Supporting InformationKlein et al. 10.1073/pnas.1209811110SI MethodsImaging Quantitation. Colocalization of two molecules was de-termined using the JACoP plugin for ImageJ. Triple colocali-zation of PSD95, Sam68, Bassoon, or β-actin (actb) mRNA wasdetermined by measuring the colocalization of two molecules(using JACoP), and determining the extent of colocalized spots(using the RG2B plugin) to the third molecule or by using theBlobprob ImageJ plugin (1). Blobprob calculates Mander’s co-efficients and their statistical significance by comparing the cal-culated colocalization with the colocalization of the total numberof spots observed, randomly placed throughout the image. Allthe reported triple colocalization values have a P value <0.005.

Lentiviral Vectors and Blocking Oligonucleotides. pTRIP contains anH1 promoter derived from pSUPER vectors to drive shRNA ex-pression and a GFP construct driven by an elongation factor 1αpromoter to identify infected neurons. We designed two shRNAstargeting the 3′ UTR of Sam68: 1,441 bp [5′- GTTATGAGCAA-ACTTGTTACT (shS68#1)] and 1,499 bp [5′- CAACAGACAA-GTAATTGTCTA (shS68#2)]. In the case of overexpression ofSam68, we subcloned either full-length Sam68 or Sam68 lackingthe KH domain (ΔKH-S68) into a FUGW vector (2). Viruses weregenerated by triple transfection of pTRIP or FUGWvector, pCMV-dR8.2, and pMD2.G into 293FT cells (Invitrogen). The media fromthese cells were harvested and concentrated. We determined viraltiters by transducing primary neuronal cortical cultures plated on12- or 24-well plates with increasing amounts of lentivirus-containing293FT-derived medium. These viruses were infected into primaryneuronal cultures at 14 d in vitro (DIV), and experiments wereperformed 7 d later. We generated two sets of oligos targeting theSam68-binding site (5′-A*GT GA*C TAU* TAA A*AA AA*CCAA* A- 3′ and 5′-A*AA CA*A TGU* ACAA*TCAA*AGTC*C-3′)(S68/actb oligo), one targeting the Zip code-binding protein1 (ZBP1)-binding site (5′-C*GC AA*G TTA* GGT U*TT GU*CAA-3′)(ZBP1 oligo), and a scrambled (scr) control (5′-G*CGTC*ACCA* ATG C*GT TA*A TGG*-3′) (scr oligo) (* indicates 2′-O-methyl base.) We generated a second set of Sam68- and ZBP1-blocking oligos identical to those above but labeled with Cy3 or Cy5fluorophores, respectively, to determine transfection efficiencies.Oligos were transfected into primary neuronal cultures at DIV18using Lipofectamine 2000 (Invitrogen) according to the manufac-turer’s instructions. Experiments were performed 4 d later.

Neuronal Imaging and Western Blots. For imaging, cells were fixedusing 4% (vol/vol) paraformaldehyde for 5 min and permeabilizedusing 0.2% Triton X-100 for 5 min. Antibodies used were Sam68(sc-333 rabbit; 1:200; Santa Cruz Biotechnology); PSD95 (K28/43; 1:2,000; UC Davis/National Institutes of Health NeuroMabFacility); MAP2 (CPCA-MAP2; 1:500; EnCor Biotechnology);actb (1:1,000; Cell Signaling Technology); N-cadherin (1:1,000;BD Biosciences); ZBP1 (polyclonal rabbit; 1:1,000; gift fromRobert Singer, Albert Einstein College of Medicine, NY); Syn-aptic vesicle 2 (SV2; 1:100; Developmental Studies HybridomaBank); Bassoon (1:1,000, Genetex); c-FOS (1:1,000; Cell Signal-ing Technology); and rpS3 (1:1,000; Cell Signaling Technology).Western blots/membranes were incubated overnight at 4 °C withprimary antibodies used at the concentrations indicated in 1%BSA/PBS and processed according to standard conditions.

