Fig. 2

BHB inhibits the stem-like properties of PCa cells. A, The suppressive effects of BHB on PCa stem cells (CSCs) were measured via tumor sphere formation assays. Cells treated with or without 15 mM BHB were prepared as single-cell suspensions and seeded into ultralow attachment 96-well plates at a density of 500 cells/well containing sphere-forming medium. Cells were cultured continuously for 7–14 days, and the spheres with a diameter of > 25 or 75 μm were counted. B, The suppressive effects of BHB on prostate CSCs were measured via ALDH assays; after BHB treatment for 48 h, ALDH substrate was added and incubated for another 30 min. Then, flow cytometry was performed to measure the percentage of ALDH-positive cells. DEAB-quenched ALDH fluorescence was considered a negative control. C, The mRNA levels of prostate CSC-associated markers were examined via real-time qPCR in cells treated with or without BHB for 48 h. D, Protein levels of prostate CSC-associated markers were measured via western blot assay in cells treated with or without BHB for 48 h. Experiments were performed in triplicate, and data were expressed as the mean ± standard deviation (SD). Paired and two-tailed Student’s t-tests were performed, and significant differences were designated as *p < 0.05, **p < 0.01, and ***p < 0.001