FIGURE 7.

Reduction of PLA signal during cell death. (A) Representative PLA assay performed with APP-CT and Trk B3 antibodies in primary septal cultures (12 DIV) untreated (Ctrl) or treated with Aβ 1–40 (20 μM), staurosporine (30 nM) and rapamycin (10 nM) for 6 h. (B) The graph represents quantitative analysis of the number of PLA fluorescent red puncta/cell counted in five different fields and for a total of 20–30 cells examined and are expressed as mean ± SEM (^∗∗p < 0.005, Student t-test was used for statistical analysis). (C) Western blot analysis for cleaved and active capsase-3, is reported for Ctrl, Aβ 1–40 and staurosporine treated cells for 12 h. (D) Representative blot for LC3 in Ctrl and rapamycin treated cells for 12 h. Post-translational modification of cytosolic LC3-I (Light Chain 3 of Microtubule Associated Protein 1A/B) to LC3-II is a useful index of autophagy. (E) APP and TrkA expression in Ctrl and staurosporine and rapamycin treated cells. Double immunofluorescence for APP (red) or TrkA (green) and DNA (DAPI, blue) in Ctrl condition and following the indicated treatments for 12 h. (F) Bar plot summarizes the APP and TrkA mean fluorescence intensity in Ctrl and treated cultures. Fluorescence intensity is reported in arbitrary unit (AU). No significant difference were observed between Ctrl condition and following staurosporine or rapamycin exposure. (G) Representative Western blot of TrkA, APP and β-CTF in neurons treated with staurosporine (30 nM) and rapamycin (10 nM) for 12 h. (H) Graph depicting quantification of immunoreactivity for TrkA, APP and β-CTF bands normalized to β-actin (used as loading control) in neurons treated with staurosporine and rapamycin for 12 h (^∗p < 0.05; ^∗∗∗p < 0.0001). Results are the means (±SEM) of duplicate determinations from three independent experiments and are reported as percentage of Ctrl cells.