Fig. 6: DBC1 regulates HSF1 stability by promoting DYRK2-mediated HSF1 phosphorylation and inhibiting CHIP-mediated HSF1 ubiquitination.

a The 22RV1 cell lysates transfected with empty or FLAG-DBC1 expression vector were immunoblotted with the indicated antibodies. b Cell lysates of 22RV1, SM1 and their D1KO counterparts were analyzed by immunoblot with indicated antibodies. c mRNA levels of HSF1 were analyzed by qRT–PCR in 22RV1, D1KO, SM1 and SM1-D1KO cells. The data are means ± standard deviation (n = 3). n.s., not significant (P > 0.05). d The expression levels of indicated proteins were analyzed by immunoblot in SM1, SM1-D1KO and 4xFLAG-DBC1-expressing lentivirus-infected SM1-D1KO cells. e SM1 and SM1-D1KO cells were treated with 50 µg/ml cycloheximide (CHX) and collected at the indicated time points. Cell lysates were analyzed by immunoblot with the indicated antibodies. f Cell lysates of SM1, SM1-D1KO and 4xFLAG-DBC1-expressing lentivirus-infected SM1-D1KO cells were immunoprecipitated with anti-HSF1 antibody and immunoblotted with indicated antibodies. The closed and open arrowheads indicate endogenous and 4xFLAG-tagged DBC1 proteins, respectively. g In vitro-translated HA-DYRK2 was incubated with recombinant GST-DBC1. The bound proteins were analyzed by immunoblot with anti-HA antibody. h The endogenous CoIP between DBC1 and DYRK2 in SM1 cells. Immunoprecipitation and immunoblot were performed using the indicated antibodies. i The 293T cell extracts transfected with expression vectors as indicated were immunoprecipitated and immunoblotted with the indicated antibodies. j The 293T cell extracts transfected with expression vectors as indicated were analyzed by immunoblot using the indicated antibodies. k In vitro kinase assays using recombinant His-HSF1, immunopurified HA-DYRK2 and FLAG-DBC1 as indicated. The reactions were analyzed by immunoblot using the indicated antibodies. l SM1 cell lysates transfected with an empty or CHIP-V5 expression vector were immunoprecipitated with anti-HSF1 antibody and immunoblotted with the indicated antibodies. The closed and open arrowheads indicate endogenous and V5-tagged CHIP proteins, respectively. m SM1 cell lysates transfected with the indicated expression vectors were immunoprecipitated with anti-HSF1 antibodies and immunoblotted with the indicated antibodies. n The role of DBC1 as a HSF1 coregulator in mCRPC progression. DBC1 inhibits CHIP-mediated HSF1 ubiquitination and enhances HSF1 trimerization and DYRK2-mediated HSF1 phosphorylation, thereby increasing HSF1 stability and activity. This coregulator activity of DBC1 contributes to the reprogramming of HSF1 cistrome and the active chromatin landscape, including SEs, and thus promotes mCRPC progression by activating metastasis-associated genes, including MMP11. n was created using bioRender.com.