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. Author manuscript; available in PMC: 2009 Aug 5.
Published in final edited form as: Science. 2008 Aug 22;321(5892):1095–1100. doi: 10.1126/science.1155998

Fig. 1. Suppression of p21-initiated senescence by HES1.

Fig. 1

(A–C) Human fibroblasts (HFs) were transduced (day 0) with a p21Cip1 expression cassette flanked by loxP sites (p21) or a loxp vector (vector). On day 4, p21 expressing cells were transduced with a vector expressing cre recombinase and green fluorescent fusion protein (p21-cre). On day 10, bromodeoxyuridine (BrdU) was added for 6 hours. (A) The percentage of BrdU positive cells is indicated. (B) Abundance of p21 protein and (C) expression of senescence-associated β-galactosidase (SA-β-gal). (D) HFs were transduced with an empty vector, contact inhibited for four days (contact), then infected with cre-GFP and re-plated at lower density (release). The percentage of BrdU positive cells is shown. (E–I) HFs were transduced with wild-type HES1 (wt), wild-type HES1-estrogen receptor fusion protein (ER), DNA-binding mutant HES1 (Δb), or dominant-negative HES1 (dn), and then transduced with p21-loxP. 4-hydroxytamoxifen was added to wtHes1-ER cells prior to p21 transduction (pre +), nine days after p21 transduction (post+), or not applied (−). Four days after p21 transduction, cells were either transduced with cre-GFP (F,G) or maintained with high p21 levels (H,I). (E) Expression levels of p21; lanes 1: control; 2: p21; 3: p21 + cre. (F,H) Cells were incubated with BrdU for 6 hours. (G,I) expression of SA-β-gal. (J) Cells were stained with DAPI and an antibody to HP1γ. (K) HFs were transduced with dnHes1 or an empty vector, serum deprived for ten days (SF) and then re-stimulated with full-serum medium (SP). BrdU was addded for 24 hours. The percentage of BrdU positive cells is indicated. (L) Expression of SA-β-gal.