FIG 5.
Active PKC-θ is required for activity of the NF-κB family. (A) Transcription factor motifs significantly overrepresented in the promoters of the genes increased and decreased in expression in CSCs (compared to both NCSC and NS) compared to a randomly chosen set of 974 promoters. (B and C) ELISA-based NF-κB activity assays using p50 antibody or p65 antibody, respectively. Raji cell extract was used as a positive control. Nuclear and cytoplasmic extracts were prepared from MCF-IM cells, and 5 μg/well protein was used in triplicate wells. (D) PKC ELISA-based kinase assays for PKC-θ specific activity inhibitor compound 27 (C27). Kinase activity was calculated after subtracting blank wells. (E) PKC ELISA-based kinase assays of nuclear extract or cytoplasmic extract from MCF-IM cells without (−) or with (+) C27 inhibitor. Assays were performed without specific immunoprecipitates for global PKC activity. (F) FACS analysis of CD44high/CD24low CSC subpopulation in MCF-IM. MCF-7 cells were stimulated with PMA (0.65 ng/ml for 60 h) either without pretreatment of PKC-θ specific activity inhibitor compound 27 (−C27) or with pretreatment with compound 27 (+C27) (1 μM for 24 h). (G) mRNA expression by real-time PCR with samples as described for panel F. (H and I) Nuclear p50 or nuclear p65 activity in samples as described for panel B after treatment of MCF-IM cells either with vehicle alone or with PKC-θ-specific inhibitor compound 27 (C27). (J) Immunoblotting of MCF-IM nuclear extracts for phosphorylated NF-κB p65 at serine 468 (p468) after treatment with (+) or without (−) compound 27. Fifteen micrograms of the protein was used for Western blots, and histone H3 antibody was used as a nuclear control. (K and L) Immunoblotting of MCF-IM cell nuclear fraction (K) and cytoplasmic fraction (L) for global NF-κB p65 after treatment with (+) or without (−) compound 27. Densitometric analyses using Image J software are provided below Western blots in panels J, K, and L. (M) Halfway ChIP of stimulated MCF-IM cells with anti-PKC-θ antibody. WCL, whole-cell lysate utilized for the immunoprecipitation (IP) experiments. WCL is immunoblotted with anti-PKC-θ antibody. −AB or +AB, absence or presence, respectively, of antibody. Immunoblotting was done with either anti-RNA Pol II, anti-p65 antibodies, or anti-PKC-θ antibody. All results either represent the means ± the standard errors from three independent experiments or, for panels J, K, L, and M, are the individual results from a representative experiment of three replicates (n = 3). *, P < 0.05; ns, not significant.