Figure 1.
PS dKO MEFs are vulnerable to serum deprivation-induced cell death.
(A) Western blot analysis of nicastrin and PS1 showing that PS dKO MEFs prevent nicastrin maturation. hPS1 MEFs were rescued PS1 and nicastrin maturation. Calnexin served as a loading control for membrane fractions. Filled and blank arrows indicate mature and immature nicastrin, respectively. Filled and blank arrowheads indicate full-length PS1 and PS1-CTF, respectively. (B) LDH release assay showing that PS dKO MEFs are injured by serum deprivation-induced stress. All MEFs were incubated DMEM media without serum for 36 hr. The graph represents (%) of positive control. Data are means±SEM values of three independent experiments. * represents significant differences from PS WT MEFs. ***P<0.001. # represents significant differences from PS dKO MEFs. ###P<0.001.
Figure 2.
ROS-mediated serum deprivation-induced cell death in PS dKO MEFs.
(A) ROS generation of MEFs was detected using the DCFDA fluorophore. All MEFs were incubated in DMEM w/wo 200 µM trolox or 10 mM LiCl for 36 hr. PS dKO MEFs showed higher signal intensities, but hPS1-rescued MEFs and trolox- or LiCl -treated PS dKO MEFs had lower signals. The white bar represents 50 µm. (B) PS dKO MEFs were incubated in the presence or absence of 200 µM trolox or 10 mM LiCl in DMEM for 36 hr. LDH release assay showed that both trolox and LiCl efficiently protected against serum deprivation-induced cell death. The graph represents (%) of positive control. Data are means±SEM values of three independent experiments. * represents significant differences from untreated PS dKO MEFs. ***P<0.001.
Figure 3.
Phospho-β-catenin was accumulated in PS dKO MEFs under serum deprivation conditions.
(A) β-catenin was accumulated in PS dKO MEFs, not in PS WT under normal and serum deprivation conditions. β-actin served as a loading control. (B) PS WT and PS dKO MEFs were incubated in growth media or DMEM for 30 hr, followed by immunofluorescent staining. PS dKO MEFs in DMEM labeled more brightly against anti-phospho-β-catenin (S33, 37, T41) antibody. The white bar represents 200 µm. (C) PS dKO MEFs were incubated w/wo 10 mM LiCl or 200 µM trolox in DMEM for 30 hr and labeled with phospho-β-catenin (S33, 37, T41)-specific antibody (Red). Many cells contained phosphorylated forms of β-catenin in DMEM- and trolox-treated PS dKO MEFs by fluorescence microscopy. But, LiCl-treated PS dKO MEFs stained weakly. DAPI staining was used to visualize cell nuclei (Blue). The white bar represents 200 µm. (D) Western blot analysis showed that LiCl effectively decreased phosphorylation of β-catenin at the S33, 37, and T41 sites without altering total β-catenin or β-actin levels. However, trolox-treated cells showed similar levels of phospho-β-catenin compared with DMEM alone.
Figure 4.
Accumulation of phospho-β-catenin induces cytotoxicity in H4 neuroglioma cells.
(A) H4 cells were transiently transfected with EYFP vector, WT, and mutant β-catenin constructs in DMEM for 16 hr. K19/49R β-catenin-transfected cells showed higher immunoreactivity against anti-phospho-β-catenin (S33, 37, T41) without affecting endogenous β-catenin and β-actin levels. Filled and blank arrows indicate exogenous β-catenin-EYFP fusion protein and endogenous β-catenin, respectively. Arrowhead indicates nonspecific bands. (B) LDH release assay showed that K19/49R β-catenin induced cytotoxicity in the transfectants. The graph represents (%) of positive control. Data are means±SEM values of three independent experiments. * represents significant differences from EYFP vector-transfected H4 cells. *P<0.05, **P<0.01, ***P<0.001. # represents significant differences from WT β-catenin-EYFP-transfected H4 cells. ###P<0.001. (C) DCFDA staining showing ROS generation of mock and β-catenin-transfected H4 cells. Although fewer cells remained among K19/49R β-catenin tranfectants, the DCFDA signal was much higher than in other transfectants. The white bar represents 200 µm.