*2.3. Western Blot Analysis*

Western blots were performed as previously described in Reference [26]. The primary antibodies used were: AR (1:1000; Santa Cruz Biotechnology), AR-V7 (1:400; Precision), GR (1:1000; BD transduction laboratories), PSA (1:1000; Santa Cruz Biotechnology), FKBP5 (1:1000; Santa Cruz Biotechnology), UBE2C (1:1000; Boston Biochem), NSE (1:1000; Merck), Mdr-1 (1:1000; Santa Cruz), Aurora A (1:1000), BRN-2 (1:1000), total-STAT3 (1:1000), p-STAT3Tyr705 (1:1000), total-AKT (1:1000), p-AktSer473 (1:1000), total-S6 (1:1000), p-S6 (1:2500), total-p44/42MAPKErk1/2 (1:1000), p-p44/42MAPKErk1/2 (1:1000), 110 α (1:1000), 110β (1:1000), 110 γ (1:1000), PI3KClass III (1:1000), p85 (1:1000), 4EBP1 (1:1000), and p-4EBP1 (1:1000), from Cell Signaling Technology. Beta-actin (1:1000, Abcam and Cell Signaling Technology) was used as a loading control.

### *2.4. Real-Time Quantitative Reverse Transcription PCR (Real-Time RT-qPCR)*

Total RNA was isolated using the RNAqueous Total RNA Isolation Kit (Life Technologies, Waltham, MA, USA) and it was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). Real-time RT-qPCR was performed in triplicate for each biological sample. Transcript levels for each gene were normalized to levels of the GAPDH transcript. Primers were purchased from Applied Biosystems: AR (Hs00171172\_m1), KLK3 (Hs02576345\_m1), FKBP5 (Hs01561006\_m1), UBE2C (Hs00964100\_g1), CDC20 (Hs00426680\_mH), GAPDH (Hs00266705\_g1), AR-V7 (forward, 5-CCATCTTGTCGTCTTCGGAAATGTTA-3; reverse, 5-TTTGAATGAGGCAAGTCAGCCTTTCT-3).

### *2.5. AR-Driven PSA(6.1kb)-Luciferase Reporter Gene Assay*

PSA (6.1kb)-luciferase reporter plasmid encodes nucleotides −6000/+12 relative to the transcription start site of the human PSA/KLK3 gene and it includes the PSA promoter, with AREII (−395 to 376) and AREI ( −170 to −156), and enhancer regions with AREIII ( −4148 to −4134), as described in References [27,28]. LNCaP95-C and LNCaP95-DR cells seeded in 24-well plates were transfected using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA), with a plasmid encoding the prostate-specific antigen (PSA) (6.1 kb)-luciferase reporter gene construct. The next day, the cells were pre-treated with vehicle (DMSO), enzalutamide (10 μM), docetaxel (5 nM), or cabazitaxel (10 nM) for 1 h before adding R1881 or EtOH (vehicle) under serum-free, phenol red-free conditions. After 48 h of incubation, the cells were harvested and lysed using the lysis buffer that was provided with the Luciferase Assay System (Promega). PSA-luciferase activity was measured using with the Wallac 1420 ARVOsx multi-label plate reader (PerkinElmer, Waltham, MA, USA) and normalized to protein concentration by the Bradford method as explained in Reference [29].
