Figure 1

Autophagy regulates ABP expression in the primary Sertoli cells and rat testes.

(A), Cells isolated from 20-day-old rat testis were stained with oil red O, bodipy 493/503 or alkaline phosphatase detection kit and pictures were taken with a fluorescence microscope. Black arrow: germ cell. (B–E), Rat primary Sertoli cells were treated with CQ (50 uM) or rapamycin (10 nM) for 24 h and ABP expression in the cells was assessed with Western blot (B) and immunocytochemistry (D). ABP in the supernatants was determined by ELISA (E) (n = 3, *p < 0.05). Densitometric analysis of the bands in ABP is shown in (C) (mean ± SD of independent experiments, n = 3, *p < 0.05). LC3B was assessed by Western blot (B). (F–G), Cells were treated with scrambled siRNA, ATG7 siRNA, or MTOR siRNA for 48 h and ABP, LC3B, ATG7, MTOR and Beta actin were determined by Western blots (F) and densitometric analysis of ABP immunoblots is shown (G) (n = 3, *p < 0.05). (H–J), The rat testis was directly injected with 50 ul mixed liquid of in vivo-jetPEI and methylation modified siRNA (5 nmol) as indicated; primary Sertoli cells were isolated after 2 days and ABP was determined by Western blot (H) and immunohistochemistry (J) using adjacent tissue. Densitometric analysis of ABP immunoblots is shown (I) (n = 3, *p < 0.05). Haematoxylin was used to stain the nuclei (blue). Black arrows: Sertoli cells.