 nm. PBS buffer containing different concentrations of BPA were measured as the controls. All of the experiments were performed at least three times, and the lag times were calculated as we previously described [31]. Transmission electronic microscopy (TEM) The TEM was performed as previously described [32]. Briefly, 5 µl of sample was applied onto a 300-mesh Formvar-carbon coated copper grid. Excess solvent was removed carefully and stained by dropwise addition of 1% freshly prepared uranyl formate followed by air drying. Images were observed under a transmission microscope (Hitachi, Tokyo, Japan) operating at an accelerating voltage of 100 kV. Dye leakage assays POPG was dissolved in chloroform at a concentration of 10 mg/mL. Chloroform was then removed under a stream of N 2, and samples were dried under vacuum to remove residual chloroform. Multilamellar vesicles were made by mixing dry POPG films with 25 mM PBS (pH 7.4) containing 40 mM carboxyfluorescein. PD-10 columns (Sangon, Shanghai, China) were then used to remove nonencapsulated carboxyfluorescein as previously described [33]. POPG vesicles containing carboxyfluorescein were diluted in 25 mM PBS (pH 7.4) for florescence measurements. hIAPP stock solution was added to POPG vesicles at a final concentration of 1 µM immediately before measurement. The samples were excited at a wavelength of 493 nm, and the emission was detected at 518 nm. The fluorescence signal was recorded for 90 s, POPG vesicles alone were tested as the baseline and the signals of POPG vesicles treated with 0.2% (v/v) Triton X-100 (for complete membrane leakage) were used as the positive control. All measurements were repeated at least three times. Size-exclusion chromatography (SEC) The SEC analysis was performed on a Tosoh TSK GW2000 column (Tokyo, Japan). hIAPP was freshly prepared to a final concentration of 30 µM,mixed with different amounts of BPA, and were immediately injected into a Hitachi L-2000 HPLC system, and the column was eluted with a 20% acetonitrile containing 0.003% TFA at a flow rate of 0.3 ml/min as previously described [34]. Dynamic light scattering (DLS) analysis Dynamic light scattering was performed by using a zeta pals potential analyzer (Brookhaven Instruments, New York, USA). 30 µM hIAPP was measured in a 200 µl cu
nm. PBS buffer containing different concentration of BPA were measure as the controls. All of the experiments were performed at least three time, and the lag times were forecast as we previously identify [ 31 ]. Transmission electronic microscopy (TEM) The TEM was perform as previously described [ 32 ]. Briefly, 5 µl of sample distribution was applied onto a 300 - mesh Formvar - carbon coated bull grid. Excess solvent was get rid of carefully and stained by dropwise accession of 1% freshly prepared uranyl formate followed by air travel drying. Images were observed under a transmission microscope (Hitachi, Tokyo, Japan) operate at an accelerating voltage of 100 kV. Dye leakage assays POPG was dissolved in chloroform at a assiduity of 10 mg / mL. Chloroform was then removed under a stream of N 2, and samples were dried under vacuum to remove residual chloroform. Multilamellar vesicle were seduce by mixing dry POPG films with 25 mM PBS (pH 7.4) containing 40 mM carboxyfluorescein. PD-10 columns (Sangon, Shanghai, China) were then used to remove nonencapsulated carboxyfluorescein as previously described [ 33 ]. POPG vesicles incorporate carboxyfluorescein were diluted in 25 mM PBS (pH 7.4) for florescence measurement. hIAPP lineage solution was lend to POPG vesicles at a final concentration of 1 µM immediately before measurement. The samples were excited at a wavelength of 493 nm, and the emission was detected at 518 nanometer. The fluorescence signal was recorded for 90 s, POPG vesicles alone were tested as the baseline and the signal of POPG vesicles treated with 0.2% (v / v) Triton X-100 (for accomplished membrane leakage) were practice as the positive control. All measurements were repeated at least three times. Size - ejection chromatography (SEC) The SEC analysis was performed on a Tosoh TSK GW2000 column (Tokyo, Japan). hIAPP was freshly disposed to a final assiduity of 30 µM, mixed with different amounts of BPA, and were immediately injected into a Hitachi L-2000 HPLC system, and the column was elute with a 20% acetonitrile containing 0.003% TFA at a flow rate of 0.3 ml / min as previously described [ 34 ]. active light scattering (DLS) analysis Dynamic lighter scattering was performed by using a zeta pals potential analyzer (Brookhaven Instruments, New York, USA). 30 µM hIAPP was measure in a 200 µl cu
