Supporting InformationBieschke et al. 10.1073/pnas.0910723107SI TextMaterials and Methods. EGCG and other catechins were obtainedfrom Sigma–Aldrich and aqueous stock solutions were freshlyprepared from lyophilized powder.

Preparation of Recombinant αS Protein. Recombinant His-taggedαS was expressed in Escherichia coli and purified as describedin ref. S1 under denaturing conditions with 6 M guanidinehydrochloride in the lysis buffer and 8 M urea in the other buffersby Ni-NTA affinity chromatography. After purification, theurea-containing buffer was exchanged for Tris buffered saline(TBS) via a PD10 desalting column (GE Healthcare). Purifiedprotein solutions were kept on ice for immediate use.

To confirm the results obtained with His-tagged αS, untaggedαS was expressed in E. coli and purified via ion exchange and sizeexclusion chromatography as described in (S2). Briefly, E. colicells were grown at 37 °C in 2L LB media containing ampicillinto an OD600 of approximately 0.5, induced with IPTG (1 mM)and grown overnight at 30 °C. Cells were pelleted for 30 minat 3; 000 × g, frozen at −80 °C and then resuspended in TBS(10 mM, pH 8.0) containing EDTA (1 mM) and PMSF(1 mM). Benzonase (1250 U) was added and cells were lysedby sonication on ice (3 × 1 min, Branson Sonifier 450, Branson),boiled for 20 min and then centrifuged for 30 min at 20; 000 × g.αS was pelleted from the supernatant by ammonium sulfate(360 mg∕mL) treatment for 15 min followed by centrifugation(20; 000 × g, 30 min). The pellet was resuspended in TBS(25 mM, pH 7.7) and purified on a Resource-Q anion exchangecolumn using a NaCl gradient from 0–500 mM. αS fractionswere pooled, run on a PD-10 gel chromatography column in10 mM ammonium carbonate buffer, and eluted fractions werelyophilized.

Lyophilized αS was dissolved in water, sonified for 10 min in awater bath sonifier (TK-52, Bandelin) and centrifuged for 20 minat 200; 000 × g. Protein concentration was determined by OD280

(E280 ¼ 6410 M−1) in a Nanodrop 8000 photometer (ThermoFisher).

In Vitro Aggregation of αS and Aβ42. Fibrillar αS aggregates wereformed by incubating His-tagged or untagged αS protein(100 μM) in microcentrifuge tubes placed in a rotating mixer(20 rpm) for 7 d at 37 °C in TBS buffer (20 mM Tris, 150 mMNaCl, pH 7.2). Each tube contained a small glass bead (approxi-mately 2 mm diameter) to increase agitation. Kinetic aggregationassays were performed by incubating monomeric αS (50 μM) inlow-binding 96-well plates (#3651, Corning) at 37 °C in TBS[20 μM Thioflavin T (ThT)] and measuring fluorescence(440 nm excitation, 485 nm emission, top read) using a fluores-cence plate reader (InfinitE M200) at 10 min intervals precededby 5 s shaking.

Synthetic Aβ42 peptide (R. Volkmer, Institute for Medical Im-munology, Charité) was dissolved in hexafluoro-isopropanolovernight, lyophilized, and stored at −20 °C. The lyophilizedAβ42 peptide was redissolved in DMSO to Aβ42 concentrationsof 1 mM. DMSO solutions were ultracentrifuged (30 min,200; 000 × g) and the supernatants were diluted into PBS to finalAβ42 concentrations of 15 μM, and incubated in microcentrifuga-tion tubes in a rotating mixer (20 rpm) for 4 d at 37 °C.

To produce oligomeric aggregates HFIP-treated Aβ42 wasdissolved in DMSO to a concentration of 2.5 mM and sonicatedin a water bath sonifier for 10 min. 40 μL solution was diluted infour subsequent steps into 4 × 240 μL DMEM (Biochrome,

w/NaHCO3, w/Glc, w/o Glutamate, w/o Pyruvate) and the solu-tion was mixed by vortexing after each step. The final solution wasincubated at 4 °C for 24 h, centrifuged at 14; 000 × g for 10 min at4 °C, and the supernatant was collected and analyzed by atomicforce microscopy (AFM).

