Figure 5
BDNF-mediated reduction of the survival of GFP-p75 STHdh111/111Q mutant cells is associated with activation of apoptotic pathways. Wild-type (ST7/7Q) and mutant (ST111/111Q) huntingtin striatal cells transfected with GFP or GFP-p75 were treated with BDNF (50 ng/ml, 30 min) and cell survival was evaluated 24 h later by scoring the percentage of Hoechst-stained nuclei. (a) Photomicrographs of ST7/7Q and ST111/111Q cells stained with Hoechst showing a significant reduction of cell survival in BDNF-treated GFP-p75 ST111/111Q cells. Scale bar, 10 μm. Quantification of surviving cells is shown as the percentage of total cells in control conditions. The results are representative of seven independent experiments performed in triplicate and are expressed as the mean±S.E.M. Data was analyzed by one-way ANOVA followed by Student’s t-test *P<0.05 versus vehicle-treated GFP-p75 mutant cells. (b) Representative immunoblot showing the levels of JNK, p-JNK and α-tubulin as a loading control in striatal extracts obtained from mutant ST111/111Q cells incubated with BDNF (50 ng/ml, 3 h). The histogram represents the relative p-JNK/JNK ratio considering 100% the ratio obtained in control conditions (vehicle). Values are given as mean±S.E.M. Data was analyzed by Student’s t-test. ***P<0.001 versus vehicle-treated GFP-p75 cells. (c) Representative immunoblots showing the levels of Ser-473-Akt (p-Akt), Akt, phosphoERK1/2 (p-ERK1/2), ERK1/2 and α-tubulin as a loading control in striatal extracts obtained from wild-type (ST7/7Q) and mutant (ST111/111Q) cells transfected with GFP or GFP-p75 and treated with BDNF (50 ng/ml) for the indicated time points. Blots are representative of four independent experiments. The histograms represent the relative p-Akt/Akt and p-ERK/ERK ratios considering 100% the ratio obtained in control condition (vehicle). Values are given as mean±S.E.M. Data was analyzed by Student’s t-test. +P<0.05, ++P<0.01 versus vehicle-treated GFP cells, *P<0.05, **P<0.01,***P<0.001 versus vehicle-treated GFP-p75 cells, $P<0.05 versus BDNF-treated GFP cells