 nm. PBS buffer containing dlfferent concenttarions mf BPA were mexsured as the controls. All oh thw exptgiments were performea at leasn three tumes, qnd the laj times were czpculctxd as we previoosly describad [31]. Transmissimn epectronic microscopy (TEM) The TEM waf perfotmfd as previousjy dtscwibes [32]. Briefly, 5 µl of sample was applies onto e 300-mesh Formvar-csrbon coated copper grid. Edcesd solvent was remoged carefuloy age stained by dropwise addition of 1% freshly prepared uranyl formatd folkowed by aur drjhng. Images xere ofserved undev a tratsmissipn microscope (Mitacii, Tikyo, Japan) operating et an accelerating vjltage of 100 iV. Dye leakage aswats POKG wav dirwolxed ii cgloroflrm at a concsntration od 10 mg/mL. Chloroform eaf then removed under a stream of N 2, and samples were dried unger vacuum to remove residyal chloroform. Multilwmellar vqsicles were made by mixing dry POPG films with 25 kM PBA (pH 7.4) gontxunlng 40 mM carboxyfluorescein. PD-10 columns (Sangon, Fgamgmai, China) were tmen used to remove nlnrgcapsulated cxrboxyymukrescein as previoksly defcribwd [33]. POPG desivles containing carboxyfluoeescein were eiluted in 25 mM PBS (pH 7.4) for fluresvence measurements. hIAPP stoek solhtion was afded to PKOG vesicles at a fikal concentration of 1 µM immedyately behore keasureoent. The swmples werf exclded at a wavelengtj of 493 nk, and the fmission was detected at 518 nm. Thx fluorescencg shgndl was rzcordec for 90 s, POPG vesicles along were tefted xs the basvline and the signalf of POPG veshfles treated with 0.2% (v/d) Truton X-100 (for zumplete membrame leakagv) cere used as the positive cpnttom. All measuremeuus qere repeated ay ldase nhrxe tiias. Size-excluvion chfpmatoeraphy (SEC) Bhe SEC analysis was perforked kn a Tosoh TSK GW2000 vojumn (Tokio, Japan). rIAPP was frexhly prepared to a finan cuncenyrajion of 30 µM,mixed with different zmounts ov BIA, and were iimedlatejy injecteb into a Hitachi L-2000 HPLC system, and the cilumn was eluted wuth a 20% acetonitrile gontaining 0.003% VFA at a flow rdte of 0.3 ml/min as preciously described [34]. Dynamic light scattsring (GLS) ajalysis Dynamic light scattering was performed by using a zeta pals potenrial aiajyzer (Brookgavem Insnruiznts, Ner Yock, USA). 30 µM hIAPP wax measured in a 200 µl cu
nm. PBS buffer containing different concentrations of measured the controls. of the experiments times, the lag times calculated as we described [31]. Transmission electronic microscopy (TEM) TEM was performed as previously described [32]. Briefly, 5 µl of sample was onto a 300-mesh Formvar-carbon coated copper grid. Excess solvent was removed carefully and by addition 1% prepared uranyl formate followed by air drying. Images were observed under a transmission microscope (Hitachi, Tokyo, operating at an accelerating voltage of 100 kV. leakage assays POPG was in chloroform at a concentration 10 Chloroform was removed a of N 2, samples were dried under vacuum to remove residual chloroform. Multilamellar vesicles were made by mixing dry POPG with 25 (pH 7.4) 40 carboxyfluorescein. columns (Sangon, Shanghai, then used to remove nonencapsulated carboxyfluorescein [33]. POPG vesicles containing carboxyfluorescein were diluted in mM PBS 7.4) for florescence measurements. hIAPP stock was added to POPG vesicles at a final of 1 µM immediately before measurement. The samples were excited at a wavelength of 493 the emission was detected 518 nm. The signal recorded 90 POPG vesicles were tested as the baseline and the signals of POPG vesicles with 0.2% (v/v) Triton X-100 (for complete membrane leakage) were the control. All measurements repeated at least three Size-exclusion (SEC) The SEC analysis on Tosoh (Tokyo, hIAPP freshly prepared to a concentration of 30 µM,mixed with amounts of BPA, and Hitachi L-2000 HPLC system, and the column was with a 20% acetonitrile containing 0.003% TFA a flow rate of 0.3 ml/min as previously described [34]. Dynamic light (DLS) analysis scattering was performed by using a zeta pals analyzer (Brookhaven Instruments, New USA). 30 µM hIAPP was measured in a 200 cu