Subcellular Fractionation for Postsynaptic Densities and Nuclei andSynaptopolysomes. Postsynaptic densities (PSDs) were purified asdescribed (3). Brain tissue was Dounce homogenized in a 0.32-M

sucrose solution and centrifuged at low speed to remove heavycellular components. The supernatants then were centrifuged at30,000 × g to collect the light membrane fraction. After re-suspension, the pellets were layered on top of a 0.85-M, 1-M, and1.2-M sucrose step gradient and centrifuged at 82,500 × g for 2 h.The synaptosomal fraction was collected at the 1-M/1.2-M su-crose interface and pelleted down at 45,000 × g for 30 min. Thepellet was resuspended in pure ice-cold water to induce hypo-osmotic shock and then was repelleted. The lysed synaptosomesthen were resuspended in an equal volume of a 1% Triton X-100, 0.32-M sucrose solution to remove membranes and thenwere repelleted by centrifugation. The resuspended pellet waslayered on a final sucrose step gradient of 1-M, 1.5-M, and 2-Msucrose and centrifuged at 200,000 × g for 2 h, and the PSDfraction was collected at the 1.5-M/2-M sucrose interface. ThePSDs were extracted once more with 0.5% Triton X-100 to ob-tain the “two-Triton” PSD fraction. Nuclei were purified by ex-tracting the initial heavy cellular fraction using 0.5% Nonidet P-40, layering this fraction on top of a 2.0-M sucrose solution, andcentrifugation at 45,000 × g. The pellet contained purified nuclei.To isolate synaptopolysomes, hippocampal synaptosomes were

isolated as described above and then lysed in hypoosmotic buffer[10 mM NaCl, 3 mMMgCl2, 0.05 M sucrose, 0.3% Triton X-100,1 mM DTT, 20 mM Tris·HCl (pH7.5), 100 μg/mL cycloheximide,2 mg/mL heparin]. Lysate was adjusted to 170 mM NaCl with 13mM MgCl2 and layered on top of linear sucrose gradients (15–55%) [25 mM Tris·HCl (7.5 pH), 170 mM NaCl, 13 mM MgCl2,1 mg/mL cycloheximide, 2 mg/mL heparin]. The gradients werespun at 37,000 × g for 2.5 h at 4 °C in an ultracentrifuge, and1 mL fractions were collected. Protein was isolated from thefractions using a standard deoxycholate/trichloroacetic acidprotocol or RNA was isolated using RNeasy (Fermentas) ac-cording to the manufacturer’s instructions.

RT-PCR. Isolated RNA was subjected to DNase (Fermentas) treat-ment for 30 min at 37 °C and then was heat inactivated for 10 minat 65 °C. cDNA reactions were prepared using Maxima RT (Fer-mentas) according to the manufacturer’s instructions with randomhexamers (100 pM). Taq polymerase (Fermentas) was used forsubsequent PCRs with actb-specific primers (Fwd: 5′-tga acc cta aggcca acc gtg a-3′ and Rev: 5′-cga cca gag gca tac agg gac a-3′)

Golgi Staining. In compliance with the Albert Einstein College ofMedicine Institutional Animal Care and Use Committee, KO andWT Sam68mice (a gift from Stéphane Richard, McGill University,Montreal, Canada) (C57/BL6 background) were killed using CO2.Whole brains were removed quickly and immersed in Golgi stainsolution (Golgi–Cox Variant Kit from Cornell Center for Tech-nology) for 10 d. The brains then were incubated in 30% (wt/vol)sucrose for 3 d, followed by sagittal sectioning at 150 μm usinga vibratome (Leica Microsystems). Sections were mounted ontoslides, developed for 10 min, and visualized using an AxioImager(Carl Zeiss, GmBH). Approximately 30–40 z-plane sections wereimaged for primary dendrites in CA1 region of the hippocampus.Contrast for each z-section was enhanced using ImageJ, and theimage stacks were delivered to Imaris (Bitplane) software. Den-dritic spines were reconstructed and counted using the FilamentTracer module.

