SUPPLEMENTAL FIGURE LEGENDS

Transcription

1 SUPPLEMENTAL FIGURE LEGENDS Fig. S1. SDSPAGE of crosslinked Aβ42 oligomers after SEC. After crosslinking of Aβ42 with or without Myr or RA, APS and Ru(bpy) were removed by SEC. The resulting products were analyzed by SDSPAGE on a 1020% gradient SDS gels. Bands were visualized using silver staining. Lane 1, uncrosslinked Aβ42; Lane 2, crosslinked Aβ42 before SEC; Lane 3, crosslinked Aβ42 after SEC; Lane 4 crosslinked Aβ42 with Myr before SEC; Lane crosslinked Aβ42 with Myr after SEC; Lane, crosslinked Aβ42 with RA before SEC; Lane 7, crosslinked Aβ42 with RA after SEC. The gel is representative of each of three independent experiments. Fig. S2. The effects of Myr, FA, NDGA, Cur, or RA on Aβ oligomerization. PICUP, followed by SDSPAGE and silver staining, was used to determine the effects of 0,, 10, 2, 0, 100, and 00 µm Myr (A, B), FA (C, D), NDGA (E, F), Cur (G, H), and RA (I, J) on oligomerization of Aβ40 (A, C, E, G, I) or Aβ42 (B, D, F, H, J). (K and L) Densitometric analysis of Aβ oligomerization. Densitometry was performed with a luminescent image analyzer (LAS 4000 mini, Fujifilm, Tokyo) and image analysis software (Multi gauge v. 3.2., Fujifilm). The intensity of each band in a lane from the SDS gel was normalized to the sum of the intensities of all the bands in that lane, according to the formula x 100 (%), where R i is the normalized intensity of band i and I i is the intensity of each band i. R i varies from To calculate the oligomer ratio, the sum of oligomer intensities of Aβ40 (K) or Aβ42 (L) with, 10, 2, 0, 100, and 00 µm Myr ( ), FA ( ), NDGA ( ), Cur ( ), or RA ( ) was divided by the sum of oligomer intensities without each compound. Each points represents mean±s.e. of three independent experiments. Fig. S3. [Met(O) 3 ]Aβ42 oligomerization. PICUP, followed by SDSPAGE and silver staining, was used to determine the effects of 10 and 100 µm Myr or RA on oligomerization of [Met(O) 3 ]Aβ42. Lane 1, molecular weight markers; Lane 2, Met(O) 3 ]Aβ42 alone (uncrosslinked); Lane 3, Met(O) 3 ]Aβ42 alone (crosslinked); Lane 4, Met(O) 3 ]Aβ42 with Myr (10 µm); Lane, Met(O) 3 ]Aβ42 with Myr (100 µm); Lane, Met(O) 3 ]Aβ42 with RA (10 µm); and Lane 7, Met(O) 3 ]Aβ42 with RA (100 µm). The gel is representative of each of three independent experiments. Fig. S4. Expanded HSQC spectra of the Aβ42 with or without FA, NDGA, or Cur. The control spectrum of the Aβ42 alone (2 µm, ph 7.2, C, black crosspeaks) is overlaid with the spectra of the Aβ42 (2 µm) containing FA (A), NDGA (B), or Cur (C) at 00 µm concentrations (red crosspeaks). Fig. S. Upfield 1 H NMR spectral regions of Αβ40 alone (2 µm, ph 7.2, C) plus RA or Myr. Reference spectrum of Aβ40 alone obtained with offresonance irradiation at 30 ppm (A), STD spectrum obtained by subtracting spectrum in (A) from that obtained with irradiation at 0.2 ppm (B). Reference spectrum for Aβ40 and RA (0 µm) (C), and STD spectrum for Aβ40 and RA (D). Reference spectrum for Aβ40 and Myr (0 µm) (E), and STD spectrum for Aβ40 and Myr (F). Fig. S. MTT metabolism. MTT assays were performed on HEK293 cells incubated 20 h with uncrosslinked Aβ42 (UnXL Aβ42), crosslinked Aβ42 (XL Aβ42), crosslinked Aβ42 with Myr (XL Aβ42 + Myr), and crosslinked Aβ42 with RA (XL Aβ42 + RA) of Aβ42 at final nominal concentrations of 1 and 10 µm. Each column represents means ± S.E. (n=3). Differences reaching statistical significance are noted by line segments between samples, along with their associated pvalues, where * signifies p < 0.0 and ** signifies p < Fig. S7. Mechanism of polyphenolic inhibition of Aβ aggregation. The pathway of Aβ aggregation that starts with random structured monomers and ends with amyloid fibrils. Intermediate stages include 1

2 loosely associated disordered oligomers, soluble βstrand aggregates (with parallel orientations), and laterstage protofibrils. Solution NMR detects only monomeric peptide, while CD detects laterstage βsheet oligomers. Myr shows strong binding with the monomer, while RA does not bind to the monomer and probably prevents neurotoxicity by binding to a nonnmr detectable structural motif of the earlyformed oligomers. All polyphenols disrupt laterstage βsheet fibril formation, consistent with the PICUP data. 2

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4 Fig. S2 E Myr NDGA F.3 Myr NDGA FA Cur 100 Oligomer Ratio (%) FA Myr FA NDGA Cur RA 40 L.3 Aβ H RA D Oligomer Ratio (%).3 K G RA C J B.3 I.3 A 80 Aβ42 0 Myr FA NDGA Cur RA Cur Concentration (log M) 4 3

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6 Fig. S4 A B C

7 Fig. S (A) Aβ40 (B) Aβ40 STD (scale x) (C) 1D reference Aβ40 +RA (D) STD Aβ40 +RA (scale x) (E) 1D reference Aβ40 +Myr (F) STD Aβ40 +Myr (scale x)

8 Fig. S MTT reduction (%) * * * ** ** UnXL XL Aβ42 XL Aβ42+Myr XL Aβ42+RA µm 10 µm

9 Fig. S7

10 Table S1. Morphological analysis of Aβ assemblies a Assembly Aβ40 Aβ42 Uncrosslinked (4) (89) Crosslinked (34) (4) Crosslinked with Myr (3) (80) Crosslinked with RA (3) (88) a Mean height + S.E., in nm, is listed for (n) Aβ assemblies visualized by AFM.