Supporting Information Chen et al. 10.1073/pnas.0807991106 SI Methods Sucrose Gradient Fractionation. Fractionation by sucrose gradient was performed as described by Gasparini et al. [(2001) J Neurosci 21:2561 2570]. After 24 h of incubation in the absence or presence of 10 mm metformin, N2a695 cells were homogenized by using a stainless steel ball bearing homogenizer in 5 vol of 0.25 M sucrose, 10 mm Tris HCl, ph 7.4, 1 mm MgAc 2, and a protease inhibitor mixture. The homogenate was loaded on top of a step gradient comprised of 1.5 ml of 2 M sucrose, 4 ml of 1.3 M sucrose, 3.0 ml of 1.16 M sucrose, and 2.0 ml of 0.8 M sucrose. All sucrose solutions contained 10 mm Tris HCl, ph 7.4, and 1 mm MgAc 2. The gradients were centrifuged for 2.5 h at 100,000 g in a Beckman SW41Ti rotor. Fractions were collected and assayed for total protein with BCA assay. Proteins from each fraction were analyzed by Western blot with antibodies against APP, BACE1, ARF (Santa Cruz), -adaptin (Santa Cruz), calnexin (Cell Signaling) or Na, K-ATPase (Abcam) respectively. A Degradation Assay. N2a695 cells were starved with DMEM (GIBCO) without methionine and labeled for 4 h with 750 Ci/ml [ 35 S] methionine. Medium was collected to serve as the source for labeled A proteins to be added to naive N2a cells, which were pretreated with metformin (10 mm) for 24 h, and incubated at 37 C up to 4 h. At various time points as indicated, the media were collected and the amount of labeled A in each sample was determined by IP-Western blot analysis, using antibody 6E10. 1of5
Fig. S1. Intracellular A measured from cell lysates does not include the membrane-associated extracellular A. Metformin modulates subcellular compartmentalization and distribution of APP but not BACE1 in N2a695 cells. (A) N2a695 cells were pretreated with metformin for 24 h and then treated with trypsin briefly to remove the membrane-associated extracellular A and then with trypsin inhibitors prior to lysis. Intracellular A production was measured by ELISA of lysates. (B) Representative autoradiographic analysis of intracellular APP and BACE1 subcellular distribution after metformin treatment is shown. Cells were treated for 24 h in the absence or presence of 10 mm metformin, homogenized, and fractionated on an equilibrium flotation sucrose gradient. Markers for subcellular compartments: anti-calnexin (ER), anti- -adaptin (TGN), anti-arf (post-tgn vesicles, cytosol), anti-na, and K-ATPase (plasma membrane). Fraction 1 0.25 M sucrose solution (loading, cytosol). Fractions 2 5 correspond, respectively, to interfaces between 0.25/0.8 M (post-tgn vesicles), 0.8/1.16 M (Golgi/TGN), 1.16/1.3 M, and 1.3 M/2 M (heavy membranes such as ER, plasma membranes) sucrose solutions. (C) Quantification of data from 3 independent experiments. 2of5
Fig. S2. Metformin has no effect on 2 A -degradating enzymes and A degradation. (A) Unchanged protein levels as measured by Western blot analysis. (B) Unchanged enzyme activities of IDE and neprilysin from cell lysates and from culture conditioned media (data not shown) after metformin treatment as measured by using Calbiochem IDE kit and a standard procedure described by Li et al. [(1995) Methods Enzymol 248:253 263]. (C) Metformin has no effect on A degradation in pulse chase experiment performed as described by Gasparini et al. [(2001) J Neurosci 21:2561 2570]. 3of5
Fig. S3. Metformin activates AMPK/BACE1 and increases A level in triple Tg mice. (A) Western blots of frontal brain lysates from the triple Tg mice after drinking metformin (2 mg/ml) for 3 months. (B) Effect of metformin on increasing soluble A as measured by quantitative ELISAs. (C) Immunohistochemistry staining with monoclonal anti-a 6E10 shows increased A immunoreactivity in both cortex and hippocampal regions of the triple Tg mice. (D) Thioflavin-S staining of insoluble A, showing increased small aggregates. n 4 animals in each group. *, P 0.05; **, P 0.01. 4of5
Table S1. LC-MS Analysis of Metformin Concentration Exp. group Met, mg/ml Plasma, M Brain, M 1 0 BLOQ* BLOQ* 2 1 1.195 0.006 0.093 0.003 3 2 2.189 0.397 1.093 0.188 Table S1. Plasma and brain (frontal region) were collected from WT C57/BL6 mice after receiving metformin in drinking water for 6 days and the concentration of metformin was measured by liquid chromatography (LC)-MS analysis. The data show means SD (n 3 animals in each group). BLOQ*, below limit of quantitation. 5of5