In Vivo Injections. All animal handling was performed in compli-ance with the Albert Einstein College of Medicine InstitutionalAnimal Care andUse Committee. Self-inactivating lentiviral virus

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encoding GFP in addition to Sam68-specific or scr shRNAs wasdelivered into the DG of live Sprague–Dawley rats [age, postnatalday (P)21–22] by stereotaxic injection. Animals were anes-thetized with 4% isoflurane and maintained on 2% isofluraneanesthesia. Rat hair was clipped, the incision site was scrubbedwith Betadine (Medline) antiseptic, and eye lubricant was ap-plied to prevent corneal dehydration. Throughout the injectionprocedure supplemental heat was provided with a heating pad.The animal’s head was placed in a stereotactic frame, and theanimal, frame, and anesthesia tubing were placed inside a bio-safety cabinet. The location of the DG was determined using astereotactic atlas (from bregma; x, 4.0 mm; y, 3.6mm; z, 2.7 mm),and bilateral holes were opened in the skull at the appropriatecoordinates to allow the passage of the injection needle. Con-centrated viral solution (1.5 μL) was injected into the hippo-campus by means of a 10-μL Hamilton syringe with a 28-guageneedle at a flow rate of 0.15 μL/min. The needle was left in placefor an additional 3 min after injection and then was withdrawngently. The incision was closed with sutures, and lidocaine oint-ment was applied topically as local anesthetic. Animals were in-jected s.c. with a single dose of flunixin (2.2 mg/kg) to limit in-flammation. After injection, animals were placed in a clean cagewith a hand warmer for supplemental heat and were monitoreduntil they recovered from anesthesia. The health of the animalswas monitored continually until they were killed 5–7 d after sur-gery for electrophysiological experiments.

Electrophysiology.Acute brain slices were made using a DTK-2000microslicer (Dosaka EM) or VT1200 S microtome (Leica) in ice-cold cutting solution containing (in mM) 215 sucrose, 20 glucose,26 NaHCO3, 4 MgCl2, 4 MgSO4, 1.6 NaH2PO4, 1 CaCl2, and 2.5KCl. Slices then were transferred to a holding chamber with 50%cutting solution and 50% extracellular artificial cerebrospinalfluid (ACSF) recording solution containing (in mM) 124 NaCl,26 NaHCO3, 10 glucose, 2.5 KCl, 1 NaH2PO4, 2.5 CaCl2, and 1.3MgSO4. After 30-min incubation at room temperature, the me-dium was switched gradually to recording solution for at least 1 hbefore recording. All solutions were equilibrated with 95% O2and 5% CO2 (pH 7.4). Recordings were performed at 32 °C(CA1), or 25 °C (DG), in a submersion-type recording chamberwith temperature-controlling elements (Warner Instruments)and perfusion with ACSF at 1.5–2 mL/min using a peristalticpump (Rainin). ACSF was supplemented with reagents asneeded. Patch-type pipettes (2.7–3.5 MΩ) were filled with in-tracellular recording solution containing (in mM) 131 Cs-glu-conate, 8 NaCl, 1 CaCl2, 10 EGTA, 10 Hepes, 10 glucose, 5 Mg-ATP, and 0.4 Na-GTP (pH 7.25, 291 mmol/kg). Series resistance[typically <10 MΩ for CA1 pyramidal cells or <20 MΩ fordentate granule cells (DGCs)] was monitored throughout ex-periments with a −5 mV, 80 ms voltage step, and cells that ex-hibited a significant (>10%) change in series resistance wereexcluded from analysis. Output signals from whole-cell and fieldrecordings were filtered at 2.4 kHz, acquired at 5 kHz, and storedonline in IgorPro (Wavemetrics).To isolate miniature excitatory postsynaptic currents (mEPSCs),

CA1 pyramidal cells and DGCs were voltage-clamped at −60 mV,and NMDA receptors [25 μM D-(-)-2-amino-5-phosphonopenta-noic acid], GABAA receptors (100 μM picrotoxin), and voltage-gated sodium channels (1 μM; tetrodotoxin) were blocked. Therms noise (typically < 4 pA) was measured in whole-cell mode atthe beginning of each experiment. DGCs with high input re-sistance (>1,000 MΩ) were excluded from the analysis.

Extracellular field potentials were recorded in the CA1 regionof the hippocampus or the DG by stimulating Schaffer collateralsin stratum radiatum or themedial perforant path using monopolarsquare-wave voltage pulses (200 μs) delivered through a stimulusisolator (Digitimer Ltd; DS2A). The stimulating pipette con-tained ACSF, and the recording pipette contained 1 M NaCl.Stimulation and acquisition were controlled by IgorPro customsoftware.

