F and HN cooperate synergistically to disturb mitochondrial fusion–fission homeostasis and induce nuclear aggregation. A Representative images of F and HN co-operation that changes the subcellular distribution of mitochondria in A549 cells, which were mock transfected or transfected with F, HN, or both at the indicated time. At 0, 12, 18 and 24 hpt, coverslips were examined via IFA in accordance with previously described protocol . After transfection, the treated cells were fixed, and processed for IFA. Image J was used to analyze the mitochondrial network morphological characteristic. B Results from mitochondrial network analysis performed in A549 cells. The mitochondrial network morphology was analyzed by using Image J. The workflow for the pre-processing and analysis of the targeted images was established in accordance with previously Ref. . C Detection of fusion–fission proteins through Western blot analysis in A549 cells. D Intensity ratio of mfn2 to β-actin in response to F and HN co-expression in A549 cells. Protein band intensities were quantified using Image J. 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 the activation of ER UPR and UPRmt. 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 corresponding to the marked point times were collected and analyzed. F Intensity ratio of Mfn1 to β-actin in response to the co-transfection cleavage mutant of F and HN in A549 cells at 24 hpt. Protein band intensities were quantified using Image J.