In Vitro Seeding Experiments. αS-aggregates were treated with equi-molar concentrations of EGCG for different time intervals (0, 1,4, 24, and 72 h) to obtain seeds. EGCG-treated or untreated αSseeds were sonified for 1 h and added to 50 μM monomeric αS(10% seeds (wt/wt) of total αS solution). Triplicate aggregationreactions (100 μL) were incubated at 37 °C in TBS containingThT (20 μM). ThT fluorescence was monitored every 5 min pre-ceded by 20 s shaking using a plate reader as described above.

Circular Dichroism Spectroscopy. Recombinant His-tagged αS(100 μM) was aggregated for 7 d in vitro and then treated withEGCG at a molar ratio of 1∶1 as described above. Samples werediluted to 12.5 μMwith 1x TBS, sonicated for 1 h, and CD spectrawere recorded on a J-720 spectrometer (Jasco). Solutions con-taining corresponding amounts of EGCG but no αS were incu-bated in parallel. Reference CD spectra were recorded andsubtracted from the CD signals to isolate the αS-specific changesin the CD spectra.

Transmission ElectronMicroscopy.Fibrillar αS-aggregates were trea-ted with equimolar concentrations of EGCG for different timeintervals (1, 4, 24, and 72 h) in TBS. Aliquots of untreatedand EGCG-treated αS-aggregates were adsorbed onto carbon-coated copper grids, negatively stained with 2% uranyl acetate,blotted dry, and viewed on a Philips CM100 transmission electronmicroscope.

Atomic Force Microscopy. Sheet mica (Nanoworld) was glued to amicroscope slide and samples (10 μL) were adsorbed for 10 minonto the freshly cleaved mica, washed with freshly filtered deio-nized water (4 × 30 μl) and dried overnight. Dry AFM imageswere recorded on a Nanowizard II/Zeiss Axiovert setup (JPK)using intermittent contact mode and FEBS cantilevers (Veeco).

Fluorescence Microscopy. Fluorescently labeled αS monomers wereprepared by conjugating αS with amino reactive Alexa 488-NHS(Invitrogen) or Cy5-NHS dyes (GE Healthcare) overnight at 4 °Cat pH 8.8 at a 1.5∶1 molar ration of dye:protein and via gelfiltration according to the manufacturer’s instructions. The de-gree of labeling was quantified by dye and protein absorptionand was found to be approximately 1 dye/protein. UnlabeledαS monomers (100 μM) were mixed with 5% fluorescentlylabeled monomers and incubated for 7 d at 37 °C in TBS toproduce labeled fibrils. Fibril morphology was confirmed byAFM prior to use (Fig. S3A).

Fluorescence microscopy samples were prepared by pipetting10 μL of fluorescently labeled αS aggregate solution onto amicroscope slide and covering the droplet with a coverslip.Fluorescent images were recorded on an AxioImager (Zeiss)using a 100 x oil immersion objective and FITC and Cy5 standardfluorescence filter sets, respectively.

Filter Retention Assay. Cell lines were plated out at a density of18 × 105 cells∕mL in 96-well plates. Incubation was carried outat 37 °C with 5% (volume) CO2. The adherent HEK-293 wt αScells, CHO, and 7PA2 cells were either transfected with

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αS-aggregates (final concentration 1.0 μM) or Aβ42 aggregates(final concentration 1.0 μM). After 6 h cells were rinsed carefullywith DMEM and further incubated for 1, 2, or 3 d with the in-dicated concentrations of EGCG.

Amyloid quantification of cellular or in vitro samples wascarried out as described (S4). Lysis was performed either with1% NP-40 at 4 °C to determine total aggregates or with 2%SDS and incubation at 97 °C to determine SDS-stable aggregates.Samples were filtered through a cellulose acetate membranefilter (BioRad) using a 96-well vacuum apparatus. Membranebinding was visualized by NBT staining or membranes wereblocked in milk and stained using anti-Aβ mAb 6E10 (1∶2000;Covance) or anti-αS mAb 211 (1∶1000; Santa Cruz). Nonspeci-fically bound primary antibodies were washed off with tris buf-fered saline þ0.05% Tween-20 (TBST) before the addition ofthe secondary antibodies antimouse IgG peroxidase conjugate(1∶2000; Sigma) or antirabbit HRP conjugate (1∶2000; Chemi-con). Filters were developed with ChemiGlow (Alpha InnotechCorporation); the signal was quantified with the TotalLab100densitometric software (Nonlinear Dynamics).

