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Fig. 4. The JNK signaling pathway might be responsible for the subcellular distribution of hSlu7. (A) HeLa cells were incubated with either SB205823 (an inhibitor of the p38 pathway; p38-I) or SP600125 (an inhibitor of the JNK pathway; JNK-I) for 1 hour at the indicated concentrations (bottom right corner of each image; units are in µM). Cells were then UV-C irradiated (0.9 J cm–2) and allowed to recover for 3 hours, followed by fixation and staining with anti-hSlu7. (B) Proportion of hSlu7 exported out of the nucleus. Inhibitor concentration (low, high and medium) refers to 5 µM, 10 µM and 40 µM for SB205823 (p38 inhibitor; shaded bars) and to 0.1 µM, 1 µM and 10 µM for SP600125 (JNK inhibitor; white bars). (C) Inhibition of p38 and JNK phosphorylation by SB205823 and SP600125, respectively, is effective. HeLa cells were UV-C irradiated (0.9 J cm–2) and then allowed to recover for 1 hour or 3 hours, as indicated (lanes 1,2 and 3,4, respectively). To some cells, either SB205823 (40 µM; p38-I; top) or SP600125 (10 µM; JNK-I; bottom) was added. Total proteins were extracted, separated by SDS-PAGE and probed with a monoclonal anti-phospho-p38-specific antibody (p38-P) or a polyclonal anti-phospho-Jun-specific antibody (JNK-P). (D) Inhibition of JNK phosphorylation reverses the UV-C irradiation effect on DDO alternative splicing. Cells were mock treated (lane 2) or UV-C irradiated (lanes 3,4) and then incubated for 24 hours at optimal conditions followed by RNA isolation and RT-PCR. One sample was pretreated with 1 µM SP600125 (JNK inhibitor; JNK-I) for 1 hour before irradiation (lane 4). (E) Active JNK1 affects DDO alternative splicing. Cells were mock treated (lane 2) or UV-C irradiated at a low dose (0.01 J cm–2; lanes 3,4) in the absence or presence of a JNK1-cDNA-containing plasmid (lanes 3,4, respectively). Following 24 hours at optimal conditions, RNA was isolation and RT-PCR performed. (F) Active JNK1 increases the hSlu7 nuclear to cytoplasmic shift. 293T cells were either UV-C irradiated (i) or transfected with a JNK1-cDNA-containing plasmid (ii; detected using an HA tag) and then UV-C irradiated at a low dose (0.01 J Cm–2; iii). hSlu7 and JNK1 cellular localization was observed by indirect immunofluorescence by fixing the cells and using anti-hSlu7 and anti-HA antibodies. Superimposed images are presented where nuclear DNA DAPI staining is colored blue (i-iii), anti-hSlu7 staining is colored red (i and iii) and anti-HA-tag (fused to the JNK1 cDNA; ii) is colored green.
