Fig 1: Testosterone up-regulates ABP expression through promoting its synthesis and suppressing autophagic degradation.(A–E), Rat primary Sertoli cells were cultured in CM and treated with testosterone for 24 h, and ABP expression in the cells was determined with Western blot (A), immunocytochemistry (C) and qPCR (D) (n = 3). The ABP expression level in the supernatant was assessed by ELISA (E) (n = 3, **p < 0.01). The densitometric analysis of ABP immunoblots is shown in (B) (n = 3, **p < 0.01). (F–H), Cells were cultured in CM, MCSS, MCSS+T1 and MCSS+T2 for 24 h, and LC3B, P62 and ABP were determined by Western blots (F). LC3B was also assessed by immunofluorescence (H). Densitometric analysis of the bands in ABP immunoblots is shown in (G) (n = 3, *p < 0.05). The blots of LC3B and Beta actin in Fig. 3F were cropped and full-length blots are presented in Supplementary Figure S4. The average number of LC3 puncta per cell was calculated in Supplementary Figure S5. (I), Cells were treated with CQ (50 uM) in the presence or absence of testosterone (10 nM), and the ABP and LC3 expression levels were assessed with double immunofluorescence after 24 h. The average number of ABP-LC3 colocalizations per cell was calculated in Supplementary Figure S7. (J–K), Cells were treated with cycloheximide (150 uM) in the presence or absence of testosterone (10 nM) as indicated (T: Testosterone) for different time periods, the ABP and LC3 expression were determined by Western blots (J). The relative ABP levels were determined by measuring the density of protein band, and normalized to Beta actin (K) (n = 3, *p < 0.05). The relative ABP protein level at time zero was termed as 1. The blots of ABP and Beta actin of the testosterone (+) group were cropped and full-length blots are presented in Supplementary Figure S10. (L), Cells were cultured in CM and treated with testosterone (10 nM), CQ (50 uM) or rapamycin (10 nM), as indicated. ABP, LC3B, P62 and Beta actin were determined by Western blots after 24 h. CM: Complete medium (medium with 10% foetal bovine serum); MCSS: Medium with 10% charcoal stripped serum. T1: Testosterone (10 nM); and T2: Testosterone (50 nM).
Fig 2: ABP colocalises with LC3 in primary Sertoli cells.(A), Rat primary Sertoli cells were treated with CQ (50 uM) or rapamycin (10 nM) for 24 h (n = 3), or treated with ATG7 or MTOR siRNA for 48 h (n = 3), ABP mRNA was determined by qPCR. (B–C), Cells were treated with CQ or rapamycin for 24 h, and ABP and LC3 were assessed with double immunofluorescence (B). The average number of colocalisations per cell was calculated from 10 random fields (approximately 200 cells) (C) (n = 3, **p < 0.01).
Fig 3: 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.
Fig 4: Stress-induced autophagy does not degrade ABP.(A–C), Primary Sertoli cells were cultured in hypoxia (1%) or serum deprivation medium for 24 h, LC3B and P62 were determined by Western blots (A). LC3B was also assessed by immunofluorescence (B). A high concentration of CQ (100 uM) was used before the immunofluorescence assay to promote the accumulation of LC3. The average number of puncta per cell was calculated from 10 random fields (C) (n = 3, **p < 0.01). (D–G), Cells were treated with CQ (50 uM) or rapamycin (10 nM) in the presence or absence of hypoxia for 24 h. ABP, LC3B and Beta actin were determined by Western blots (D). The bands in ABP immunoblots were analysed (E) (n = 3, *p < 0.05, **p < 0.01). ELISA was used to determine the ABP expression in the supernatants (F) (n = 3, *p < 0.05). ABP was also assessed by immunocytochemistry (G). (H–I), Cells were exposed to hypoxia and treated with testosterone (10 nM), CQ (50 uM) or rapamycin (10 nM) as indicated, ABP and LC3B were determined by immunoblots (H). The densitometric analysis of the bands in ABP immunoblots is shown in (I) (n = 3, *p < 0.05 when compared with any other group).
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