KLF9 directly interacts with NOXA promoter in bortezomib-dependent manner. (A) Schematic representation of human NOXA promoter. Vertical lines above the central bar indicate potential KLF9 binding sites. Arrowheads correspond to the position of primers used for Q-PCR. The transcription start site (+1) is shown by the arrow. (B) MM1.S cells untreated or treated with 5nM bortezomib for 24 hours were cross-linked, lysed, and chromatin was immunoprecipitated with 2 different KLF9-specific antibodies (KLF9₁ or KLF9₂) or nonspecific (IgG) antibodies followed by the reversal of the cross-linking and DNA isolation. DNA was used in quantitative PCR with GAPDH or NOXA promoter-specific primers positions of which are designated by the numbers. All PCR signals were normalized by GAPDH-specific PCR signals and by the corresponding PCR signals obtained in reactions on DNA precipitated with IgG antibodies. (C) Control pGL3-basic vector “[0]” or vectors containing indicated regions of NOXA promoter were mixed with pRL-SV40 plasmid, expressing renilla luciferase gene in 1÷10 proportion, followed by transfection into HEK293 cells along with vector expressing KLF9 cDNA or empty vector. Forty-eight hours after transfection, cells were collected, and firefly and renilla-dependent luciferase activities were determined using the dual-luciferase assay kit (Promega). Measurements of firefly-dependent luciferase activity were normalized with respect to renilla signals. (D) Cells were infected with control (Cl) or KLF9-specific shRNAs (K9-1 or K9-2). Cells were collected 48 hours after infection and the total protein extracts were probed in Western blotting with antibodies shown on the left. *Note that an enhanced assay was used for detecting basal levels of NOXA (see“Immunoblotting”).