Fig. 4. Degradation of Pol II LS is required for transcriptional restart following irofulven-exposure and influences cell viability. (A) HeLa cells were incubated for 30 minutes in the presence (, ) or absence () of 150 nM velcade before addition of 1 µg/ml irofulven (time –1). After 1 hour of irofulven exposure, cells were post-incubated for the indicated times in the presence () or absence () of 150 nM velcade. Cells continuously exposed to velcade alone were included as control (). Bromo-uridine was added for the last 15 minutes of incubation and the cells were fixed. Nascent RNA was revealed by immunolabeling, and the intensities were measured. All curves represent the average of at least three independent experiments in which the intensities of bromo-uridine labeling were assessed for more than 100 cells per time-point. Error bars represent standard errors and are indicated when they exceed symbol size. (B) The same conditions as above. The relative contents of H14 (left panel) and H5 (right panel) species were assessed by immunoblot and the relative intensities of each band measured and standardized versus actin. All curves represent an average of three independent experiments. Error bars represent standard errors and are indicated when they exceed symbol size. Filled symbols: cells treated for 1 hour with 1 µg/ml irofulven and post-incubated in drug-free medium; open symbols: cells pre-, co- and post-incubated with 150 nM velcade. (C) HeLa cells were preincubated for 30 minutes in the absence (left panel) or presence (right panel) of 10 µg/ml cycloheximide (CHX) followed by coincubation with 1 µg/ml irofulven for 1 hour. Cells were then post-incubated for the indicated times in the absence or presence of 10 µg/ml cycloheximide, and the relative content of Pol II LS forms was assessed by immunoblotting. Actin was included as a control for equal loading (lower panels). (D) HeLa cells were treated with irofulven for 1 hour, incubated in drug-free media for 5 days, and cellular viability was assessed by MTT assay. , cells treated with irofulven and post-incubated in drug-free medium; , cells pre-, co- and post-incubated with 500 nM velcade; , cells pre- co- and post-incubated with 100 µM DRB. All curves represent an average of at least three independent experiments, each done in duplicate. Error bars represent standard errors and are indicated when they exceed symbol size.