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. 2012 May 9;7(5):e36562. doi: 10.1371/journal.pone.0036562

Figure 4. c-Abl-MST2 Signaling Mediates Rotenone-induced Neuronal Cell Death.

Figure 4

(A). Neuro2A cells were left untreated or treated with 400 μM Rotenone for 1.5 hours with or without 10 μM STI571. Lysates of cells were immunoprecipitated with anti-MST2 antibody and analyzed by immunoblotting with anti-pan-tyrosine phosphorylation antibody. (B). Cerebellar granule neurons were transfected with the pEGFP alone or together with the FLAG-MST2 expression plasmid, and c-Abl RNAi or control vector as indicated. 72 hours after transfection, neurons were left untreated or treated with Rotenone for 20 hours. Transfected neurons were subjected to immunofluorescence analysis using GFP antibody together with the DNA dye Hoechst 33258 to reveal neuronal nuclei. Representative images of neurons are shown in the upper panel. White arrowheads indicating healthy transfected neurons and red arrowheads indicating transfected neurons that are undergoing apoptosis. The percentage of cell death of GFP-positive neurons is represented as the mean ± SEM. Exposure of MST2-transfected neurons to Rotenone induced neuronal cell death (ANOVA; p<0.01, n = 3), and the cell death was dramatically reduced by c-Abl knockdown (ANOVA; p<0.01, n = 3).(C). Cerebellar granule neurons were transfected with the pEGFP alone or together with the MST2 shRNA, c-Abl shRNA or control vector as indicated. 72 hours after transfection, neurons were treated and analyzed as in B. The percentage of cell death of GFP-positive neurons is represented as the mean ± SEM. Knockdown MST2 or c-Abl protects neurons from Rotenone induced cell death (ANOVA; p<0.01, n = 3). (D). The upper panel shows the expression of the MST2 rescue constructs in Neuro2A cells. Lower panel: Lysates of HEK 293T cells transfected with FLAG-MST2 or FLAG-MST2R expression plasmids together with MST2 RNAi or the control vector, were immunoblotted with the FLAG or ERK1/2 antibody. MST2 RNAi effectively knockdown endogenous MST2 in Neuro2A cells, but not the rescue form of MST2. (E). Cerebellar granule neurons transfected with pEGFP and MST2 RNAi or control vector, alone or together with MST2, MST2R, and MST2R-Y481F expression plasmids, were treated and analyzed as in B. MST2R but not Y81F mutants of MST2R enhanced Rotenone-induced cell death (ANOVA; p<0.01, n = 3). The percentage of cell death in GFP-positive neurons is represented as the mean ± SEM.