Figure 2.

Expression of c-Myc protein in HaCaT lines blocks TGFβ-mediated cell-cycle arrest and subsequent up-regulation of p21^CIP1 mRNA. (A) Ectopic expression of c-Myc in stable lines. Stable clonal HaCaT lines vector-cl1 (clone 1), Myc-cl1, and Myc-cl2 were analyzed for ectopic c-Myc expression by Western blot. Blots were probed with anti-av-myc 12C, which recognizes only exogenous c-Myc protein. (B) Down-regulation of c-Myc after TGFβ treatment. HaCaT clonal lines vector-cl1 and Myc-cl1 were left untreated or treated for 20 hr with 1 ng/ml TGFβ. Whole-cell lysates were analyzed by Western blot by using anti-Mycfl. (C) Inhibition of entry into S phase after TGFβ treatment. Cell-cycle inhibition by TGFβ treatment was determined by [³H]thymidine incorporation. Clonal HaCaT lines were plated in duplicate and re-fed the next day with fresh media with or without 1 ng/ml TGFβ. Cells were treated for 16 hr, then labelled with 1 mCi/ml [³H]thymidine for an additional 4 hr with or without TGFβ. [³H]thymidine incorporation was determined by scintillation counting. Inhibition of DNA synthesis is represented as the percent difference between untreated and treated cells. Error bars indicate standard deviation of the average of three independent experiments. (D) Up-regulation of p21^CIP1 after TGFβ treatment is blocked by ectopic c-Myc expression. Clonal HaCaT control or c-Myc-expressing lines as indicated were left untreated or treated with 1 ng/ml TGFβ for 20 hr. PolyA+ mRNA was isolated, and 2 μg mRNA per sample was analyzed by Northern blot analysis. The blot was sequentially probed for both p21^CIP1 (Top) and cyclophilin (Bottom).