Fig 2: Myo5b KO mice have microvilli-lined inclusions associated with a-actinin and Rab11a. (A) Immunofluorescence staining for the basolateral protein p120 (magenta) and the plasma membrane and actin cytoskeleton linker Ezrin (green) shows the presence of numerous intracellular inclusions in Myo5b KO mice. (B) Micrograph of a-actinin-4 (magenta), which delineates the terminal web immediately below the apical brush border and the lateral membrane of intestinal epithelial cells and ?-actin (green), which identifies the actin-rich apical membrane immunostaining. No intracellular inclusions were observed in the proximal small intestine of neonatal control mice. Myo5b KO mice had numerous ?-actin–positive inclusions that also were ringed by a-actinin–4. (C) The small intestine of littermate control and Myo5b KO mice were stained for Rab11a (magenta) and ?-actin (green), which showed altered Rab11a localization in Myo5b-deficient enterocytes. Moreover, Rab11a surrounded numerous inclusions at the apical membrane and inclusions that were fully internalized. (D) Z-stack projections of the proximal small intestine of control and Myo5b KO mice to visualize the 3-dimensional nature of inclusions after loss of Myo5b. (E) Fracture SEM was performed on neonatal small intestine from Myo5b KO mice to view microvilli-lined inclusions. A completely internalized inclusion (pseudocolored magenta) and a forming inclusion (pseudocolored purple) are shown. n = 4–6 mice per group. Scale bars (immunofluorescence images): 50 µm. Scale bars (SEM from left to right): 10 µm, 5 µm, 5 µm.

Fig 3: Graphical illustration of the molecular roles of SEPT14, ACTN4, and ACTIN in sperm head formation. During sperm head formation, the manchette structure, which consists primarily of microtubules and actin, assists with sperm head shaping. Filamentous SEPT14 binds to ACTN4/ACTIN complexes, which are involved in sperm head shaping. In sperm with SEPT14 mutations (e.g., A123I or I333T), mutated SEPT14 affects ACTN4-ACTIN function, resulting in abnormal sperm head morphology.

Fig 4: TP73-AS1 depletion inhibits the xenograft tumor growth of NSCLC. (a) Bright field tumor samples to reflect the tumorigenesis of SPC-A1 treated with sh-TP73-AS1. (b) Tumor growth curves of mice after injected with different treated SPC-A1 cells. (c) Statistical graph of tumor weight. (d) The mRNA level of TP73-AS1, miR-125a-3p, and ACTN4 in subcutaneous tumors was measured via qRT-PCR assay. (e) Representative images of Ki67-and ACTN4-positive sections of subcutaneous tumor tissues by IHC assay (top panels: magnification = 200x; Scale bar = 100 µm; bottom panels: magnification = 400x; scale bar = 50 µm). Compared to the relevant sh-NC group, **P < 0.01, ***P < 0.001.

Fig 5: ACTN4 targets on miR-125a-3p via TP73-AS1. (a) Venn diagram to show predicting targets mRNAs of miR-125a-3p. (b) Potential binding sites between miR-125a-3p and ACTN4. (c) QRT-PCR assay to confirm overexpression or knockdown of miR-125a-3p. (d) The luciferase reporter experiment indicates the interaction of miR-125a-3p with ACTN4. (e) MRNA expression of ACTN4 after overexpression or knockdown of miR-125a-3p. (f) Immunoblot bands and statistical analysis to show the protein level of ACTN4. (g) MRNA expression of ACTN4 in SPC-A1 cell line and A549 cell line treated with sh-TP73-AS1 and miR-125a-3p inhibitor. Compared to the relevant miR-NC group, anti-miR-NC group or sh-NC group, *P < 0.05, **P < 0.01, ***P < 0.001; compared to the sh-TP73-AS1group, ##P < 0.01.