Fig. 4: Coregulator function of DBC1 for HSF1 and requirement of DBC1 for transcriptional regulation of genes involved in mCRPC progression.

a The 22RV1 cells were treated with or without HS at 42 °C for 1 h. The cell lysates were immunoprecipitated with normal IgG or an anti-HSF1 antibody. The input and immunoprecipitated proteins were analyzed by immunoblot with indicated antibodies. b Endogenous CoIP experiments were performed as described in a in 22RV1 and SM1 cells. c A pulldown of FLAG-HSF1 untreated or treated with λ-phosphatase with GST-DBC1. The bound and input proteins were analyzed by immunoblot with indicated antibodies. d SM1 cell extracts were treated with or without λ-phosphatase and immunoprecipitated with anti-HSF1 antibody. The input and immunoprecipitated proteins were analyzed by immunoblot with indicated antibodies. e The 293T cells were transfected with the indicated constructs, and the cell lysates were immunoprecipitated with FLAG-M2 agarose beads. The input and immunoprecipitated proteins were analyzed by immunoblot with indicated antibodies. f The 22RV1 cells were transfected with 6xHSE-LUC reporter and an increasing amount of HA-DBC1 vector and collected for luciferase assays. The data are means ± standard deviation (s.d.) (n = 3). g 22RV1 and D1KO cells were transfected with indicated reporters and collected for luciferase assays. The data are means ± s.d. (n = 3). h SM1 and SM1-D1KO cells were transfected with indicated NanoLUC reporters, along with pRL-SV40, and collected for dual-luciferase assays. The data are means ± s.d. (n = 3). i The 293T cells were transfected as indicated, and FLAG-HSF1 immunoprecipitates were analyzed by immunoblot with the indicated antibodies. To strengthen the transient interaction between HSF1 and HSP90β, the client-trapping mutant of HSP90β (HSP90β E42A) was used. j Cell extracts from 293T cells transfected with the indicated expression plasmids were subjected to in vitro cross-linking with 2 mM ethylene glycol bis(succinimidyl succinate) (EGS) and analyzed by immunoblot with the indicated antibodies. k Volcano plots showing the differential gene expression between 22RV1 and D1KO cells and between SM1 and SM1-D1KO cells. log₂(fold change) (log₂FC) ≥0.58 (FC ≥1.5) and P < 0.05. l A Venn diagram showing the overlap of DBC1 target genes identified in 22RV1 and SM1 cells. m A Venn diagram showing the overlap between HSF1 and DBC1 target genes in SM1 cells. n Scatter plots of Pearson correlation between log₂FC in expression levels of HSF1–DBC1 cotarget genes in SM1 cells upon H1KO and D1KO. o A bar plot shows the enriched hallmark and canonical pathways for HSF1–DBC1 positive cotarget genes in SM1 cells.