NBT Binding Assay. NBT staining of proteins electroblotted ontonitrocellulose membranes has been described by Paz et al.(S3). Briefly, quinones and related quinonoid substances, suchas the polyphenol EGCG, catalyze redox cycling at an alkalinepH in the presence of excess glycine as reductant. NBT is reducedto formazan in the presence of oxygen, which stains membrane-bound proteins purple (S3). EGCG staining was quantifieddensitometrically using a photoimager (LAS-3000, Fuji) andAIDA image analysis software (Raytest). Binding saturation datawere normalized and fitted nonlinearly by a single binding sitemodel: Y ¼ ½EGCG�∕Kdþ ½EGCG� to obtain the apparentbinding constant.

Monomer Quantification. Fibrillar αS (50 μM) aggregates wereincubated with EGCG (50 μM) at 37 °C in TBS for 0.2–24 h.Insoluble aggregates were removed by ultracentrifugation(200; 000 × g, 20 min). Supernatants were loaded onto a Superose6 (3.2∕30) gel filtration column (Amersham/Pharmacia) andeluted in TBS using a Smart System FPLC (Pharmacia). Amountsof αS monomers were normalized to peak height of the mono-meric αS protein. Note that unfolded αS monomer eluted at1.75 mL, whereas residual αS adopted a more compact confor-mation in the presence of EGCG (2.2 mL elution vol.)

Generation of Stable Cell Lines. For generation of stable, tetracy-cline-inducible HEK-293 cell lines, wild-type αS protein was sub-cloned into a gateway compatible pcDNA5/FRT/TO vector,according to the manual of the Flp-In system (Invitrogen).

Flp-In HEK-293 host cells contain a single integrated Flprecombination (FRT) site and stably express the tetracyclinerepressor. Flp-In HEK-293 host cells were cotransfected withthe pcDNA5/FRT/TO-αS plasmid and the pOG44 plasmid ex-pressing the Flp recombinase using Lipofectamin 2000 (Invitro-gen) following the manufacturer’s protocol. Transfected cellswere selected with 100 μg∕mL Hygromycin B and Blasticidin Sfor 2 weeks. Five to 10 individual clones were pooled andscreened for tetracycline (4 μg∕mL) inducible regulation of αSprotein using the Western immunoblotting method.

Cell Culture. Stably transfected Flp-In HEK-293 wt αS cells, CHOcells, stably APP-transfected CHO cells (7PA2, D. Walsh), andNeuro2A (N2a) cells were cultured in DMEM (Biochrome)containing 10% FBS and 100 units∕mL penicillin, 100 μg∕mLstreptomycin. HEK-293 wt αS media were additionally supple-mented with Hygromycin B (100 μg∕mL), Blasticidin(15 μg∕mL), and tetracycline (4 μg∕mL); 7PA2 and CHO cellmedia with 11 μg∕mL L-Prolin.

Internalization of Amyloid Fibrils and Fluorescence Microscopy. Aβ-aggregates (final concentration 0 or 1 μM) were internalized into7PA2 and CHO cells using jetPEI transfection agent (Polyplus)according to the manufacturer’s protocol. After 6 h cells wererinsed carefully with DMEM and control samples were taken re-presenting the 0 d time point. Further samples were incubated for3 d in DMEM containing 0 or 20 μM EGCG. After washing withPBS, cells were fixed in 4% paraformaldehyde in PBS, washedagain and permeabilized with 0.2% Triton-X in PBS. After addi-tional washing steps cells were blocked for 40 min in blockingbuffer (1% BSA in PBS) and incubated with the primary β-amy-loid monoclonal antibody 6E10 (1∶500 in blocking buffer, Cov-ance; sig-39320). Nonspecifically bound primary antibodies werewashed off thoroughly with PBS before the addition of AlexaFluor 568 phalloidin (1∶40 in blocking buffer; Molecular ProbesA12380) for actin staining and the secondary antibody Cy5-Affi-niPure Goat AntiMouse IgG (1∶200 in blocking buffer; JacksonImmunoResearch 115-175-003) for 40 min. Cells were washedagain thoroughly with PBS and mounted with prolong gold anti-fade reagent with DAPI (Invitrogen P36935). All images wererecorded with a Zeiss Axiovert 200 Apotome using standardfluorescence filters.

