Fig 2: SPATA33 and ATG16L1 are colocalized in germline cells in mice.a Immunofluorescence of SPATA33 and ATG16L1 proteins in mouse testis. Anti-SPATA33, anti-ATG16L1 (Alexa Fluor®488), and TRITC-conjugated goat anti-rabbit IgG (H + L) antibodies were used to detect SPATA33 (red) and ATG16L1 (green). The nuclei were stained by Hoechst (blue). Images were taken by confocal fluorescence microscopy (SP8, Leica, Wetzlar, Germany). Positive signals were detected in Leydig cells (LC), Sertoli cells (Sn), spermatogonia (Sg), spermatocytes (Sc), and spermatids (Sp). Scale bar, 25 μm; scale bar in enlarged panels: 20 μm. b Cell sorting of spermatogonia (Sg), spermatocytes (Sc), and the round spermatids (Sp) from testis by cell flow sorter. Serial charts of cells were indicated. c Expression of Spata33 was detected in spermatogonia (Sg), spermatocyte (Sc), the round spermatids (Sp), and spermatozoa (sperm). The sperm cells were isolated from epididymis of adult mouse. RT-PCR was performed from mRNAs isolated from these cells. Actin was used as an internal control. d SPATA33 was colocalized with ATG16L1, LC3B, and VDAC2 in the mid-piece region (mitochondria region) of the sperm cells. The spermatozoa were extracted from epididymis of adult mice. The cells were stained with MitoTracker (mitochondria matrix marker, red). Immunofluorescence analysis were performed with anti-SPATA33, anti-ATG16L1 (Alexa Fluor®488), anti-VDAC2, anti-LC3B (Alexa Fluor®488), TRITC-conjugated goat anti-rabbit IgG (H + L), and FITC-conjugated rabbit anti-goat IgG (H + L) antibodies. The enlarged images were originated from the squares in the merged panels. The nuclei were stained by Hoechst (blue). Images were taken by confocal fluorescence microscopy (SP8, Leica). The graph on the right indicates sperm ultrastructure, highlighting cross-section of the mid-piece region, including axoneme, outer dense fiber, and mitochondrial sheath (red). Scale bar: 25 μm; scale bar in enlarged panels: 6 μm.

Fig 3: MYBL2 and GATA1 are expressed in the developing ovaries of postnatal mice. (A) MYBL2 immunolocalization in the developing ovaries of postnatal mice using the anti- MYBL2 antibody. MYBL2 is expressed in the nuclei of stromal cells and oocytes at 2 dpp; MYBL2 expression gradually increases from 6 dpp to 18 dpp in oocytes, theca and granulosa cells. After 18 dpp, MYBL2 maintains high expression in oocytes and granulosa cells but less in theca cells. The nuclei were stained with Hoechst reagent. The enlarged images originated from the white squares. The arrows indicate granulosa cells, whereas arrowheads indicate the oocytes. Scale bar: 50 µm. See also Figure S1. (B) Western blot analysis showing the GATA1, MYBL2, ATG5, and ATG16L1 protein levels in the developing ovaries of postnatal mice. The MYBL2 expression pattern is similar to VDAC2, with a peak at 16 dpp in the ovary. The ATG5 and ATG16L1 expressions in postnatal ovaries increased till 12 dpp and then decreased obviously after 14 dpp. The A and B isoforms of ATG16L1 were detected. ACTB was used as an internal control.

Fig 4: Mutation analysis of the mouse Vdac2 promoter. (A) Schematic diagram of the Vdac2 promoter, which includes a CpG island with a GC box, a GATA-like element and 2 MBS sites. The short vertical bars indicate CpG dinucleotides. (B) Luciferase assay shows the activities of a series of deleted constructs in both CHO and NIH-3T3 cells. Left panel shows each deleted mutant linked with the luciferase gene in the pGL3-basic vector. Right panel indicates the relative activities of these deleted constructs, as determined by luciferase assays. (C) Point mutation analysis of the core promoter using luciferase assays. The 259 bp LS7 construct was used as a basic construct for the point mutation analysis. Luciferase assays were used to determine the relative activities. The intact binding sites of the GC box, GATA-like element MBS1 and MBS2 are indicated by open boxes and by circles, respectively. The filled boxes and circles show corresponding mutations. The pGL3-basic vector was used as a negative control. The data are based on the luciferase activity of pGL3-LS7. The mean ± SD are from 3 independent experiments.*, P < 0.05; **, P < 0.01 compared with the LS7 wild-type construct. See also Figure S3.

Fig 5: Mitochondria damaged severely in immortal fibroblast (iFB)-LHON cells with more variants. (A) ROS level was detection in different fibroblast cell lines: iFB-NC, iFB-III-4, and iFB-I'-2. *p = 0.05, ***p = 0.001. (B) JC-1 monome and aggregate were pictured. The ratio of fluorescence intensity between monomer and aggregate characterized mitochondrial membrane potential. *p = 0.05. (C) oxygen consumption rate (OCR) value of three fibroblast cells was detected using seahorse. The basal (OCR value = OCR value at the beginning, meaning the level of energy metabolism rate in basal state), max (OCR value = OCR value at the highest point after FCCP treatment, meaning the maximum respiration rate that the cell can achieve) and ATP-linked (OCR value = OCR value decreased after oligomycin treatment, meaning the respiration part used for ATP synthesis) OCR values were calculated. ***p = 0.001. (D) Mitochondrial morphology in iFB-NC, iFB-III-4, and iFB-I'-2 cells was photographed by electron microscope. (E) The protein levels of MT-ND1, MT-ND4, MT-CO IV, and VDAC2 were detected by Western blot. The ß-actin was used as reference.