Expression of IL6 and its receptor is up-regulated in bladder cancer spheres with cancer stem cell (CSC) properties. (A) Morphology of bladder cancer cell lines RT4 and J82 grown as adherent cells (scale bar =75 µm) and spheres (scale bar =50 µm). (B) Expression of stem-like associated genes (CD133, CD44, ABCG2, Oct4 and Nanog) as assessed by qRT-PCR in the adherent cells and spheres, which were incubated in 6-well ultra-low-attachment culture plates for 4 days. The results are expressed as the fold change relative to the adherent cells. (C) The conditioned media from the adherent and sphere RT4 and J82 bladder cancer cell lines cells after 4 days of culturing were collected for the IL6 ELISA, which was performed to measure the IL6 level in the supernatants of the adherent cells and spheres (***P<0.001). (D) IL6 and IL6R expression were assessed by qRT-PCR in adherent and sphere RT4 and J82 cells. (E) Western blots were performed to detect IL6R expression in the adherent and sphere RT4 and J82 cells. Representative results from three independent experiments are shown, and all the values were expressed as the means ± SD, *P<0.05, **P<0.01 and ***P<0.001. qRT-PCR, real-time quantitative reverse transcription polymerase chain reaction; IL6, interleukin 6; IL6R, IL6 receptor; ELISA, enzyme-linked immunosorbent assay.