LDH Cell Viability Assay. HEK-293, HEK-293 wt αS, and CHO and7PA2 cells were plated out at a density of 18 × 105 cells∕mL in 96-well plates. Incubation was carried out at 37 °C with 5% (volume)CO2. αS-aggregates (final concentration 1.0 μM) or Aβ42 aggre-gates (final concentration 1.0 μM) were internalized into cellsusing jetPEI transfection agent (Polyplus) according to the man-ufacturer’s protocol. After 6 h cells were rinsed carefully withDMEM and further incubated for 1, 2, or 3 d with the indicatedconcentrations of EGCG. Clear medium solution was transferredinto an optically clear 96-well plate. The cytotoxicity of Aβ42 andαS-aggregates and EGCGwas quantified with the LDH-Cytotoxi-city Assay Kit II (BioVision) and the Tecan plate reader infiniteM200 at OD450.

MTT Metabolic Assay. PC12 cells (rat pheochromocytoma, Amer-ican Type Culture Collection) were cultured in DMEM (GibcoBRL) medium supplemented with 5% FBS, 15% horse serum,100 units∕mL penicillin and 100 μg∕mL streptomycin in a 5%CO2 humidified environment at 37 °C. Cells were plated at adensity of 10,000 cells per well on 96-well plates in 90 μL freshmedium. After 24 h, the αS or Aβ42 protein aggregates wereadded and cells were further incubated for 3 d at 37 °C. Cytotoxi-city was measured utilizing a modified MTTassay kit (Promega).Absorbance values of formazan were determined at 590 nm withan automatic plate reader after 1 h. P-values were calculatedusing the two-tailed T-test.

Western Blotting.CHO and 7PA2 cells were plated out at a densityof 18 × 105 cells∕mL in 96-well plates. Adherent cells weretransfected with Aβ42 aggregates (final concentration 1.0 μM).After 6 h cells were rinsed carefully with Dulbecco’s PhosphateBuffered Saline (DPBS; Gibco) and either directly lysed in 1%NP-40 representing the 0 d time point or further incubated for1, 2, or 3 d in cell culture medium with the indicated concentra-tions of EGCG before cells were washed again with DPBS andlysed in 1% NP-40.

Aliquots of the cell lysates were separated on 15% SDS-PAGEand electrophoretically transferred to a nitrocellulose membrane(Protran; Whatman). Membranes were blocked in milk andstained using anti-Aβ mAb 6E10 (1∶2000; Covance). Nonspeci-fically bound primary antibodies were washed off with TBSTbefore the addition of the secondary antibody antimouse IgG per-oxidase conjugate (1∶2000; Sigma). Filters were developed withChemiGlow (Alpha Innotech Corporation). After washing themembranes were restained with a polyclonal rabbit antiGAPDH

Bieschke et al. www.pnas.org/cgi/doi/10.1073/pnas.0910723107 2 of 12

antibody (1∶2000; produced in house). After several washingsteps the membrane was incubated with the secondary antibodyantirabbit IgG alkaline phosphatase (1∶10000; Sigma) and subse-

quently developed with Attophos reagent [10 mM Attophos(Europa Bioproducts) in 100 mM Tris pH 9.0] and quantifiedusing a photoimager (LAS-3000, Fuji).

1. Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S,

Pastore A, Wanker EE (2008) EGCG redirects amyloidogenic polypeptides into

unstructured, off-pathway oligomers. Nat Struct Mol Biol 15:558–566.

2. Hoyer W, Antony T, Cherny D, Heim G, Jovin TM, Subramaniam V (2002) Dependence

of alpha-synuclein aggregate morphology on solution conditions. J Mol Biol

322:383–393.

3. Paz MA, Fluckiger R, Boak A, Kagan HM, Gallop PM (1991) Specific detection ofquinoproteins by redox-cycling staining. J Biol Chem 266:689–692.

