Fig. 3: Depletion of Kindlin-2 increases apoptosis and inhibits phagocytosis in Sertoli cells.

A Left: Ki67 was detected in 4 and 8 W WT and KO testes by immunohistochemical analyses. Scale bar, 50 μm. Right: The quantification of Ki67 positive cells in one Seminiferous tubule. Data are mean ± s.e.m. The variance was similar between the groups. *p < 0.05, **p < 0.01 and ***p < 0.001 by Student’s t-test. B TUNEL detection for apoptosis (red) in WT and KO testes samples at 2, 4 and 12 W. The nuclei were stained blue with DAPI. KO testes showed increased apoptosis signaling at 2 W testes compared with WT testes. Scale bar, 50 μm. C Flow cytometry analysis were used to analyze the apoptosis rate in TM4 and 15P-1, transfected with control or Kindlin-2 siRNA separately. Knock down of Kindlin-2 induced more apoptosis in both TM4 and 15P-1 cells. D The Sertoli cell lines TM4 and 15P-1 were transfected with control or Kindlin-2 siRNA for 48 h. Cells were lysed and immunoblotted for Cleaved Caspase 3. β-actin was used as a loading control. Experiments were repeated three times. E Immunofluorescence staining of EZ-link sulfo-NHS-LC-biotin tracer was performed in wild type and KO mice testis. WT mice show restriction of biotin tracer to the basal compartment, while KO mice show spreading of biotin tracer dye in seminiferous tubules. Scale bar, 50 μm. Circles indicate the seminiferous tubules, and arrows showed the biotin entering seminiferous tubules through blood testes barrier. F Immunofluorescence of α-tubulin (green) was performed 3 h after the microbeads phagocytosis test in Kindlin-2 siRNA compared with control siRNA in 15P-1 cells. Scale bar, 50 μm. G Quantification for the phagocytosis ratio in control and Kindlin-2 siRNA in 15P-1 cells. Cells treated with Cytochalasin D were used as negative control. Statistical analyses were performed using Student’s t-test. The variance was similar between the groups. ***p < 0.001. H Immunohistochemistry staining for ABP in testes from 4 W WT and KO mice testis. Scale bar, 50 μm. Magnifications were shown on the right panel. I Western blot analyses of ABP in testes from 4 W KO and WT mice. GADPH acts as an internal reference. J Representative western blot analyses of ABP and Kindlin-2 using specific antibodies through loss of function of Kindlin-2 in two cell lines TM4 and 15P-1. GADPH acts as an internal reference.