Functional role of the GMCSF-mediated NF-κB activation. (A) Effect of NF-κB inhibition or GMCSF withdrawal on the proliferation of TF-1 cells. Cells were incubated for 3 days in the presence or absence of GMCSF (2 ng/mL), in the presence of GMCSF and the specific NF-κB inhibitor Bay 11-7082 (BAY; 5 μM), or in the absence of GMCSF but in the presence of TNF-α (200 U/mL). Cell numbers were recorded by flow analytic counting of cells at defined flow rates. (B) Cell cycle analysis of TF-1 cells. Cells were GMCSF-starved for 24 hours followed by readdition of GMCSF in the absence or presence of the NF-κB inhibitor Bay 11-7082. Alternatively, cells were further incubated in the absence of GMCSF with or without addition of TNF-α. After 24 hours, cells were fixed and permeabilized, followed by propidium iodide staining in the presence of RNAse to label DNA. Cells containing less than the diploid DNA content (M₁; sub-G₀/G₁) represent apoptotic cells. The positions of G₀/G₁ (M₂), S-phase (M₃), as well as G₂/M-phase cells (M₄) are indicated. (C) Apoptosis of TF-1 cells after inhibition of NF-κB. Cells were incubated in the presence of GMCSF with or without addition of the NF-κB inhibitor Bay 11-7082 for 27 hours. Apoptotic cells were detected by flow analysis after binding of FITC-labeled annexin V.