4. Wanker EE, Scherzinger E, Heiser V, Sittler A, Eickhoff H, Lehrach H (1999) Membranefilter assay for detection of amyloid-like polyglutamine-containing protein aggre-gates. Method Enzymol 309:375–386.

5. Chafekar SM, Baas F, Scheper W (2008) Oligomer-specific Abeta toxicity in cell modelsis mediated by selective uptake. Biochim Biophys Acta 1782:523–531.

BA

C

500 nm

00

4

8

0.5 1.51.0Offset (µm)

110 nm

100 nm

t (h)

1

0.2

0.4

0.6

0.8

00

20 40 60 80 100 120

Tht

Flu

or.

/Flu

or.(

t=11

0h)

λ (nm)200 220 240 260

monomer (0 h)

fibril (168 h)

fibril (168 h)

+ 72 h incubation no EGCG

0

1

0.5

0 50 100 150 200

F/F

(175

h)

Time [h]

-15

-10

-5

0

23

[Θ] (

deg

cm/d

mol

)×10

5

Fig. S1. αS-aggregates have β-sheet rich fibrillar structure and bind ThT. (A) Analysis of αS amyloid fibril morphology. αS (50 μM)was incubated for 168 h at 37 °C; aggregates were imaged by negative staining EM (αS-His) and AFM (untagged αS). Identical aggregation products were observed for both His-tagged anduntagged αS preparations. (Inset) Height profile of a single αS fibril reveals helical morphology with a repeat length of approximately 110 nm. (B) αS-aggre-gates that bind ThT are formed over time. αS (50 μM) in TBS (20 μM ThT) was incubated at 37 °C in a fluorescence plate reader and shaken for 5 s every 10 min.(Inset) Aggregation kinetics of αS in TBS at 37 °C with continuous agitation in an overhead rotating shaker (20 rpm). At different time points, aliquots of theaggregation reaction were mixed with 80 ng∕μL ThTand fluorescence was measured. (C) αS-aggregates have a β-sheet-rich secondary structure. αS (50 μM) wasincubated for 7 d in TBS at 37 °C and CD spectra were recorded after 1 h sonication. Spectra of monomeric αS are characteristic of unstructured proteins,whereas αS-aggregates adopt a β-sheet-rich structure after incubation for 168 h. The CD spectrum remains mostly unchanged after 72 h incubation inthe absence of EGCG.

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0

5

10

15

20

NB

Tst

aini

ng(a

rb.u

.)

NoEGCG 1 10 60 120 min

EGCG staining (NBT) time course

ThT fluorescence time course

B

ASolvent

1x EGCG

10x EGCG

3T

hTflu

ores

cenc

e(1

0R

FU

)

00 50

4

8

12

16

t (h)

Fig. S2. αS remodeling and quantification of NBT stained αS-aggregates. (A) Loss of ThT fluorescence after incubation of αS fibrils (50 μM) with EGCG (50 or500 μM) in TBS (20 μM ThT) at 37 °C was monitored for 72 h under intermittent agitation (5 s shaking every 10 min). (B) αS fibrils (50 μM) were analyzed afterEGCG treatment for 1–120 min by filter retardation assay. The addition of EGCG converts SDS-unstable, fibrillar aggregates into SDS-resistant, amorphousprotein aggregates. Aggregates retained on filter membranes were quantified after NBT staining by densitometry using the AIDA software package. Bargraphs show mean values� SD (n ¼ 3).

