BMI1 is a BTF3 target gene (a) BMI1 downregulated gene signatures (MSigDB Database C6) were enriched for upregulation upon BTF3 knockdown. GSEA was carried out to examine the expression of a set of BTF3-repressed genes in a microarray dataset that profiled VCaP cells with control or BTF3 knockdown. ES = -0.44, P = 0.001, FDR q = 0.02. (b-c) Western blot analysis of BTF3 and BMI1 expression in PCa cells with BTF3 knockdown or overexpression. (d) Immunofluorescence staining analysis of BTF3 and BMI1 expression in PCa cells.
(E) Representative images of HE and IHC staining of BTF3 and BMI1 in PCa patients from Qilu cohort. (f) Representative IHC images of BTF3 and BMI1 expression in xenograft tumors derived from PC3 cells. (g) Overlapped genes regulation by BTF3 and BMI1 through mRNA profiling analysis. (h) GSEA of BMI1 signatures (genes upregulated upon BMI1 inhibition) in a microarray dataset that profiled VCaP cells with control or BTF3 knockdown. ES = -0.3, P = 0.022, FDR q = 0.022. (i) Cytoplasmic and nuclear BMI1 protein levels were analyzed in PC3 and 22RV1 cells after shBTF3 or BTF3 overexpression. GAPDH and LaminA/C were used as cytoplasmic and nuclear protein-loading controls, accordingly. (j) The mRNA levels of P16, ZMYM3 and WWOX in PCa cells were detected by qRT-PCR in the presence of Scr or shBTF3 (left), and Vector or BTF3 (right). Values represent mean ± SD of technical duplicates from a representative experiment. (k) Sphere formation assay of PC3 cells. PC3 Scr/shBTF3 cells were transiently transfected with empty plasmid (P-Enter) or BMI1 expression plasmid (BMI1). Left panel: Representative images of spheres. Right panel: Quantitative results of sphere formation assays from triplicate experiments. (l-m) The mRNA level of CSC markers, CD44, CD133, SOX2 and NANOG determined by real time PCR in PC3(j) and 22RV1 (k) cells.
(n) Effect of BMI1 in Transwell migration of BTF3. PC3 Scr/shBTF3 cells with were transiently transfected with empty plasmid (P-Enter) or BMI1 expression plasmid (BMI1) as indicated. Cells were subjected to transwell migration assay. * P < 0.05, **P < 0.01, ***P < 0.001.