Fig 2: Molecular actors involved in Brucella abortus-induced mitochondrial fragmentation (a,c) FIS1 (a)/MFF (b) and DRP1 co-immunostaining in HeLa cells infected or not (control) with B. abortus 2308–24 and 48 h PI (Representative of n = 3). Green: DRP1 (Alexa488)/Red: FIS1/MFF (Alexa563)/Turquoise: Nuclei and B. abortus 2308 (Hoechst) (b,d) Quantification of the percentages of DRP1 co-localising with FIS1 (b)/MFF (d) in HeLa cells infected (white) or not (black) with B. abortus 2308. Results represent means ± SD for three independent experiments (n = 3). Statistical analysis: Rank sum test (Mann-Whitney) (**P < 0.01, ***P < 0.001). The numbers indicated in the columns represent the number of cells analysed for each condition (e). TOM20 immunostaining in DRP1+/+ or DRP1-/- MEFs infected or not (control) with B. abortus 2308–48 h PI (Representative of n = 3). Green: TOM20 (Alexa488)/Red: B. abortus 2308 (mCherry)/Turquoise: Nuclei (Hoechst) (f,g) Aspect ratio (f) and end point/branch point ratio (g) of DRP1+/+ or DRP1-/- MEFs infected (white) or not (black) with B. abortus 2308 mCherry - 48 h PI. Results represent means ± SD for three independent experiments (n = 3). Statistical analysis: two-way ANOVA on Box Cox transformed data. P value for interaction: 0.004 and 0.692 respectively. The numbers indicated in the columns represent the number of cells analysed for each condition. (h) Western blot analysis of MFN1, MFN2 and OPA1 abundance in mitochondrial enriched fractions (Mito) or total proteins from total cell lysates of RAW 264.7 cells infected (Inf) or not (Ctl) with B. abortus 2308 mCherry - 48 h PI. TOM20 abundance was assessed on the same blot as the loading control. These full-length blots are presented in Supp. Fig. S10c,e.

Fig 3: In Vivo cross-linking sites of TIA1-iCLIP at specific nuclear-encoded mitochondrial pre-mRNAs associated with mitochondrial organization/dynamics. The RNA map, corresponding to TIA1 binding on indicated human genes, was adapted using TIA1-iCLIP analysis [18]. The localization of target genes on human chromosomes and the exon and intron positions of the human pre-mRNAs are shown. The bar graph in each panel indicates the number of cDNAs identified in each TIA1 crosslinking site. The following human genes are shown: optic atrophy protein 1 (OPA1), mitofusin 1 (MFN1), mitofusin 2 (MFN2), dynamin 1 like (DNM1L), mitochondrial fission factor (MFF), mitochondrial fission 1 protein (FIS1), YME1 like 1 ATPase (YME1L1) and OMA1 zinc metallopeptidase (OMA1).

Fig 4: Effect of Mfn2 on chondrogenic differentiation of CPSCs. A Relative mRNA expression of chondrogenic genes (Sox-9, Col2, and Acan) with or without chondrogenic induction medium for 14 days. B, C The expression of chondrogenic proteins (SOX-9, Col2, and Acan) with or without chondrogenic induction medium for 14 days and quantitative analysis. D–G Safranin O and Alcian blue staining of chondrogenic differentiation in plate or pellet culture with chondrogenic medium for 21 days. H Relative expression of Col2 (red) determined by immunofluorescence on 21 days of chondrogenesis. GFP (green), DAPI (blue). Bar = 200 µM. The data are expressed as mean ± standard deviation, N = 3. *P less than 0.05 versus OE-NC. OE over-expression of Mfn2, OE-NC negative control group of Mfn2 over-expression

Fig 5: Silencing MFN1 or MFN2 reveals their distinct roles in DENV infection.(A and B) RT-qPCR (A) and western blot (B) validation of A549 cells stably expressing shRNA targeting LacZ, MFN1 or MFN2. Data in panels A and C-F are mean ± SD (n = 3 per group) and were compared by two-tailed Student’s t test. Quantification in panel B is the relative ratio of the indicated protein to actin expression. (C) LDH release assay of shLacZ-, shMFN1- or shMFN2-expressing A549 cells infected with DENV serotype 2 (moi 10) for the indicated time. (D) DENV plaque-forming assay of shLacZ-, shMFN1- or shMFN2-expressing A549 cells infected with DENV (moi 0.1) for 24 or 48 h. (E) Analysis of antiviral activity in culture media from indicated cells with DENV infection (moi 5, 48 h) against dSinF-Luc/2A virus. (F) RT-qPCR analysis of IFNß mRNA level in DENV infected (moi 10) A549-shLacZ, -shMFN1, and -shMFN2 cells for the indicated time. (G) Western blot analysis of DENV infected (moi 10) A549 cells expressing shLacZ, shMFN1 or shMFN2. The relative ratios of band intensity were quantified as indicated. (H) Flow cytometry with JC-1 staining of shLacZ-, shMFN1 or shMFN2-expressing A549 cells infected with DENV (moi 5) for 30 h. Decreased red/green fluorescence ratio of JC-1 represents disrupted MMP. CCCP, carbonyl cyanide m-chlorophenyl hydrazone, an ionophore that disrupts MMP. (I) Plaque forming assay of vesicular stomatitis virus (VSV) by using A549 cells expressing shLacZ, shMFN1 or shMFN2 as indicated. pfu, plaque-forming unit.