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A BEGCG

i

ii

αS fibrils green / red

EGCG -induced oligomers

24 h monomer co-aggregation, EGCG

24 h fibril incubation, no EGCG

24 h monomer co-aggregation, no EGCG

1h fibril incubation with EGCG

1h fibril incubation control

green (A488) red (Cy5) overlay

C

DαS-A488

500 nm

20 nm

αS

αS

-A488

αS-Cy5

αS-Cy5+

Fig. S3. EGCG directly remodels αS fibrils without their disassembly into monomers. (A) Fluorescently labeled αS forms fibrils. AFM images of αS fibrils formedfor 7 d from unlabeled, green or red fluorescently labeled monomers, or mixtures of red and green monomers (total monomer concentration 50 μM, 2.5% αS-A488, or 2.5% αS-Cy5), scale bars ¼ 500 nm. (B) Experimental setup: αS fibrils were formed in the presence of labeled αSmonomers (green or red fluorophores);total monomer concentration 50 μM, 2.5% αS-A488 (Green), 2.5% αS-Cy5 (Red). Incubation with EGCG should result in uniformly green or red labeled sphericalaggregates if EGCG directly remodels amyloid fibrils (i) or in doubly labeled aggregates if EGCG induces fibril disassembly/reassembly by binding to soluble αSmonomers (ii). (C) Fluorescence microscopy of labeled αS-aggregates. Labeled αS fibrils (equivalent 50 μMmonomer) were incubated in the presence or absenceof equimolar EGCG for 1 or 24 h (see Fig. 2A for 24 h). EGCG-induced aggregates were predominantly labeled by either the green or the red fluorophores,scale bars ¼ 5 μm. (D) Coincubation of red and green labeled αS monomers (50 μM) results in doubly labeled (Yellow) aggregates, indicating copolymerizationof red and green labeled monomers, scale bars ¼ 5 μm.

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1 h

24 h monomerco-aggregation

fibril remodelling

24 h

EGCG

EGCG

control

control

fluorescence intensity (red)

fluor

esce

nce

inte

nsity

(gre

en)

A

B

Fig. S4. Intensity histogram analysis of fluorescently labeled αS-aggregates remodeled with EGCG. (A) Red and green fluorescently labeled fibrils of αS formedfor 7 d (total monomer concentration 50 μM, 2.5% αS-A488 or 2.5% αS-Cy5) weremixed and incubated with an equimolar concentration of EGCG for 1 h or 24 has in Fig. 2A. Fluorescence microscopy images (n ¼ 30, 10–50 aggregates/image) were analyzed after appearance of aggregates. Two-color intensity histogramsfrom representative images are shown. Pearson coefficients were calculated using fluorescent threshold values shown in yellow. (B) Intensity histograms offluorescence images from αS monomer coaggregation experiments (total monomer concentration 50 μM, 2.5% αS-A488 or 2.5% αS-Cy5). Monomers incubatedfor 24 h show strong correlation of green and red fluorescence signals.

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A

B

monomer quantification

0

0.2

0.4

0.6

0.8

1

0 1 2 3

Elution vol. (ml)

OD

280n

m

unfoldedαS monomer

αS monomer

EGCG treated fibrils, 1 h (10 x OD )280

13%5% 2% 2% 3% 1%

0%

20%

40%

60%

80%

100%

αSmono

cont

rol 0

h

------

-

--

-

MWkDa

00 2424 2424 0.20.2 0.10.1 0.50.5 11 22 44 88

Supernatant Pellet

49

64

82

116

182

37

2619

15

6

agg

control controlEGCG EGCG

αS-EGCG

EGCG

EGCG 0.2

h

EGCG 1 h

EGCG 4 h

EGCG 2 h

EGCG 24

h

incubationtime

rel.

αSm

onom

erpe

akar

ea

Fig. S5. Monomer quantification during αS fibril remodeling. (A) Monomer dissociation during EGCG-induced remodeling of preformed αS fibrils is negligible.Fibrillar αS (50 μM) aggregates were incubated with EGCG (50 μM) at 37 °C in TBS for the indicated times. Insoluble aggregates were removed by ultracen-trifugation (200; 000 × g, 20 min) and supernatants were analyzed by FPLC gel filtration. Amounts of αS monomers were normalized to the monomeric protein.Note: the αS monomer retention time shifts in the EGCG-treated aggregated samples when compared to untreated unfolded αS monomer. No peaks corre-sponding to unfolded αS monomer were detected in the supernatant of EGCG-treated αS-aggregates. (B) EGCG induces the formation of soluble and insolubleSDS-resistant αS-aggregates. Fibrillar αS (50 μM) aggregates were incubated with EGCG (50 μM) at 37 °C in TBS for the indicated times. Soluble and insolubleaggregates were separated by ultracentrifugation (200; 000 × g, 20 min). Supernatant and pellet fractions were analyzed by SDS-PAGE and Western blottingusing the monoclonal antibody αS 211. We observed that EGCG treatment stimulates the formation of SDS-resistant aggregates in the supernatant fraction.This indicates that the aggregates formed in EGCG-induced remodeling reactions are soluble, although they are large in size and do not enter the separatinggel (>200 kDa). However, no SDS-soluble αS monomers were detected after incubation with EGCG.

