p97/valosin-containing protein (VCP) is expressed in many cells and plays critical functions in a broad range of diverse cellular processes. Because it is expressed in the mouse testes, predominantly in Sertoli cells, and is known to play a critical role in autophagy and apoptosis in different cell types, we set out to investigate its function in autophagosome maturation, apoptosis and cell cycle arrest in a mouse Sertoli cell line. To study the mechanism of p97/VCP action, p97/VCP siRNA and a specific p97/VCP inhibitor, N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ), were used in the mouse 15P1 Sertoli cell line. Loss of p97/VCP activity due to DBeQ exposure and silencing of p97/VCP (siVCP) expression results in autophagosome (LC3 and p62) accumulation in the cytoplasm of Sertoli cells. The coexpression of autophagosomal and lysosomal markers (LAMP1 and LAMP2) was reduced in cells in which p97/VCP expression had been inactivated. To better understand in which step of autophagy p97/VCP functions, the interaction between autophagosomal and autolysosomal markers was studied by coimmunoprecipitation and colocalization experiments. The interaction between autophagosomal markers and lysosomal markers decreased in siVCP-expressing and DBeQ-exposed cells. Moreover, the expression of siVCP and DBeQ exposure caused cytoplasmic vacuolation, induced caspase 3-7-mediated cell death and decreased cell cycle progression in mouse Sertoli cells. Taken together, the results show that p97/VCP is essential for autophagosome maturation and cell survival in mouse Sertoli cells. When these functions are prevented, impaired autophagy and apoptosis may have a detrimental effect on germ cells and cause male infertility.

中文翻译：

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抑制 p97/VCP 功能导致小鼠支持细胞自噬体成熟缺陷、细胞周期停滞和凋亡

含有 p97/valosin 的蛋白质 (VCP) 在许多细胞中表达，并在广泛的不同细胞过程中发挥关键作用。因为它在小鼠睾丸中表达，主要在支持细胞中表达，并且已知在不同细胞类型的自噬和细胞凋亡中起关键作用，我们开始研究其在小鼠自噬体成熟、细胞凋亡和细胞周期停滞中的功能支持细胞系。为了研究 p97/VCP 作用的机制，在小鼠 15P1 Sertoli 细胞系中使用了 p97/VCP siRNA 和特定的 p97/VCP 抑制剂 N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ)。由于 DBeQ 暴露和 p97/VCP (siVCP) 表达沉默导致 p97/VCP 活性丧失导致自噬体（LC3 和 p62）在支持细胞的细胞质中积累。自噬体和溶酶体标志物（LAMP1 和 LAMP2）的共表达在 p97/VCP 表达已被灭活的细胞中减少。为了更好地了解自噬 p97/VCP 在哪个步骤起作用，通过共免疫沉淀和共定位实验研究了自噬体和自噬体标记物之间的相互作用。自噬体标志物和溶酶体标志物之间的相互作用在表达 siVCP 和暴露于 DBeQ 的细胞中降低。此外，siVCP 和 DBeQ 暴露的表达导致细胞质空泡化，诱导半胱天冬酶 3-7 介导的细胞死亡并减少小鼠支持细胞中的细胞周期进程。总之，结果表明 p97/VCP 对小鼠支持细胞的自噬体成熟和细胞存活至关重要。当这些功能被阻止时，