM 23 proteins through Western blot analysis in A549 cells. Western-blot samples were collected and performed in accordance with a previously described protocol . B Detection of five membrane-bound protein complexes through western blot analysis in A549 cells. C Western blot analysis in DF-1 cells. D Intensity ratio of COX II to COX IV in response to F and HN transfection in A549 cells. The intensity of the COX II signal was normalized to the signal of COX IV subunit and calculated as the COXII: COIV ratio. Protein band intensities were quantified using Image J (National Institutes of Health, Bethesda, MD, USA). Significance was analyzed using a one-tailed Student’s t test. *p-value < 0.05; **p-value < 0.01. E Influence of the cleavage site motif of F on mitochondrial-related protein. In A549 cells, wild-type HN plasmid was co-transfected with wild-type F, F112G, F115G, F117L, F112G+115G, F112G+117L, F115G+117L and F112G+115G+117L. At 36 hpt, the Western-blot samples were collected and analyzed. F Intensity ratio of COX II to COX IV in response to the co-transfection of eight F mutants and HN plasmids in A549 cells. Protein band intensities were quantified using Image J. G Synergistic cooperation of F and HN to enhance intracellular ATP levels. At 24 hpt, the intracellular ATP levels were determined using an enhanced ATP assay kit (Beyotime Biotechnology, S20027). The number of cells of the detected samples was normalized through Western blot analysis. H Influence of cleavage site motif on the intracellular ATP levels. In A549 cells, wild type HN plasmid was co-transfected with wild-type F, F112G, F115G, F117L, F112G+115G, F112G+117L, F115G+117L and F112G+115G+117L. At 24 hpt, the ATP values of cells were determined using an enhanced ATP assay kit.