To assess whether these protein kinases participate in Spp1-induced PIN cell proliferation, we utilized our 3D culture model, in which Pr111 PIN cells were treated with or without recombinant Spp1 followed by immunoblotting to evaluate the levels of activated Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38 MAPK (Figure 4A,B and Figure S3). This evidence concerns the gene AKT1 and prostate intraepithelial neoplasia.