Journal: Journal of Cellular Physiology

Article Title: Amyloid Precursor Protein (APP) Affects Global Protein Synthesis in Dividing Human Cells

doi: 10.1002/jcp.24835

Figure Lengend Snippet: Artificial downregulation of APP causes a reduced ERKs activation but it does not affect Akt‐1 activation. APP modulates 4E‐BP1 phosphorylation through Styx. A‐C: Western blot analyses of the indicated proteins and phosphoproteins at the indicated time‐points in H1299 cells with depleted APP (siAPP) or in control siRNA‐transfected cells (c). Similar results were obtained in cell lines A549 and H1437. D: Immunoblot of the indicated protein and phosphoproteins in cell lysates obtained from cell line H1299 48 h after exposure to vehicle (c) or roscovitine (Rosc.). E: Immunoblot of the indicated protein and phosphoproteins in cell lysates obtained from cell line H1299 after exposure to vehicle (c) or UO126. In this experiment we show the 24 h time point because longer UO126 exposures caused paradoxical ERKs overactivation, as often described in scientific literature. F: Q‐PCR of the Styx mRNA in H1299 cells transfected with a control siRNA (c) or with the indicated siRNAs to Styx. Columns, averages of three independent experiments; bars, S.D. G: Western blot analysis of the indicated proteins and phosphoproteins 48 h after transfection of the indicated siRNAs. H: Q‐PCR of the Styx mRNA in cells transfected with the control plasmid pCAX (c) or with APP 695 cloned in pCAX (APP695). Columns represent averages of independent experiments performed in A549, H1299, and H1650 cells; bars represent S.D. I: Western blot analysis of the indicated protein and phosphoproteins in H1299 48 h after transfection with a variety of nucleic acids. Lanes: 1, cells transfected with siRNA control (c); 2, cells transfected with siRNA to APP (siAPP); 3, cells transfected with siRNA to APP and siRNA to Styx (siAPP + siStyx); 4, cells transfected with siRNA to Styx (siStyx); 5, cells transfected with control plasmid (pc); 6, cells transfected with a plasmid encoding AICD (AICD); 7, cells transfected with the control plasmid and siRNA to Styx (pc + siStyx); 8, cells transfected with a plasmid encoding AICD and with siRNA to Styx (AICD + siStyx). The apparent additive effect of siAPP and siStyx (lane 3) can be explained by a synergistic effect of the two treatments on the Styx mRNA expression level. APP forced expression, on the other hand, elevates the Styx mRNA expression levels (Fig. H). The fact that relatively small variations of the Styx mRNA level can produce measurably different effects on the 4E‐BP1 phosphorylation level at T37/46 is supported by the comparisons between siStyx2 and siStyx3 (Fig. F). SiStyx3 seems slightly less efficient in downregulating the Styx mRNA compared to siStyx2. This small variation yields measurable differences in 4E‐BP1 phosphorylation at T37/46 as measured using Western blot analysis (Fig. G).

Article Snippet: Insulin in solution (Sigma–Aldrich) was diluted in sterile PBS and used at the specified concentrations. pcDNA3‐RLUC‐POLIRES‐FLUC was a gift from Nahum Sonenberg (McGill University, Montreal); pCDF1‐MCS1‐EF1‐cop GFP expressing the APP C‐terminal 59 aa was a gift from Dr. Xiao Z.C. (Institute of Molecular and Cell Biology, Singapore); full length APP 695 in pCAX vector was from Addgene (Cambridge, MA).

Techniques: Activation Assay, Western Blot, Transfection, Plasmid Preparation, Clone Assay, Expressing