Mice model, histological and IHC staining

NSG male mice, 6-8 weeks old were obtained from the Jackson Laboratory and maintained in compliance with Institutional Animal Care and Use Committee (IACUC) guidelines. Mice were injected with 0.5x10⁶ cells orthotopically in the prostate (n = 5 mice/group). Where indicated, testosterone pellets (12.5 mg, 90 days release) were implanted at time of cell injections. Mice were administered with DUSP6 inhibitor BCI (10mg/kg) or PBS (as vehicle) via intraperitoneal injection for five consecutive days per week, until experimental endpoint. Bioimaging was performed at different time points/once per week starting one week post cell implantation using an in vivo IVIS (100 bioluminescence/optical imaging system (xenogen)). All studies were performed in compliance with institutional guidelines under an IACUC-approved protocol. Tumor specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Serial sections were cut on a microtome and mounted on glass slides. Histopathological examination and IHC were performed as previously described (Lignitto et al., 2019; Moro et al., 2020). All tumor burden and IHC analyses were done in a blinded fashion, in which the pathologist was unaware of the samples’ genotype. Pictures were obtained using a digital whole-slide scanner (Leica, SCN400F) and Slidepath software version 4.0.8.