Figure 3.

Characteristics of the growth stimulation. (A) EGFP-expressing HaCAT cells, seeded onto presenescent or senescent WI-38 lawns. EGFP fluorescence (in arbitrary units) was measured after 1–8 days, as indicated. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent lawns; black bars, senescent lawns. (B) SCp2 cells, seeded onto WI-38 fibroblasts (Cells), plated in the upper chambers of Millicells containing fibroblasts in the lower chambers (Soluble), or plated onto matrices deposited by fibroblasts (Matrix), as described in Materials and Methods. Cell number was assessed after 8 days by DAPI fluorescence. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent fibroblasts; black bars, senescent fibroblasts. (C) HaCAT cells, seeded onto lawns of control (Left) or p14^ARF-expressing (Right) presenescent 82-6 fibroblasts, stained 8 days later with Rhodanile (250×). (D) HaCAT cells, quantified by EGFP fluorescence after coculture with control (−) or p14^ARF-expressing (+) 82-6 fibroblasts (ARF); control (−) or p14^ARF-expressing (+) hTERT-immortalized 82-6 cells (TERT-ARF); control (−) or RAS-Ha/V12-expressing (+) WI-38 cells (RAS); 82-6 cells untreated (−) or treated (+) with H₂O₂; or replicatively senescent WI-38 cells (R). Growth was assessed after 8 days, except for RAS panels, where growth was assessed after 5 days. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent lawns; black bars, senescent lawns.