KLF9 induces NOXA-dependent apoptosis. (A) MM1.S and RPMI-8226 cells were infected with control vector (V) or vector expressing KLF9 cDNA (KLF9). 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”). The percentage of dead cells was determined by counting trypan blue positive and negative cells in multiple view fields. (B) NOXA/ACTB Q-RT-PCR signal ratios were obtained in indicated cells expressing KLF9 cDNA or empty vector. Signal ratios were normalized by the corresponding ratio in cells infected with the empty vector. (C) Cells were infected as described in (A). At the indicated days after infection, cells were stained for the activated caspase 3 using APO LOGIX “Carboxyfluorescein FAM-DEVD-FMK for caspase 3” kit. The percentage of apoptotic cells was determined by counting positive and negative cells with fluorescence microscopy in multiple view fields. (D) MM1.S and RPMI-8826 cells were infected with NOXA shRNA followed by super-infection with control vector or vector expressing KLF9 cDNA. Five days after the second infection cells were collected and total cellular protein extracts were probed in Western blotting with the antibodies designated on the left. (E) Cells described in panel D were stained for the activated caspase 3 using APO LOGIX “Carboxyfluorescein FAM-DEVD-FMK for caspase 3” kit. The percentage of apoptotic cells was determined by counting positive and negative cells with fluorescence microscopy in multiple view fields. The 2-tailed P value is shown.