FIGURE 6.

Loss of Scribble mediates drug resistance by activation of p38 MAPK pathway. A, phosphorylated p38 MAPK (p-p38) was activated in Scribble KD cells. B, the p38 MAPK inhibitor prevented Scribble KD-mediated HuR cytoplasmic translocation. Control and Scribble KD cells were treated with or without p38 MAPK inhibitor (SB203580; 10 μm, 12 h), and HuR (red) and nuclear (DAPI; blue) were detected by immunofluorescence staining. C, the translocation of HuR to the cytoplasm in Scribble KD cells was counteracted by exposure to the p38 MAPK inhibitor (SB203580, 10 μm, 12 h). Scribble KD cells were separated into cytosolic (Cyto) and nuclear (nuc) fractions, HuR, Lamin B (nuclear fraction control; loading control), and Hsp90 (cytosolic fraction control; loading control) were detected by Western blotting. D, HuR protein levels were measured in a time course by the proteasome inhibitor MG132 (20 μm) in Scribble KD and control cells. E, HuR protein levels were not significantly changed when Scribble KD cells were treated with SB203580, as assessed by Western blot. F, cytosolic and nuclear distribution detected in control cells were treated with or without p38 MAPK agonist (isoproterenol; 10 μm, 12 h) and processed as in C. G, Snail accumulation in CRL-1848 Scribble KD cells was decreased by SB203580 treatment (10 μm, 12 h). H, relative polyribosome distribution of Snail mRNA in the CRL-1848 cell line control and Scribble KD cells by SB203580 treatment (10 μm, 12 h) analyzed by sucrose gradient centrifugation as indicated above. I, analysis of RNP protein content in polyribosome fractions. Polyribosome distributions (12 fractions each) from the CRL-1848 cell line control and Scribble KD cells by SB203580 treatment (10 μm, 12 h) were isolated as above, and RNA-binding protein content was analyzed by Western blotting; β-actin served as an internal control.