αS(100nM)

control

EGCG concentration (µM)

0 0.02 0.05 0.1 0.2 0.5 1 2 5 10

Fig. S6. EGCG binds to αS-aggregates Fibrillar αS (100 nM) was incubated with EGCG (20–10 μM) overnight at 37 °C in PBS. Aggregate-bound EGCG wasdetected by NBT staining after filtration (FRA) to determine EGCG binding affinities.

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αS

αS

GAPDH

GAPDH

Protein levels HEK 293 wt-αS

αSfib

rils

+

-

3210 incubation time (d)3210

[EGCG] (µM)

0 20

LDH release HEK 293

LDH

rele

ase

(%of

untr

eate

dce

lls)

1 d

2 d

3 d100

200

300

***

[EGCG] (µM)

incubation time

0 020 20

- αS fibrils + αS fibrils

Protein levels HEK 293

αSfib

rils

+

-

3210 incubation time (d)3210

[EGCG] (µM)

0 20

A

B

C

Tot. Protein(αg/µl)

0.2 0.3 0.5 0.5 0.1 0.2 0.5 0.5

αS

αS

GAPDH

GAPDH

Tot. Protein(αg/µl)

0.2 0.3 0.5 0.6 0.1 0.2 0.5 0.6

Fig. S7. EGCG reduces and detoxifies cellular αS-aggregates Fibrillar αS (1 μM) aggregates were introduced into HEK-293 wt αS cells expressing wild-type αS(A) and HEK-293 cells (B). After 6 h cells were washed and incubated for 0–3 d in the presence of EGCG at the indicated concentrations. SDS-unstable αS-aggregates, which dissociate tomonomers in SDS gels were quantified byWestern blot after cell lysis with 1%NP-40 using anti-αS antibody. GAPDHwas used asa marker for overall protein concentration. (C) Cytotoxicity of αS-aggregates to HEK-293 cells was assayed by LDH release. LDH signals were normalized to cellsneither treated with αS-aggregates nor treated with EGCG and incubated for 3 d. Values represent means� SD, n ¼ 8; *** P < 0.0005.

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O

OH

HO R1

R2

OH

OH

Catechine (C)

R1:

R2: -OH

OH

OH

Epi-catechine (EC)

R1:

R2: -OH

Epi-gallocatechine (EGC)

OH

OH

OH

R1:

R2: -OH

O

HO OH

OH

O

OH

OH

OH

R1:

R2:

O

HO OH

OH

O

OH

OH

OH

R1:

R2:

Gallocatechine gallate (GCG)

Epi-gallocatechine gallate (EGCG)

C

GC

G

EG

CG

EG

C

EC

GEC

0

0.5

1

C

1x

5x

Rel

.ThT

sign

al(2

0h)

B

A

control

EGCG

GCG

ECG

EGC

EC

C

ThT binding SDS- res. agg.

mAb 6E10

Fig. S8. Green-tea catechins remodel Aβ42 fibrils (A) Structures of green-tea catechins. (B) Loss of ThT fluorescence of fibrillar Aβ42 (15 μM) after 24 hincubation with catechine (C), epicatechine (EC), epigallocatechine (EGC), gallocatechine-gallate (GCG), or EGCG. (C) Detection of SDS-resistant aggregatesby FRA after Aβ42 fibril remodeling for 24 h with chemical compounds. The gallate group in ECG, GCG, and EGCG is essential for inducing SDS-resistance.

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7PA2

CHO

0 days + Aβ 3 days + Aβ 3 days + Aβ + EGCG

0 days + Aβ 3 days + Aβ 3 days +Aβ + EGCG

anti-Aβ

dapi

merge

anti-Aβ

dapi

merge

50µm

B

A

50µm

Fig. S9. Analysis of Aβ42 aggregates in 7PA2 and CHO cells by indirect immunofluorescence microscopy. Aβ42 was internalized into 7PA2 or CHO cells for 6 h.Overview images of IF microscopy show loss of Aβ aggregates (mAb 6E10, Red) in 7PA2 and CHO cells after ECGG (20 μM) treatment for 3 d (Blue): DAPI staining(Green): Phalloidin staining.