RNA Immunoprecipitations. The RNA immunoprecipitation (RIP)protocol was adapted from ref. 4. Briefly, hippocampi from WTP21 mice were lysed in polysome lysis buffer [100 mM KCl,5 mMMgCl2, 10 mM Hepes (pH 7.0), 0.5% Nonidet P-40, 1 mMDTT, 100 U/mL Ribolock RNase inhibitor (Fermentas), 200 nmVanadyl Ribonucleoside Complex (Fermentas), protease in-hibitor tablet (Pierce)]. The protein concentration was de-termined using a BCA assay (Pierce), and 5 mg of total proteinin 1 mL of lysis buffer was used subsequently for the RIP. Lysatewas spun at 16,000 × g for 15 min, and the supernatant wasprecleared twice with two 200-μL aliquots of washed protein Aagarose beads. One microgram of αSam68 (ab sc-333 rabbit;Santa Cruz Biotechnology) was added to the lysate in addition to100 nm of either the Sam68- or scr-blocking oligo and was al-lowed to incubate overnight at 4 °C. After incubation with theantibodies, protein A agarose beads were added to the lysate for1 h at 4 °C; then the lysate was centrifuged briefly to collect thebeads. Beads were washed four times (5 min each washing) withpolysome lysis buffer at 4 °C and then were washed an additionaltwo times with polysome lysis buffer with 1 M Urea for 5 min at4 °C. mRNA was extracted with TRIZOL according to themanufacturer’s protocol and then was subjected to RT-PCRusing Maxima Reverse Transcriptase (Fermentas), and Dream-Taq MasterMix (Fermentas).

FISH. For FISH experiments, cells were fixed and permeabilized,washed in wash buffer [10% (vol/vol) formamide, 2× SSC] andincubated with 0.5 ng/μL of actb probe (Biosearch Technologiescoupled to tetramethyl rhodamine) or 1 ng/μL oligo(dT) (Cy3) todetect poly(A) mRNA (Gene-Link) in hybridization buffer (10%dextran sulfate, 1 μg/μL Escherichia coli tRNA, 2 mM VanadylRibonucleoside complex, 0.02% acetylated BSA, and 10% form-amide) for 2 h at 37 °C. Cells were washed twice (30 min per wash)with wash buffer and then were washed once with diethylpyr-ocarbonate-treated PBS, blocked with 1% acetylated BSA/PBSfor 1 h, and incubated with primary and secondary antibodies asper standard conditions for immunocytochemistry. Cultures weretreated with bicuculline for 2 h to increase the detectable amountof dendritic actb mRNA.

Quantification of FISH Signal.Neuronal cultures were infected withthe indicated lentiviruses at 14 DIV and were imaged 7 d later.Portions of primary dendrites (10 μm to 60 μm from the soma or10–100 μm from the soma) of infected neurons were straight-ened using the GFP or MAP2 channel (ImageJ). For quantifi-cation of actb puncta, local maxima in the actb channel of thestraightened dendrites were determined using the “find maxima”function of the ImageJ software package. After maxima selec-tion, a binary image was made, and points (puncta per mi-crometer) were counted. For quantification of oligo(dT) signal,the average intensity along the length of the dendrite (intensityper micrometer) was determined.

1. Fletcher PA, Scriven DR, Schulson MN, Moore ED (2010) Multi-image colocalization andits statistical significance. Biophysics Journal 99(6):1996–2005.

2. Dittgen T, et al. (2004) Lentivirus-based genertic manipulations of cortical neuronsand their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci USA101(52):18206–18211.

3. Jordan BA, et al. (2004) Identification and verification of novel rodent postsynapticdensity proteins. Molecular and Cellular Proteomics 3(9):857–871.

