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: NIX drives mitochondrial fragmentation via the fission GTPase DRP1(A) SDC images of DRP1 IF in NHEKs transduced with GFP-NIX(B) Diagram showing a measure of peripheral bias as the ratio between the distance (marked A) from the nucleus to each particle and the distance (marked B) from the nucleus to the cell edge.(C) Quantification of the peripheral bias of DRP1 particles in cells expressing GFP-NIX compared with neighboring non-transduced cells (mean ± SD, n = 19 cell pairs, 4 expts., *p = 0.0148).(D) SDC images of FIS1 IF in NHEKs transduced with GFP-NIX.(E) Model depicting a GFP-NIX-positive mitochondrion (green rim) with FIS1 stabilized on its surface, which recruits DRP1, leading to mitochondrial fragmentation.(F) SDC images of DRP1 and Hsp60 IF in NHEKs transduced with Halo-FIS1.(G) Quantification of mitochondrial AR and perimeter in NHEKs expressing Halo-FIS1 compared with non-transduced neighbors (mean ± SD, n = 14 cell pairs, 3 expts., ***p < 0.0001).(H) IF of FIS1 and NIX in human skin.(I) SDC images of NHEKs transduced with GFP-NIX alone (top panels) versus neighboring cells co-transduced with DRP1-K38A (bottom panels).(J) Quantification of mitochondrial AR and perimeter in NHEKs expressing GFP-NIX alone or with DRP1-K38A (mean ± SD, n = 54 cells, 4 expts., ***p < 0.0001).White scale bars, 10 µm.

Fig 4: Blockade of mitochondrial fission via DRP1 impairs epidermal differentiation(A) IF of DRP1 and TOM20 in mature epidermal cultures.(B) H&E staining of organotypic epidermis treated with DMSO or Mdivi-1, showing retained nuclei in cornified layers (yellow arrowhead) and (right) quantification of retained nuclei in DMSO- versus Mdivi-1-treated cultures (mean ± SD, n = 70 fields, 4 expts., ***p < 0.0001).(C) WB of DRP1 in lysates from organotypic cultures treated with Mdivi-1 or transduced with DRP1-K38A.(D) Quantification of mitochondrial size in upper-layer cells transduced with TOM20-mCh along with DRP1-K38A or a viral vector (mean ± SD, n = 61 fields, 4 expts., ***p < 0.0001).(E) SDC images of the upper layers of organotypic epidermis expressing TOM20-mCh alone (left) or with DRP1-K38A (right).(F) H&E staining of cultures expressing a viral vector or DRP1-K38A, highlighting KH granules (white arrowhead) and retained nuclei (yellow arrowhead) and (bottom) quantification of tissue thickness (mean ± SD, n = 103 fields, 4 expts., ***p < 0.0001) and retained nuclei (mean ± SD, n = 101 fields, 4 expts., *p = 0.0115) in vector- versus DRP1-K38A-expressing cultures.(G) IF of K10 and FLG in organotypic cultures expressing vector or DRP1-K38A and (right) quantification of K10-positive cell size (mean ± SD, n = 30 fields, ***p < 0.0001) and FLG staining (mean SD, n = 30 fields, ***p < 0.0001). Dashed lines mark the bottom of the epidermis. Black scale bars, 50 µm; white scale bars, 10 µm.(H) Model depicting the onset of NIX expression in differentiating keratinocyte layers driven by hypoxia-regulated signaling; NIX-positive mitochondria (green rim) exhibit enhanced localization of FIS1, which recruits DRP1 to produce mitochondrion fragments that are targeted for lysosomal degradation in the uppermost epidermal layers.

Fig 5: Effects of melatonin treatment on mitochondrial fission/fusion markers in the red vastus lateralis muscle of Zücker diabetic fatty rats and their lean littermates as measured by Western blot. (A–D) Densitometry quantification of fission 1 protein (Fis1), dynamin-related protein 1 (DRP1), optic atrophy protein type1 (OPA1), and mitofusin 2 (Mfn2) protein levels. (E) Ratio of DRP1 to Mfn2 expression. (F) Representative blots of Fis1, DRP1, OPA1, and Mfn2. C-ZL: control lean rats without melatonin; M-ZL: lean rats with melatonin; C-ZDF: control diabetic fatty rats without melatonin; M-ZDF: diabetic fatty rats with melatonin; ZL: Zücker lean rats; ZDF: Zücker diabetic fatty rat. All values are expressed as mean ± SD of three independent experiments performed in duplicate in each rat. The represented values were expressed relative to β-actin protein levels. ** p < 0.01; * p < 0.05; ## p < 0.01, # p < 0.05 (One-way ANOVA followed by Tukey post hoc test).