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. 2013 Jul 16;288(34):24691–24704. doi: 10.1074/jbc.M113.469692

FIGURE 6.

FIGURE 6.

siRNA-mediated reduction of SH3BP5 attenuates the neuroprotective effect of Humanin in human SH-SY5Y cells and mouse primary hippocampal neurons. A–C, SH-SY5Y cells, cotransfected with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP) together with the pRNA/U6.1-Shuttle vector (sivector) or the pRNA/U6.1-Shuttle vector encoding siRNA for human SH3BP5 (sihSH3BP5) in the presence or the absence of 10 μm HN. At 48 h after transfection, they were harvested for Trypan Blue cell death assays (A) and WST-8 cell viability assays (B). Cell lysates were subjected to SDS-PAGE and immunoblot analysis with the APP antibody (22C11) (C). D, PHNs were cotransfected with the empty pcDNA3 vector (vector) or pcDNA3-V642I-APP (V642I-APP) together with the pRNAT-U6.1-IRES-cGFP vector (sivector) or the pRNAT-U6.1-IRES-cGFP encoding siRNA for mouse SH3BP5 (simSH3BP5). At 48 h after transfection, the cells were stained with the APP antibody. cGFP was expressed in cells into which the sivector or the simSH3BP5-encoding vector had been introduced. Nuclei were stained with DAPI. The percentages of morphologically apoptotic cells in the cells that expressed both V642I-APP plus cGFP were counted for the pcDNA3-V642I-APP-transfected cells. The percentages of apoptotic cells in the cells that expressed cGFP were counted for the pcDNA3 vector-transfected cells. This study was performed with n = 3; 50–100 cells were counted per well. ***, p < 0.001; n.s., not significant.