4. Peritz T, et al. (2006) Immunoprecipitation of mRNA-protein complexes. NatureProtocols 1(2):577–580.

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Fig. S1. Sam68 antibody (sc-333; Santa Cruz Biotechnology) is specific for immunostaining of Sam68. (A) Lentiviral-mediated knockdown of Sam68 in ratprimary hippocampal neurons using shS68#1 significantly reduces Sam68 signal compared with scr control. Cultures are triple stained for PSD95 (green), Sam68(red), and MAP2 (gray). Images were taken with identical exposure times for each channel. (B) Adjacent neurons on the same coverslip, one infected withshS68#1-expressing virus and the other uninfected. Minimal Sam68 staining (red) is observable in the infected neuron (green from GFP expression). (C )Quantitation of Sam68 signal shows that knockdown greatly reduces Sam68 signal in both the soma and dendrites (somatic Sam68 signal: 18 ± 2.7% of WT,n = 9 neurons; dendritic Sam68 signal: 22 ± 2% of WT, n = 9 dendrites).

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Fig. S2. Knockdown of Sam68 reduces the amount of actb mRNA in the distal dendrite but does not affect overall poly(A) mRNA distribution. (A) Distributionof dendritic actb mRNA puncta identified using FISH along dendrites from neurons infected with control (scr), knockdown (shS68#1), or Sam68 overexpression(S68 over) viruses. The relative distribution of distal dendritic actb mRNA in all three conditions is comparable to the relative distribution of actb mRNA in theproximal dendrite. To quantify the number of actb puncta, dendrites were straightened in the GFP or MAP2 channel, and local maxima were identified andcounted in the actb channel (ImageJ). Actb puncta were binned every 20 μm to generate the graph. (B) Analysis of oligo(dT) FISH in dendrites demonstratesthat knockdown of Sam68 has no effect on the distribution of poly(A) mRNA along the length of the dendrite out to 100 μm from the soma. The intensity ofoligo(dT) signal was quantified along the length of the dendrite and binned every 20 μm. (C) Actb FISH of representative dendrites, 100 μm in length, from 21-DIV hippocampal cultures infected with lentiviruses expressing scr virus, shS68#1, or Sam68 overexpression (S68 over). (D) Oligo(dT) FISH of representativedendrites, 100 μm in length, from 21 DIV hippocampal cultures infected with lentiviruses expressing scr shRNA or shS68#1. (E) RT-PCR quantification of actblevels in hippocampal cultures infected with virus expressing an scr shRNA (shScr), an shRNA directed against the 3′ UTR of Sam68 (shS68#1), or Sam68-GFP (S68over). Levels of actb mRNA also were measured in cultures transfected with an scr 2′-O-methyl oligonucleotide (scr oligo) or with an oligo antisense to theSam68-binding site in the actb 3′ UTR (S68 oligo). Signals from RT-PCRs with actb-specific primers were quantified and normalized to GAPDH cDNA signals. Nochange in the amount of total actb mRNA in hippocampal neurons was observed under any condition. (F) Neuronal cultures were transfected (lipofectamine)with blocking oligos conjugated to Cy dyes. The Sam68-blocking oligo was conjugated to Cy3 (colored green), and the ZBP1-blocking oligo was conjugated toCy5 (colored red). Low- and high-magnification images were used to quantify the amount of neurons containing oligos (84 ± 0.04% of neurons transfected,n = 6 fields of view). If a neuron was transfected, then it almost always contained both oligos (97 ± 0.05%, n = 6 fields of view).

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Fig. S3. Sam68 targets actb mRNA to the PSD. (A) Representative dendrite showing triple colocalization of actb mRNA (red), PSD95 (green), and Sam68 (blue).Sam68 and actb mRNA are clearly visible together at the PSD (white puncta). Dendritic morphology is observed by GFP (false-colored gray). (B) Representativedendrite displaying colocalization of actb with Sam68 (purple puncta), actb with PSD95 (yellow puncta), and Sam68 with PSD95 (cyan puncta). (C) Quantifi-cation of triple colocalization as described in SI Methods (75 ± 4% of colocalized PSD95/actb colocalizes with Sam68; 60 ± 4% of colocalized Sam68/PSD95colocalizes with actb, and 36 ± 1% of colocalized Sam68/actb colocalizes with PSD95; n = 7 dendrites). These analyses indicate that Sam68 is present at themajority of spines that contain actb mRNA. (D) The percentage of Sam68 colocalized with actb mRNA and the percentage of actb mRNA with Sam68 wasquantified using Mander’s coefficients: 40 ± 1% actb colocalized with Sam68; 28 ± 2% Sam68 colocalized with actb. n = 12 dendrites. These analyses indicatethat only a portion of actb mRNA associates with Sam68. Also, the majority of Sam68 likely is not associated with actb mRNA, indicating that it may be as-sociated with other mRNA cargos.

