MPA induces NS depletion and ribosomal stress.
A, MPA triggers NS depletion and p53 activation at 10 μm. U2OS cells were treated with varying concentrations of MPA for 12 h and then harvested on ice. Equal lysates were used for Western blotting with the indicated antibodies. In each sample, 50 μg of proteins were used (true for the following figures). B, MPA induces NS depletion in a time-dependent manner. U2OS cells were treated with 10 μm MPA and harvested at the indicated time points on ice. Protein expression was analyzed by SDS-PAGE followed by Western blotting with the indicated antibodies. C, U2OS cells were treated with MPA for 12 h in the presence or absence of GTP or guanosine (Guan) and harvested on ice. Equal lysates were used for Western blotting with the indicated antibodies. D, immunofluorescence staining to show NS depletion from the nucleolus. Cells were fixed with methanol and subjected to staining with anti-NS and anti-B23 antibodies. These experiments were repeated in H1299 and HCT116 cells as well (data not shown).