E2F-1 expression and activity in M1 and M1E2F-1 cells untreated and treated with IL-6.

(A) E2F-1 mRNA expression in IL-6–treated M1 and M1E2F-1 cells. Cells were collected at the indicated times after IL-6 treatment and total RNA was extracted. The RNA (10 μg per lane) was resolved on a 1% agarose gel and transferred to a nylon membrane. The RNA blot was then hybridized to a ³²P-labeled murine E2F-1 probe. Equal loading of RNA in each lane was confirmed both by visualizing equal intensity of ethidium bromide staining of ribosomal RNA bands and by hybridization of the Northern blot with radiolabeled β-actin probe. (B) E2F-1 protein expression in M1 and M1E2F-1 cells after IL-6 treatment. Cell lysates were prepared at the indicated time points after addition of IL-6 as described in “Materials and methods.” Fifty micrograms of protein per sample were electrophoretically fractionated on a 10% polyacrylamide gel and transferred to a PVDF membrane. The blot was then probed with an antimurine E2F-1 polyclonal antibody (Santa Cruz Biotech Inc, diluted 1:1000). Signals were developed by using the enhanced chemiluminescence (ECL) Western blotting system (Amersham). (C) E2F transcriptional activity in untreated and IL-6–treated M1 and M1E2F-1 cell lines. Cells were seeded at a density of 0.5 × 10⁶ cells/mL and 24 hours later were transfected with wt-E2F-Luc or mut-E2F-Luc luciferase reporter plasmids containing 3 tandem copies of wild-type or mutant E2F-1 binding sites, respectively; described in Krek et al.11 The cells were then incubated in the absence or presence of 50 ng/mL IL-6 and were harvested at 1-day intervals (day 0 to day 3). They were lysed in reporter lysis buffer and the resulting cell extract was used to measure luciferase activity in a Lumat LB 9501 luminometer after normalizing for transfection efficiency (described in “Materials and methods”).