Figure 4

C/EBPδ knockout (C/EBP -/-) mouse embryo fibroblasts (MEFs) exhibit defective growth arrest and increased migration. (A) C/EBPδ protein levels in C/EBPδ +/+ and C/EBPδ -/- MEFs. C/EBPδ +/+ and C/EBPδ -/- MEFs were cultured in MCGM to ~80% confluence and then switched to GAM. Whole cell lysates were isolated and western blots performed at 0 (near confluence), 24 and 48 hrs. Western blots were probed with anti- C/EBPδ and β-actin antibodies. (B) [³H]-thymidine incorporation. C/EBPδ +/+ and C/EBPδ -/- MEFs were treated as described in "A" above. [³H]-thymidine incorporation assessed at 0, 24 and 48 hrs. * = significantly different from C/EBPδ +/+ treatment group at 24 and 48 hours at p < 0.05. (C) BrdU labeling. C/EBPδ +/+ and C/EBPδ -/- MEFs were treated as described in "A" and BrdU labeling detected by in situ immunocytochemisty. (D) C/EBPδ Rescue. C/EBPδ -/- MEFs were transfected with pcDNA3 (empty vector control) or with full length C/EBPδ cloned into pcDNA3. Confluent cells were cultured in MCGM and [³H]-thymidine incorporation assessed at 0, 24 and 48 hrs post-confluence. The values presented are mean ± S.E.M from three independent experiments. * = significantly different from MEFs transfected with C/EBPδ expression vector at 24 and 48 hours at p < 0.05. Migration assay. C/EBPδ +/+ and C/EBPδ -/- MEF confluent monolayers cultured in MCGM were "scratched" with a 200 μL pipet tip as described in the Methods section. Migration of cells into the open area was assessed at 0 and 24 hrs by crystal violet staining.