E2F6 is essential for HOXC9 induction of G1 arrest and repression of cell cycle genes. (A) Immunoblot analysis of E2F6 levels in BE(2)-C/Tet-Off/myc-HOXC9 cells infected with lentiviruses expressing shRNA against GFP or various coding regions of E2F6. E2F6 levels were quantified against β-actin. (B-D) Phase contrast imaging and growth assay (B) and cell cycle analysis (C, D) showing E2F6 knockdown abrogated HOXC9-induced growth arrest. Error bars, SD (n = 4). (E-F) Immunoblot analysis (E) and quantification (F) showing that E2F6 knockdown abrogated HOXC9 repression of cyclins, but not HOXC9 induction of NEFM. HOXC9, CCNA2, CCNB1 and NEFM levels were quantified against β-actin with the protein levels in GFPsh-expressing cells cultured in the presence of doxycycline (Doxy+) were defined as 1.0 (dashed lines). Error bars, SD (n = 3). Data in (D) and (F) were analyzed with unpaired, two-tailed Student’s t-test and p values are indicated.