H2A.Z overexpression and bladder cancer cell growth. (A) Normal (UROtsa) and bladder cancer (SCaBER, J82, LD611, RT4, HT1376 and T24) cells were lysed in RIPA buffer, and equal amounts of lysates were analyzed by Western blot using anti-H2A.Z antibody. Actin served as a control for equal protein loading. (B) Tissue microarrays containing 16 bladder tumor and 8 normal tissue samples were subjected to immunohistochemistry with H2A.Z antibody. Representative high-powered images are shown. (C) Immunostaining scores of H2A.Z in normal and bladder cancer tissues. The graph indicates the percentage of sections with different scores (negative, weak, moderate and strong). (D) Proliferation of UROtsa and LD611 cells was determined by MTT colorimetric assays at the indicated time points. Data shown represent mean ± SD from three replicates. (E) UROtsa cells were transfected with H2A.Z as in Additional file 1: Figure S1, and cell proliferation was measured by MTT assay over a period of four days. All reactions were performed in triplicate. (F) LD611 cells were mock-depleted or depleted of H2A.Z as in Additional file 1: Figure S2, and cell proliferation was monitored by MTT assays over a period of four days. MTT assay, 3–4, 5-(dimethyl-thyazol-2-yl)-2, 5-diphenyltetrazolium assay.