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+ +- -

1 d

1 d

2 d

2 d

3 d

3 d

incubation time

Tota

lagg

.S

DS

-res

ista

ntag

g.

[EGCG] (µM) [EGCG] (µM)0 020 20

Aβ aggregates in 7PA2 cells

Aβ fibrils

Aβ fibrils + +- -

Tota

lagg

.S

DS

-res

ista

ntag

g.

1 d

1 d

2 d

2 d

3 d

3 d

incubation time

[EGCG] (µM)0 20

Aβ aggregates in CHO cells

GAPDH

GAPDH

Protein levels 7PA2 cells

Aβ

fibril

s

+

3210incubation

time (d) 3210

APP

APP

GAPDH

GAPDH

Protein levels CHO cells

3210 3210

APP

APP

incubationtime (d)

D

C

B

A

[EGCG] (µM)0 20

Tot. Protein(µg/µl)

Tot. Protein(µg/µl)

-

Tot. Protein(µg/µl)

Tot. Protein(µg/µl)

Aβ

fibril

s

+

-

0.1 0.5 0.4 0.6 0.1 0.5 0.5 0.6

0.1 0.5 0.5 0.7 0.1 0.5 0.5 0.8

0.2 0.6 0.7 0.9 0.2 0.6 0.7 0.9

0.2 0.6 0.7 1.0 0.2 0.6 0.7 1.0

Fig. S10. EGCG treatment increases SDS resistance but reduces overall cellular Aβ42 aggregates Aβ42 (1.0 μM) aggregates were introduced into 7PA2 cells (Aand C) or CHO cells (B and D) by PEI treatment. After 6 h cells were washed and harvested or incubated for 1–3 d in the presence or absence of 20 μM EGCG. (Aand B) NP-40-resistant (total) aggregates as well as SDS-resistant aggregates were quantified by FRA after cell lysis using anti-Aβ antibody 6E10. (C andD) EGCGtreatment has little impact on cellular APP production. Levels of endogenous APP were quantified by Western blotting using anti-Aβ antibody 6E10. GAPDHwas used as a marker for overall protein concentration. Total protein concentrations of utilized cell lysates as indicated.

Bieschke et al. www.pnas.org/cgi/doi/10.1073/pnas.0910723107 11 of 12

A

C

B SDS-resistant aggregates

no EGCG

1x EGCG

5x EGCG

10 µl30µl

10 m

in

10 m

in

24 h

24 h

Aβ42 oligomers

500 nm

10 nm

3 nm

5 nm

500 nm

500 nm

0 h

control 24 h

EGCG 24 h

Metabolic activity PC12 cells

MT

Tre

duct

i on

(%of

untr

eate

dce

ll s)

70

80

90

100

oligo control

no EGCG

EGCG

**

Fig. S11. EGCG remodels Aβ42 oligomers into benign SDS-resistant structures. Aβ42 oligomers were prepared according to (S5). (A) Oligomeric Aβ42 (equivalent15 μM monomer) was incubated with EGCG (15 μM) for 24 h in PBS at 37 °C. Aggregate morphologies were monitored by atomic force microscopy. EGCG alsoprevented the aggregation of Aβ42 oligomers into protofibrillar and short fibrillar structures as observed in untreated samples after 24 h. Scale bars ¼ 500 nm(B) Oligomers (equivalent 15 μM Aβ42) were incubated with EGCG (15 or 75 μM) for 24 h at 37 °C in PBS and SDS-resistant aggregates were detected by FRAusing mAb 4G8. EGCG stimulates the formation of SDS-stable structures in a concentration-dependent manner. (C) Oligomers (equivalent to15 μMAβ42 mono-mer) were incubated with EGCG (15 μM) for 1 h at 37 °C in PBS, diluted into cell culture medium (1.5 μM Aβ42) and added to PC12 cells. Metabolic activity wasmonitored after 3 d by MTT reduction. Values represent means� SD (n ¼ 3) normalized to cells neither treated with Aβ nor with EGCG; ** P < 0.001.

Bieschke et al. www.pnas.org/cgi/doi/10.1073/pnas.0910723107 12 of 12