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. 2015 May 15;26(10):1947–1956. doi: 10.1091/mbc.E14-11-1497

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© 2015 ErLin et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — An in vivo study assessing the role of Msi-1 in BTB function and spermatogenesis by RNAi intratesticular injection. Msi-1–targeting siRNA duplexes and nontargeting duplexes were administered to each testis in adult mice as a single treatment. The knockdown efficiency, BTB-associated protein expression, biotin tracer experiment, and hematoxylin-eosin (HE) staining at 3, 5, 9, and 12 d postintratesticular injection, respectively. (A) Considerably more intense Msi-1 staining was observed in the Sertoli cells from testes transfected with nontargeting control (a) versus testes transfected with Msi-1–specific siRNA duplexes (b). Scale bars: 20 μm. (B) Histogram summarizing immunoblotting results of Msi-1 and normalized against β-tubulin. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05. (C) Paraffin sections were used to study changes in the expression of β-catenin (a and b) and claudin-11 (c and d) after Msi-1 knockdown in vivo. The boxed area was randomly selected. Scale bars: 20 μm. (D) The protein levels of β-catenin and claudin-11 in nontargeting control and Msi-1 knockdown testes. β-tubulin served as a loading control. (E) Histogram summarizing selected immunoblotting results in D and normalized against β-tubulin. Each bar represents the mean ± SD of n = 3 experiments. *, p < 0.05. (F) We injected a biotin tracer into the testes of live anesthetized mice (n = 3) and examined the subsequent changes in BTB integrity after siRNA duplexes injection. (G) HE staining showed that the continuous process of spermatogenesis was seriously disrupted after Msi-1 knockdown in vivo. Scale bars: 80 μm.