Fig. 3: Mff regulates MAVS-mediated antiviral response independent of mitochondrial fission.

a Scheme of murine Mff deletion constructs. The amino acid numbering scheme used in the constructs corresponds to splice isoform 8 of Mff¹³. TM: transmembrane domain. b Confocal microscopic images of Mff and mitochondria. Mff KO MEFs were transfected with expression vectors for FLAG-tagged Mff (WT), Mff (ΔC20), or Mff (ΔN100), stained with MitoTracker Red (mitochondria; red), and immunostained with an anti-FLAG antibody (green). Nuclei were stained with DAPI (blue). Scale bars: 10 μm. c qRT-PCR analysis of IFN-β mRNA in WT MEFs, Mff KO MEFs, and Mff KO MEFs transfected with an empty vector or expression vectors for FLAG-tagged Mff (WT), Mff (ΔC20), or Mff (ΔN100). Cells were transfected with or without poly(I:C)-LMW (1 μg/mL) for 8 h. Data represent means ± SEM from three independent experiments (vector, WT, ΔC20, ΔN100: n = 6, 4, 4, 5, P value; two-tailed unpaired t test). d Immunoblot analyses of endogenous and exogenous Mff and Mff phosphorylation in the cells listed in c. The indicated letters (a, b, c) correspond to the endogenous Mff bands observed in WT MEFs. e Confocal microscopic images of MAVS and Mff. Mff KO MEFs transfected with expression vectors for Mff (WT), Mff (ΔC20), or Mff (ΔN100) were immunostained with anti-MAVS (green) and anti-Mff (red) antibodies. Nuclei were stained with Hoechst 33342 (blue). A magnified view of the boxed region is shown below. Scale bars: 10 μm (whole) and 2 μm (magnified). f Magnified images of MAVS on mitochondria in Mff KO MEFs co-transfected with each Mff mutant and a mitochondrial marker mitoRFP (red). Cells were immunostained with anti-MAVS antibody (green). Scale bars: 2 μm. g Co-localization ratio between MAVS and mitoRFP in WT MEFs or Mff KO MEFs co-transfected with mitoRFP and an empty vector or each Mff mutant. Data represent means ± SEM (WT-vector, Mff KO-vector, Mff KO-WT, Mff KO-ΔC20, Mff KO-ΔN100: n = 15, 13, 16, 16, 16, P value; two-tailed unpaired t test). h Working hypothesis for the coupling of the distribution of MAVS and mitochondrial fission by Mff. mito: mitochondria. Immunoblot and microscopic analyses were repeated independently three times with similar results. Source data are provided as a Source data file.