Figure 7.

PPM1B reduces serum-deprivation-induced cytotoxicity in H19-7 cell stably overexpressing DYRK1A through inhibition of toxic tau aggregate formation.A, H19-7 cells stably overexpressing DYRK1A (H19-7/DYRK1A) or control H19-7/pTK cells were incubated in N2 medium for 0 to 72 h, as indicated. Cytotoxicity was measured by assay of LDH release into the supernatant. The graph represents the mean of six independent experiments ± SD (∗∗∗p < 0.001). B‒D, the H19-7/DYRK1A and H19-7/pTK cells were either mock-transfected or transfected for 24 h with plasmid encoding Myc-PPM1B and then incubated in fresh N2 medium for 48 h. B, cytotoxicity was measured by LDH release into supernatant. The graph represents the mean of 12 independent experiments ±SD (∗∗∗p < 0.001). C, cell lysates were analyzed by western blotting with the indicated antibodies. The proper expression of stably and transiently transfected proteins in cell lysates was identified with immunoblot analysis using anti-DYRK1A, anti-tau, or anti-Myc antibodies, respectively. Tubulin served as a loading control. D, cells were washed with PBS, fixed with 3.7% formaldehyde, permeabilized with 0.2% Triton-X-100 in PBS, blocked with 1% BSA in PBS, and then stained with anti-Tau (green) and anti-Myc antibody (red). Nuclei were counterstained with DAPI (blue). Representative confocal images of immunostained cells are shown with the low and high magnification. The bottom panels showed enlarged views of the white-boxed area in the upper panels. Scale bar: 20 μm (the upper panel) and 10 μm (the lower panel). The arrowheads indicate tau aggregation dots. E, the percentage of tau-inclusion-positive cells was calculated by dividing the number of cells showing one or more intracellular tau inclusions by total number of tau-stained cells. Tau-inclusion-positive cells were chosen as the cells containing aggregated tau-foci, and the percentage of cells with foci was determined by visual inspection of at randomly chosen fixed areas (n = 9), which have approximately 5 to 10 cells. The three different symbols in the graph (i.e., filled circles, squares, and triangles) represented the data obtained from three independent experiments, respectively. The graph values are expressed as mean ± SD and represents at least three independent experiments (∗∗∗p < 0.001).