Figure 5.

NEDD1 depletion causes premature entry of MEFs into senescence. Passage 1 MEFs were transfected with a control (scrambled) siRNA or NEDD1 siRNA and then stained for NEDD1 (green) and γ-tubulin (red) on day 5 when the siRNA was removed and again on day 8 and day 11. (a–c) The MEFs transfected with the control siRNA show no reduction of NEDD1 or γ-tubulin protein. (d) After 5 days of NEDD1 siRNA treatment, the levels of NEDD1 and γ-tubulin are dramatically reduced at the centrosome. (e and f) The levels of these proteins gradually increases 8 days after the initial transfection (3 days after siRNA removal) and again 11 days after the initial transfection (6 days after siRNA removal). (g) Cells treated with the control siRNA maintain low numbers of SA-β-gal-positive cells on day 5, 8 and 11. Cells treated with NEDD1 siRNA have relatively low levels of SA-β-gal staining after 5 days of NEDD1 depletion, but SA-β-gal-positive cells become more abundant 8 days after initial depletion, and again after 11 days. (h) The number of SA-β-gal-expressing cells was counted. At days 8 and 11, there is a significant increase in the number of senescent cells in the NEDD1 siRNA-treated cells compared with control siRNA-transfected cells. There is also a significant increase in the number of senescent cells between days 5 and 11 of the NEDD1 siRNA-treated group. Approximately 300 cells were counted for each group. Error bars show S.E.M. from three independent experiments. Scale bars=40 μm. (i) Immunoblotting shows protein levels of NEDD1 in MEFs from passage 1 transfected with control (scrambled) or NEDD1 siRNA for 72 h, and again for another 48 h before harvesting. β-Actin serves as a loading control