Figure 4. Chronic canonical NF-κB activation by IKKβca induces a pronounced, short-term cell proliferation block, whose relief correlates with the dissipation of cell cycle effector repression.

A: Comparisons of proliferation rates of 7 and 14 dpi IKKβca MEFs, 7 dpi IKKβca IBIN-MEFs and 7 dpi BIP control MEFs. Cells were seeded at low density (∼1000 cells per well) in quadruplicate in 96 well plates and the relative amounts of cell growth were determined by quantifying the increase in DNA (in ng) every 24 hr over a 5 day period as described in materials and methods. All DNA values are expressed as the mean ± s.e.m. Proliferation rates for 7 dpi IKKβca vs. 7 dpi BIP control cells were statistically significant on days 2, 3, 4 and 5 (p values of 0.009 and 0.005 on days 2-3 and 4-5 respectively) as were the results for 14 dpi IKKβca vs. 7 dpi BIP control cells on days 2, 3 and 5 (p values of 0.020). B: Sybr green real time PCR results for 4 induced and 8 repressed genes are shown as fold change values obtained with total cell RNAs prepared from 21 dpi IKKβca vs. 21 dpi BIP control MEFs. Results were quantified as described in Materials and Methods and in the above legends of Figures 2 and 3 and are averages of duplicate samples from one representative experiment out of two independent experiments producing similar results. C: Immunoblot analysis of nuclear extracts of control vs. IKKβca-BIP immortalized MEFs (5 dpi and 15 dpi) for Phospho-NF-κB p65 (Ser536), total p65 protein and lamin B as a protein loading reference control.