Figure 2

APP knock-down increases neurite length in a cell-autonomous manner. (a, b) E17 primary cortical neurons were plated and transfected with plasmids encoding GFP alone (a) or GFP with APP shRNA-active (b) or with APP shRNA-inactive (not shown). Three days later, neurons were fixed and immunostained for βIII-tubulin. (c) Neurite length was quantified in GFP+, transfected cells. Error bars represent standard error of the mean; *p < 0.05; ***p < 0.001. (d-i) E17 (d, e) or E14 (f-i) cortices were electroporated with GFP (d, f, h) or GFP + APP shRNA-active (e, g, i) and harvested at postnatal day 5. Images in (d-g) are of the upper half of the cortical plate in coronal sections. (h, i) Coronal sections immunostained for Tbr1 (red), marking layer VI of the cortical plate (CP), and MAP2 (blue), marking the entire cortical plate. GFP positive, electroporated regions of E14 cortices were dissected 48 h after electroporation, dissociated, and plated. Three days later cells were fixed and immunostained for βIII-tubulin. IZ; intermediate zone; white lines delineates noted regions of the cortex (j, k) Neurite lengths (j) or the number of primary neurites (k) were quantified for GFP+ cells. Error bars represent standard error of the mean; *p < 0.05; ***p < 0.001. (l, m) FLAG-tagged murine APLP1 (l) or APLP2 (m) constructs were co-transfected into CHO cells with an empty vector or with three different shRNA constructs targeting rodent APLPs. Transfections (tf) of duplicate wells are shown. Forty-eight hours post-transfection, cells were lysed, and the western blots for FLAG on protein normalized samples are shown.