Figure 4.

Proteasome activity is required for survival of ATM-depleted fibroblasts. (A) Chymotrypsin-like proteasomal activity measured in either cytoplasmic or nuclear cell extracts from TIG1 cells. Protein extraction was carried out 72 hours after transfection with the indicated siRNA (N = 3). (B) Representative Western blot showing accumulation of carbonylated proteins in the nuclear compartment of fibroblasts depleted of ATM, upon inhibition of proteasomal activity. MG132 treatment (10 μM, 6 h) was carried out 72 h after siRNA transfection. Equal amounts of cell extract were loaded in each lane. (C) Densitometric quantification of protein carbonylation in the experiment showed in panel B (N = 8). (D) Viability assay showing fibroblast sensitivity to proteasome inhibition. TIG1 fibroblasts were transfected with the indicated siRNA; 48 hours after transfection cells were incubated with MG132 (500 nM) for further 24 h. Cell viability was measured using a Trypan Blue exclusion assay (N = 4). (E) Representative FACS profiles of cells stained with Annexin V and propidium iodide (PI). Cells were treated as in panel D before staining. (F) Percentage of apoptotic cells obtained from FACS analysis of cells treated as in panel E (N = 3). Results are expressed as mean ± SD from the indicated number (N) of independent experiments: *P < 0.05; **P < 0.01.