BTF3 expression is correlated with stem phenotype in PCa (a-b) Sphere formation assay of PC3 (a) and 22RV1 (b) cells. Left panel: Representative images of spheres. Right panel: Quantitative results of sphere formation assays from triplicate experiments. Scr/shBTF3: BTF3 was knockdown in PC3 cells by transfection of shRNA targeting BTF3 (shBTF3) or a negative control (Scr). Vector/BTF3: ectopic expression of BTF3 in 22RV1 cells by transfection of plasmid including BTF3 gene sequence (BTF3) or empty plasmid (Vector). (c-d) Clonogenic assays in PC3 (c) and 22RV1 (d) cells. Cells transfected as above were mixed with Matrigel and plated in 24-well plates and colonies counted in two weeks. Left panel: Representative images of clonogenic assay. Right panel: Quantitative results of clonogenic assay from triplicate experiments. (e) Expression of CD133 and BTF3 in holoclones and paraclones derived from VCaP and PC3 cells by immunofluorescence analysis. (f) Flow cytometry assay with CD133-PE antibody of indicated PCa cells. Left panel: FACS for CD133 indicated PCa cells. Right panel: Quantitative results of FACS from triplicate experiments. (g) Gene sets significantly enriched in high-BTF3 tumors grouped accordingly to the original annotation in the MSigDB Database C2 (http://software.broadinstitute.org/gsea/msigdb/index.jsp). Normalized enrichment score (NES) and P-value (P) are reported. TCGA-PRAD, the Cancer Genome Atlas Prostate Adenocarcinoma dataset (N = 493); VCaP, gene expression microarray assay of VCaP Scr/shBTF3 cells. (h) Circos plot displaying the interconnectivity among genes related to stemness phenotype and BTF3. The thickness and color of the ribbons correlate to the correlation of genes expression in Tomlins Prostate. (Additional file 4: Table S2) (i) The mRNA level of CSC markers, CD44, CD133, SOX2 and NANOG determined by real time PCR in indicated PCa cells. PC3 and 22RV1 cells transfected as above were subjected to real time PCR assay. (j) Representative IHC images of BTF3, SOX2 and CD44 expression of PCa tissue with different intensity in Qilu cohort. Each row represents one case. * P < 0.05, **P < 0.01, ***P < 0.001.