Fig. 3

INMT promotes the stemness of PCa cells through SOX2. A, The effects of INMT overexpression on prostate CSCs were detected via tumor sphere formation assays. LNCaP cells were transfected with INMT or vector plasmids and tested for their tumor sphere formation ability. B, The effects of overexpressed INMT on prostate CSCs were determined via the ALDH assay. LNCaP cells transfected with INMT or vector plasmids were subjected to flow cytometry to measure the proportion of ALDH-positive cells. C, The mRNA levels of prostate CSC-associated markers were examined via real-time qPCR after INMT overexpression in LNCaP cells. D, Protein levels of prostate CSC-associated markers were measured via western blotting after INMT overexpression in LNCaP cells. E and G, The effect of INMT knockdown on prostate CSCs was determined by performing ALDH assay in LNCaP and PC3 cells. F and H, The protein levels of prostate CSC-associated markers were measured via western blot assay after INMT knockdown in LNCaP and PC3 cells. I, The effects of INMT on SOX2 promoter activity was detected using dual luciferase reporter. Cells were cotransfected with an INMT expression plasmid and a SOX2 promoter reporter plasmid. Firefly luciferase activity, which represents SOX2 promoter activity, was measured and normalized to renilla luciferase activity. Experiments were conducted 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