Fig. 6

Identification of β1-integrin as a putative interactive molecule between SPCs and Sertoli cells. Thy1⁺ SPCs were co-cultured with Sertoli cells infected with control shRNA or β1-integrin shRNA for 48 h (a), bar = 20 μM, and the relative ratio of SPCs number of β1-integrin shRNA transfected group compared to control was statistically analyzed (b). The expression levels of germline markers of SPCs were detected using Western blot, n = 3 (c). RT-PCR detected expression profiles of integrins and cadherins in SPCs at mRNA level, (1-18 in integrin α family represent α1~α11,αD, αE, αL, αM, αV, α2b, αX; 1-12 in integrin β family and cadherins represent β1-β8, E-cadherin, N-cadherin,  P-cadherin and Gapdh, respectively) (d). The binding of E-cadherin and β1-integrin was detected in adult mouse testis cell lysates using immunoprecipitation, of which the binding of β-catenin and E-cadherin was used as positive control, n = 3 (e). Thy1⁺ SPCs co-cultured with Sertoli cells were transfected with control siRNA or E-cadherin siRNA for 48 h (f), bar = 20 μM, and the relative ratio of SPCs number was statistically analyzed (g). The expression levels of germline markers of SPCs were detected using Western blot, n = 3 (h). Expression of β1-integrin (i) and E-cadherin (j) in SPCs and co-cultured Sertoli cells was determined by IF, and β1-integrin signal was detected in Sertoli cells (red frames), bar = 20 μM. Purified Sertoli cells or Thy-1⁺ SPCs were lysed to detect the expression of β1-integrin and E-cadherin using western blot, n = 3 (k). Thy-1⁺ SPCs were lysed for co-IP to detect the endogenous interaction of β1-integrin and E-cadherin, n = 2 (l)