Fig 1: Apoptosis is associated with induction of cleaved caspase-3 and cleaved caspase-7 in primary cortical neurons.Primary neurons were cultured from E18 embryonic rat cortices for seven days, followed by treatment with 10 µM CPT to induce apoptosis. A and B show immunocytochemistry with MAP2 as a neuronal marker (green) and cleaved caspase-7 (A) or cleaved caspase-3 (B) antibodies (red) (Magnification: 63X). Hoechst staining, shown to the right of panels A and B, shows condensed or fragmented nuclei (indicative of apoptosis) in cells positive for cleaved caspases, whereas MAP2 positive neurons in the untreated samples show intact nuclei. (C) Western blot analysis of the lysates from neurons treated with CPT for 12 hours showed a significant induction in cleaved caspase-3 (top panel) and cleaved caspase-7 (second panel) levels. Activation of caspases was partially attenuated in the presence of 10 µM Z-DEVD-FMK and Z-VAD-FMK. Analysis of the lysates with an antibody generated against caspase-cleaved APP detected a single band of ∼25 kDa size. The level of this fragment was attenuated slightly in the presence of caspase inhibitors (Figure 4C, third panel). A reprobe of this blot using an antibody against β-Actin was performed as a control for protein loading (Figure 4C, bottom panel).
Fig 2: Activation of caspase-3 and -7 is associated with APP proteolysis in apoptotic cells.(A) H4-APP cells were treated with CPT in the presence and absence of caspase inhibitors for six hours and analyzed by western blot using antibodies directed against cleaved caspase-3 or cleaved caspase-7. Cleavage of caspase-3 (topmost panel) and caspase-7 (second panel) was observed in cells treated with CPT. Cleavage of these caspases was associated with an induction in the formation of the fragments detected by caspase-cleaved APP antibody (third panel). Blots were probed for β-actin as a loading control (bottom panel). Immunocytochemical analysis of H4-APP cells also showed significant induction in cleaved caspase-3 (B) and cleaved caspase-7 (C) after one and six hours of exposure to CPT. Cells were co-immunostained with 6E10 antibody (green) to show colocalization of both active caspase-3 and active caspase-7 (red) with APP (Magnification: 63X). Cells showing increased levels of cleaved caspases also showed a reduction in the level of APP signal intensity, consistent with the decrease in full length APP observed by western blot in Figure 2A and 2B.
Fig 3: Down-regulation of caspase-3 and caspase-7 reduces the generation of ∼25–35 kDa caspase-cleaved APP fragments in CPT treated H4-APP cells.(A) H4-APP cells were transiently transfected with shRNA to caspase-3 or shRNA to caspase-7 and the down-regulation of the respective caspases analyzed after 48 hours using antibodies against caspase-3 (top panel) and caspase-7 (second panel). The lower panel in (A) shows an actin probe of the blot to show protein loading. (B) H4-APP cells stably transfected with shRNA to caspase-3 or caspase-7 were analyzed using the respective caspase antibodies. Reprobing of the blot with β-actin antibody shows protein levels on the blot (bottom panel). (C) Quantification of the levels of caspase-3 and caspase-7 in stably transfected cells from three independent experiments (as represented in B), normalized to β-actin. An approximately 86% decrease in full-length caspase-3 levels were observed in cells transfected with shRNA to caspase-3 or shRNA to caspase-7. Similarly, an approximately 49% decrease in caspase-3 levels was observed with shRNA to caspase-3, and a 56% decrease in caspase-7 levels was observed with shRNA to caspase-7. Induction of cleaved caspase-3 was observed with shRNA to caspase-7, but no induction of cleaved caspase-7 was observed with shRNA to caspase-3. (D) Western blot analysis of the lysates with antibody to caspase-cleaved APP showed a reduction in the levels of the cleaved fragment in the shRNA transfected cells after three hours of treatment with 10 µM CPT. The figure shows data from two experiments treated with CPT (E) Quantification of data from three independent experiments (representative figure shown in panel D), normalized to actin, shows a 31% reduction in caspase-cleaved APP with shRNA to caspase-3 and a 90% reduction in cells transfected with shRNA to caspase-7. Asterisks indicate significant differences, p<0.05.
Supplier Page from OriGene Technologies for Caspase 3 (CASP3) Human shRNA Plasmid Kit (Locus ID 836)