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Fig. S4. Sam68 is not required for somatic translation of actb mRNA. (A) RT-PCR of actb mRNA extracted from polysome fractions taken from hippocampallysates from Sam68-KO and WT littermates. (B) Actb cDNA in each fraction was quantified by densitometry and graphed as a percentage of total actb cDNAfrom all fractions. (C) Real-time PCR for actb of pooled ribonucleoprotein (RNP) and polysomes from Sam68-KO andWT littermates. The first five fractions werepooled as the RNP fraction, and the last five fractions were pooled as the polysome fraction. No appreciable difference between the two genotypes wasobserved.

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Fig. S5. Loss of Sam68 reduces the number of synapses in vivo. (A) Measurement of paired-pulse ratio (PPR) by extracellular field potential recordings fromthe CA1 region of Sam68-KO and WT littermates. (Left) Sample traces from each genotype are displayed. (Right) Quantification of PPR for five differentinterstimulus intervals (ISI) of 10, 20, 30, 100, and 300 ms. No significant difference in PPR was detected between the two genotypes (10-ms ISI: WT, 1.1 ± 0.02,KO, 1.02 ± 0.05; 20-ms ISI: WT, 1.3 ± 0.04, KO, 1.3 ± 0.03; 30-ms ISI: WT, 1.4 ± 0.04, KO, 1.4 ± 0.04; 100-ms ISI: WT, 1.4 ± 0.05, KO, 1.4 ± 0.04; 300-ms ISI: WT,1.2 ± 0.03; KO, 1.2 ± 0.02. n = 5 slices from 3 paired littermates. (B) Whole-cell voltage-clamp recordings of mEPSCs from individual DGCs. (Left) Samplesweeps of mEPSCs from each genotype. (Inset) Composite average of hundreds of mEPSC events reveals similar kinetic profiles between genotypes. (Right)mEPSC frequency is significantly reduced in Sam68-KO animals compared with WT littermates (WT: 0.55 ± 0.03 Hz, n = 12 cells, three animals; KO: 0.41±0.05 Hz, n = 13 cells, three animals). In contrast, loss of Sam68 does not significantly alter mEPSC amplitude (WT: −6.7 ± 0.23 pA; KO: −6.7± 0.29 pA). (C)Measurement of PPR by extracellular field potential recordings from the medial perforant path of paired littermates. (Left) Sample traces from each ge-notype. (Right) Quantification of PPR for five different ISIs (10, 20, 30, 100, and 300 ms). No significant difference in PPR was detected between the twogenotypes (10-ms ISI: WT, 0.94 ± 0.02, KO, 0.96 ± 0.01; 20-ms ISI: WT, 0.9 5 ± 0.02, KO, 0.92 ± 0.06; 30-ms ISI: WT, 0.82 ± 0.06, KO, 0.77 ± 0.02; 100-ms ISI: WT,0.74 ± 0.05, KO, 0.71 ± 0.05; 300-ms ISI: WT, 0.62 ± 0.04, KO, 0.52 ± 0.03; n = 7 slices from 3 paired littermates.

Fig. S6. RT-PCR of Neurexin 1 (Nrxn1) splice isoforms from hippocampal and cerebellar lysates. Primer sequences were taken from Iijima et al. (1). Nosignificant difference was seen in the ratio of spliced to unspliced Nrxn1 mRNA in hippocampal lysates from 3- to 4-wk-old Sam68-KO and WT animals (WT,0.97 ± 0.08; KO, 1.07 ± 0.09; n = 6 animals, P > 0.05. two-tailed Student t test). A decreased ratio was observed in cerebellar lysates from KO animals (WT, 0.18 ±0.02; KO, 0.27 ± 0.02; n = 4 animals, P < 0.05 two-tailed Student t test).

1. Iijima T, et al. (2011) SAM68 regulates neuronal activity-dependent alternative splicing of neurexin-1. Cell 147(7):1601–1614.

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