热休克蛋白（HSP）是分子伴侣蛋白，能协助蛋白质的折叠、定向运输和代谢过程。HSP的细胞内分子伴侣功能已得到广泛研究，但最近发现了细胞外和外泌体中的HSP。外泌体中的HSP可以在细胞间传递，而细胞外的HSP则发挥细胞因子样的作用，称为分子伴侣因子（chaperokines）。我们已经发现外泌体中的HSP在舌癌和肿瘤相关巨噬细胞之间的肿瘤微环境中发挥关键的细胞间通讯作用。值得注意的是，HSP90亚型包括HSP90α、HSP90β、线粒体TRAP1和内质网的GRP94。此外，许多HSP90的假基因可以转录为RNA。此外，HSP90的功能通过其共伴蛋白（如CDC37或AHA1）来定义。因此，需要使用特异性小干扰RNA（siRNA）以精确靶向每个HSP90亚型和共伴蛋白。然而，在沉默研究中，我们经常遇到HSP90亚型的补偿表达。在这里，我们提供了用于挑战细胞内、细胞外和外泌体HSP的特异性作用和补偿表达的双重和三重敲除方法。© 2023. 作者（们）在Springer Science+Business Media, LLC（Springer Nature的一部分）的独家许可下发表。

Heat shock proteins (HSPs) are molecular chaperones that assist in protein folding, trafficking, and metabolism. Intracellular chaperone functions of HSPs had been well-investigated, but extracellular and exosomal HSPs have been recently found. Exosomal HSPs are intercellularly transferred, while extracellular HSPs play cytokine-like roles called chaperokines. We have shown that exosomal HSPs play key roles in intercellular communication between tongue carcinoma and tumor-associated macrophages in the tumor microenvironment. Notably, HSP90 isoforms consist of HSP90alpha, HSP90beta, mitochondrial TRAP1, and GRP94 in the endoplasmic reticulum. Moreover, many pseudogenes of HSP90 can be transcribed into RNA. Besides, the function of HSP90 is defined by their cochaperones, such as CDC37 or AHA1. Therefore, isoform-specific small interfering RNA (siRNA) is necessary for precisely targeting each HSP90 isoform and cochaperone. Nevertheless, we often encountered compensatory expression of HSP90 isoforms in the knockdown studies. Here, we provide dual and triple knockdown methods to target multiple RNA for challenging isoform-specific roles and compensatory expression of intracellular, extracellular, and exosomal HSPs.© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.