Figure 1. Downregulation of p18^INK4c at senescence.

A. HDFs (TIG3) were passaged continuously until they reached replicative senescence (Sen). Cell lysates prepared at the indicated population doublings (PD) were fractionated by SDS-PAGE and immunoblotted for p16^INK4a, p21^CIP1 and p18^INK4c. Right panel shows a direct comparison between proliferating (P) and senescent (S) cells. B. HDFs (TIG3) were infected with a retrovirus encoding H-RAS^G12V or the empty vector control (Vec). Following drug selection, the levels of p18^INK4c and p16^INK4a were assessed by immunoblotting. C. Primary MEFs were infected with a retrovirus encoding H-RAS^G12V or the empty vector control (Vec). The levels of p18^Ink4c and p16^Ink4a were assessed by immunoblotting. D. HDFs (Hs68) were infected with a retrovirus encoding HA-tagged p16^INK4a. The levels of p18^INK4c and p16^INK4a were assessed by immunoblotting. E. MG132 had no effect on p18^INK4c levels in cells transduced with HA-p16^INK4a or empty vector. F. The Leiden strain of p16^INK4a-deficient fibroblasts were passaged until they reached senescence. Samples taken at the indicated PDs were immunoblotted for p18^INK4c, with CDK4 as a loading control.