p21 expression increases adenovirus activity. 2A: p21 expression increases adenoviral cytotoxicity. Hct116 p21+/+ and p21-/- cells were infected with dl 922-947 (upper right), dl 309 (lower left) and Ad5 WT (lower right) (MOI 0.0001 - 100 pfu/cell). Cell survival was assessed 144 hours later. Absence of p21 expression in uninfected p21-/- cells was confirmed by immunoblot (top left). 2B: p21 increases E1A expression. Hct116 p21+/+ and p21-/- cells were grown on poly-L-lysine-coated coverslips, infected with dl 922-947 (MOI 0.5) and fixed up to 72 h pi with 5% formaldehyde. Following permeabilisation, E1A and tubulin expression was assessed by immunofluorescence. 2C: p21 undergoes proteasomal degradation following adenovirus infection. Protein was harvested from Hct116 p21+/+ cells up to 72 h post-infection with dl 922-947 (MOI 10) and analyzed by immunoblot for p21 expression (Ci). Cells were treated with 50 μM MG132 for 6 hours prior to harvest (Cii). Hct116 p21+/+ cells were also subjected to cell fractionation 24 h post-infection. C = cytoplasmic fraction, N = nuclear fraction (Ciii). p21+/+ cells were also harvested 6 h following exposure to 5Gy X-irradiation (X-IR) and blotted for p21 expression (Civ). 2D: p21 expression increases dl 922-947 activity in vivo. 5 × 106 Hct116 p21+/+ and p21-/- cells were injected subcutaneously in the flanks of CD1 nu/nu female mice. Once tumours reached approximately 100 mm3, dl 922-947 or Ad CMV GFP was injected intratumorally into (1 × 1010 particles in 50 μl PBS; n = 4-5 per group) on three separate occasions. Tumours were measured using callipers. Points represent mean ± s.e.m. *; p < 0.05 by unpaired, one-tailed Student's t test.