Fig 2: Pnldc1 mutation causes mitochondria disorder. a Expression of TDRKH, MILI, MIWI, GASZ, and MVH in WT and Pnldc1 Mut (Mut-1) testes revealed by western blotting. ß-actin is a loading control. b Aggregation of TDRKH, MILI, MIWI, and GASZ in Pnldc1 Mut spermatocytes. Immunostaining was performed using indicated antibodies on adult testis sections from WT and Pnldc1 Mut (Mut-1) mice. DNA (blue) is stained with DAPI. Protein aggregations are indicated by arrows. Scale bar, 10 µm. c Conglomeration of mitochondria in Pnldc1 Mut testes. Immunostaining of WT and Pnldc1 Mut (Mut-1) testis sections with an antibody against AIF, a mitochondrial marker. DNA (blue) is stained with DAPI. Conglomeration of mitochondria is indicated by arrows. Scale bar, 10 µm

Fig 3: NPRL2 overexpression induces oxidative stress. (a) ROS production was estimated by measuring DHE level in non-treated HEK293, H1299 or U2OS cells (NT), and in cell transformed with 1 μg pFLAG or pFLAG-NPRL2 plasmids for 24 h. (b) Analysis of ATP production in HEK293 cells expressing pFLAG and pFLAG-NPRL2 plasmids at indicated concentrations during 24 h. (c) Analysis of mRNA expression levels of indicated genes in the HEK293 cells transformed with 1 μg of plasmids expressing pFLAG or pFLAG-NPRL2 during 24 h. (d) Analysis of mRNA expression levels of NOX1, NOX2, NOX4 and NOS2 in the HEK293 cells transformed as in (c). (e) Analysis of NOX2 expression level in the HEK293 cells transformed as in (c). An increase of NOX2 expression detected by Western blot is marked by a number at the bottom of an appropriate blot. (f) Analysis of ROS production (as in (a)) in the HEK293 cells treated with different antioxidants during 3 hours prior to pFLAG or pFLAG-NPRL2 overexpression. **p-value < 0.001; *p-value < 0.05; #p-value > 0.05. For experiments in (a–d,f) error bars indicate standard deviations of the mean in three independent experiments (see also Supplementary Table S4 for all the statistics corresponding to the graphs in a–d and f). (g) Schematic representation of the effects of the NPRL2 overexpression. NPRL2 overexpression enhances mTORC1 suppression and induces ROS production via NOX2 activation. NPRL2 interacts with AIF and translocates to the nucleus, where AIF is loaded to the sites of DNA damage initiated by ROS. NPRL2 in the nucleus might also interact with other yet unknown factors (marked by “?”). DNA damage leads to the cell cycle arrest and eventual apoptosis.

Fig 4: SDC4 coordinates PKC activation. HTR8 EVTs were transduced with an empty vector (EV) or a plasmid encoding V5-tagged SDC4 (SDC4-V5). (a) Quantitative RT-PCR analysis showing increased SDC4 mRNA expression in SDC4-V5 transduced cells compared to cells transduced with EV. (b) Western blot showing increased expression of SDC4 and V5 in SDC4-V5 transduced cells compared to cells receiving EV. Tubulin was used as a loading control. (c) Co-immunoprecipitation of V5 and PKCα. Input was used as a positive control for western blotting; IgG was used as a negative control for immunoprecipitation. (d, e) Western blot showing levels of phospho-PKCSer660 (pPKC) in membrane lysates from HTR8 EVTs expressing control shRNA (CTRL1) and cells expressing shRNAs targeting SDC4 (SDC4 KD1 and SDC4 KD2) after plating on Matrigel for 3 h (d) or following multiple wounds (e). AIF was used as a loading control for membrane lysates. Graphs present results from densitometric analysis comparing pPKC levels relative to AIF of three different western blots. Graphs represent means (SEM). Uncropped images of western blots are provided in Fig. S5. Data significantly different from controls are indicated with an asterisk (**P < 0.01; ***P < 0.001; ****P < 0.0001).

Fig 5: DNM1L loss leads to release of cytochrome c into the cytosol resulting in low-level caspase cleavage. a Percentage of annexin V+MCAS cells transduced with a control targeting sgRNA or a sgRNAs targeting DNM1L (n = 3). Data are mean ± SEM. *p < 0.05 by Student’s t-test. b Immunoblot of cytochrome c or AIF in whole-cell lysate, membrane fraction, cytosolic fraction, and nuclear fraction of MCAS cells transduced with a control sgRNA or a representative sgRNA targeting DNM1L. Immunoblots are representative of three independent experiments. AIF is used as a control for contamination of the cytosolic fraction. c Left, immunoblot of vinculin, Drp1, and c-caspase 9 in MCAS cells transduced with control sgRNA or two independent sgRNAs for DNM1L. Immunoblots are representative of two independent experiments. Right, raw luminescence units of caspase 9 activity in MCAS cells transduced with control sgRNA or two independent sgRNAs for DNM1L (n = 3). Data are mean ± SEM. *p < 0.05 by one-way ANOVA. d Ratio of JC-1 fluorescence (595 nm/535 nm) in MCAS cells transduced with control sgRNA or three independent sgRNAs for DNM1L (n = 3). Data are mean ± SEM. *p < 0.05 by one-way ANOVA. e Left, DCFDA raw fluorescence units minus no stain fluorescence units in MCAS cells transduced with control sgRNA or three independent sgRNAs for DNM1L (n = 3). Data are mean ± SEM. *p < 0.05 by one-way ANOVA. Middle, DAF-FM raw fluorescence units minus no stain fluorescence units in MCAS cells transduced with control sgRNA or three independent sgRNAs for DNM1L (n = 3). Data are mean ± SEM. *p < 0.05 by one-way ANOVA. Right, mitosox raw fluorescence units minus no stain fluorescence units in MCAS cells transduced with control sgRNA or three independent sgRNAs for DNM1L (n = 3). Data are mean ± SEM. *p < 0.05 by one-way ANOVA. f Log2(sgDNM1L/sgCTRLA1) BV6 GI50 value in MCAS cells treated with either vehicle or one of the following: NaC (10 mM), Tiron (1 mM), Carboxy-PTIO (100 µM) (n = 3). Data are mean ± SEM. *p < 0.05 by one-way ANOVA