Fig. 4

Purified Sertoli cells were used for co-culture and ChIP-seq analysis to screen target genes of AR in Sertoli cells. Bright field (a), WT1 staining (b), DAPI (c) and merge (d) of purified Sertoli cells were exhibited. WT1⁺ ratio of purified Sertoli cells was higher than 95%, n = 5 (i). Purified Sertoli cells were examined for β1-integrin expression by IF (e-h), and then were infected with β1-integrin shRNA lentivirus to interfere in endogenous expression of β1-integrin before co-culture with SPCs. Morphology of Sertoli cells 72 h post infection (j), and infection efficiency was examined by expression of red fluorescence protein reporter (k). Western blot revealed the relative expression level of β1-integrin was approximately decreased by 80% after β1-integrin shRNA interfering, 1, Itgb1 siRNA, 2, control siRNA. Data represent means ± SD (*p < 0.05, **p < 0.01), n = 3 (l). The representative view of AR binding sites (visualized by Integrative Genomics Viewer [http://software.broadinstitute.org/software/igv/]) at Gata2 promoter region (m). ChIP-qPCR validated the binding of AR in the promoter region of Gata2: fold enrichment by antibodies against AR relative to control IgG was presented as mean value ± SD of two replicates (n). Overexpression of AR or DHT treatment caused down-regulated expression levels of GATA2, WT1 and β1-integrin in Sertoli cells, n = 3 (o). Knockdown of Gata2 using siRNA led to decreased expressions of WT1 and β1-integrin, n = 3 (p)