Fig 2: Mutated SEPT14 affects the filament structure of ACTN4. (A–C) Immunofluorescence staining of cells transfected with plasmids encoding WT FLAG-SEPT14 (WT panel), FLAG-SEPT14A123T (A123T panel), and FLAG-SEPT14I333T (I333T panel) with FLAG and ACTN4 antibodies. From left to right: Images of FLAG antibody (FLAG-SEPT14), ACTN4 antibody (ACTN4), and DAPI staining and merged images with FLAG antibody, ACTN4 antibody, and DAPI staining (Merge), as well as an enlarged image (Enlarge). Magnification, 1000×. Scale Bar: 10 μM.

Fig 3: Validation of proteomics data and identification of specific markers of large extracellular vesicles (lEVs) and small extracellular vesicle (sEVs). (A,B) Western blot analysis of selected proteins identified by the quantitative analysis of proteomic data. Equal amounts of protein (8 µg) for lEVs or sEVs were loaded on a sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel and immunoblotted for selected proteins found to be enriched in either lEV or sEV preparations, based on the statistical analysis of quantitative proteomic data. For each selected protein, a representative immunoblot is presented. Quantifications of signal intensity are represented as dot plots and calculated from three to seven independent experiments as described in Figure 2(B). The colours used represent different biological replicates used for the immunoblots. These results were confirmed by immunoblotting the specific enrichment of ap2/FABP4, 14-3-3, annexin A2 and endoplasmin in lEV fractions, whereas adiponectin, major vault protein (MVP) and FAS are specifically enriched in sEV fractions. (C,D) Western blot of actinin-4 (C) and syntenin-1 (D) in lEV and sEV subpopulations. Actinin-4 is found to preferentially associate with lEVs, whereas syntenin-1 expression is equally distributed in lEV and sEV preparations.

Fig 4: Immunofluorescence staining of ACTN4 in sperm from donors with SEPT14 mutations. (A–C) From left to right: bright field (BF), ACTN4 antibody staining (green), and merged DAPI and ACTN4 antibody staining. The figures from top to bottom show spermatozoa from donors with (A) WT-SEPT14 (Control), (B) SEPT14A123T (A123T), and (C) SEPT14I333T (I133T). The black and red dashed lines around the sperm indicate normal and abnormal sperm head morphologies, respectively. The red arrows indicate fragmented ACTN4 signals. Scale Bar: 50 μM.

Fig 5: SEPT14 interacts with ACTN4 in male germ cells. (A) Co-IP of FLAG-SEPT14 with ACTN4. Lysates from cells transfected with a pFLAG-SEPT14 vector were immunoprecipitated with either a nonspecific control IgG (Lane 2) or FLAG antibody (Lane 3), followed by immunoblotting with an ACTN4 or FLAG antibody. Light chain was used as a loading control. Input protein (5%) was used as an immunoblotting control (Input). (B) SEPT14 was co-localized with ACTN4 in murine spermatids. DAPI staining (blue), SEPT14 (green), ACTN4 (red), and a merged image of the DAPI, SEP14, and ACTN4 signals in murine spermatids are shown. SEPT14 signals are near the perinucleus rim (white arrows) and manchette structure (red arrows). More than 10 elongating spermatids have been stained. Magnification, 1000×. Scale Bar: 10 μM.