Figure 5: Inhibition of p300 HAT activity induces a p53/p21CIP1/p16INK4A - independent induction of senescence.

(a,b) TIG3(et) indicated knockdown cells and TIG3(et)/(–) for empty vector synchronized in G0 by serum starvation during 5 days with medium containing 0.5% FBS, then released in 10% FBS medium in presence of 9 μM of curcumin (Curc.) or vehicle (0) as control and analysed 8 days after release. (a) Immunoblot analysis with β-actin as loading control and (b) SA-β-gal activity. (c) Percentage of cells in G2/M phase analysed by FACS in TIG3(et) knockdown cell lines after 3 days of curcumin treatment. (d) Synchronized TIG3(et) cells in G0 by serum starvation during 5 days with medium containing 0.5% FBS, then released in 10% FBS medium in presence of 9 μM of curcumin (Curc.) or vehicle (0) as control and analysed 8 days after release. Nuclear (Nucl.) and cytoplasmic (Cyto.) fractions were analysed by western blot using H3 and tubulin as loading control for the nuclear and the cytoplasmic fractions, respectively. (e–g) Synchronized osteosarcoma SAOS-2 cell line in G0 by serum starvation during 5 days with medium containing 0.5% FBS, then released in 10% FBS medium in presence of 9 μM of curcumin or vehicle (0) as control. (e) Immunoblot analysis with β-actin as loading control and curcumin-treated TIG3(et) cells as immunoblot control, and (f) SA-β-gal activity (left panel) and coomassie staining (right panel) analysed 8 days after release. (g) Cell cycle distribution analysed by FACS after 3 days of curcumin treatment. (b,f) Percentage of SA-β-gal positive cells is indicated in insert. (c,g) Shown are the results of two independent experiments performed in duplicate. Error bars show the range. (b,f) Scale bars are equal to 40 μm.