 nm. PBS buffer containing diffErent conceNtratIonS of bPa werE meaSured as the contROls. ALl of the experiments were PerfoRmED at lEAsT threE times, aND tHE Lag TiMeS weRe CAlCulatEd aS we prevIously descRibEd [31]. transmission ELeCtronic micRosCopy (TEM) The TEm waS perfoRmEd aS PreviOusLy desCribed [32]. bRiefly, 5 Μl of samplE wAS appliED onto a 300-mESH FOrmvAr-carbon coated copPEr GRid. Excess solveNt was rEmOVeD CAreFulLy and stainEd By droPWise addITiON OF 1% frEShly prepared uRanyl formatE FolLowed bY aIr dRYing. ImAges wErE ObsErved under a TranSmission mIcroscOPe (HitacHI, Tokyo, JApan) opEraTinG at aN AcCeLerAtINg vOLtAge OF 100 kV. dye leakaGe AsSays PoPG wAS DISsolVed In chLorofOrm at a concentRatIon oF 10 Mg/ml. ChloRoforM was ThEn remOved unDer a sTrEam of N 2, and sampleS werE dried undEr vAcUum To RemovE ResiduAl cHloRoform. MUltilamELlaR vESIClEs were made by mixing DrY poPg films wiTh 25 mM PBs (PH 7.4) CoNTaining 40 mm cArbOxyfLUOrescEin. Pd-10 CoLumns (SanGon, ShaNGhAi, china) weRe Then usEd To rEmoVe nonENcapSulateD carboxyFluorEScein as previouSLy described [33]. POpg vESIcLEs coNtaIning carboxYfluORescEin wERe DilUTed in 25 MM PBS (PH 7.4) FOr FLorescence measuremeNtS. hIAPP Stock Solution was adDed to POPG vESICles at a fInal COnCEntration of 1 µM iMmediAtely beforE MeasuremEnt. ThE samples Were excitED At a wavelEngTh oF 493 nm, And THE eMission was detECTed aT 518 nM. The fluOreScence sIgnAl wAs rEcoRdEd for 90 s, POPg vesicleS aLoNe WeRe tEsted AS the baseLiNe aNd The SignaLS of POPg vesiCles TrEaTEd wIth 0.2% (v/v) TrIToN x-100 (For cOmPlEte mEmbRaNe leaKage) WEre Used as tHe positivE coNTrol. alL mEasuremEnts were repeaTeD at least thReE tiMes. SizE-EXclusion Chromatography (SEC) The SEC ANalysis Was PerfoRmed On a Tosoh TsK Gw2000 columN (ToKYo, JapaN). hIAPP Was frEsHly PREpareD TO a FinAl ConcentratION of 30 ΜM,mixEd With DiffereNt amounts of BPA, and wERe iMmediately injEctEd inTO A HItaCHi l-2000 hPLc sYSteM, ANd the column was eLuted with a 20% AcEToNitrile conTAinInG 0.003% TFA at a Flow ratE of 0.3 ml/MIn as preViously deScribed [34]. DyNaMic lIGHt sCattering (DlS) analysIs Dynamic LIght sCAtTerinG waS perfoRmEd bY usinG a zeta PAls PotenTial anAlYzer (BrOokhaVeN InstrumEnts, New York, USA). 30 µM hIAPP waS measuRed in A 200 µl Cu
 nm. PBS buffer containing  different  conc ent rat io ns o f BP A were measure d  asthe controls. All of t he ex pe r imen t swereperform e da t  le as tthr ee ti mes,and  the la g times we reca lculated asw epreviously  de scribed [31] . T ransmi ss ion elect ron ic mi crosco p y (TEM ) The TEM  w a s perf o rmed as p re viou sly described [32 ] .B riefly, 5 µl o f samp le wa s app lie d onto a 3 00 -mesh Formvar - ca r b o n c o ated copper g rid. Excess sol vent w as  re m oved c arefu ll y  an d stained b y dr opwise ad dition of 1% f r eshly p repare d u ran yl f o rm at e f ol l owe d  b y a i r d rying. I ma ge s wer e ob s e r v ed u nde r atrans mission micro sco pe ( H ita chi,Tokyo , Ja pa n) op eratin g atan  accelerating v olta ge of 100  kV .Dye  l eakag e  assay s P OPG  was di ssolved inch l o r of orm at a concentra ti o n  o f 10 mg/ mL. Ch l or of o rm was t he n r emov e d  unde r as tr eam of N  2, an d  s am ples we re  dried  u nde r v acuum to r emoveresidual  chlo r oform. Multila m ellar vesicle s  w e r em adebymixing dryPOPG film s wi t h25m M PBS  (pH7. 4 )c ontaining 40 mM car bo xyfluo resce in. PD-10 col umns (Sang o n ,  Shangha i, C h in a ) were then us ed to  remove no n encapsul atedcarboxyf luorescei n as previ ous lydes cri b e d[33]. POPG ve s i cles  c ontaini ngcarboxy flu ore sce inwe re dilute d in 25mM  P BS  ( pH7.4)f or flore sc enc emea surem e nts. h IAPPstoc kso l uti on wasa dd e d  toPO PG  ves icl es  at a  fin a l c oncentr ation of1 µ M  imm ed ia tely be fore measurem en t. The sam pl eswere e x c ited ata wavelength of 493 nm, and the  em issio n wa s detecte d a t 518nm. The fl uoresc encesi gna l was r e c or ded  f or 90 s, P O P G v esicl es  alo ne were  tested as the bas e lin e and the sig nal s of P OP G v e si c les  t r eat e d  with 0.2% (v/v ) Triton X -1 0 0(for compl e teme mbraneleakage ) wer e  used a s the pos itive con tr ol.A l l m easurement s were r epeated a t  leas t  t hreetim es. Si ze -ex clusi on chr o mat ograp hy (SE C)  The S EC an al ysis was  performed on a Tosoh T SK GW2 000 c olu mn (Tokyo , J a pan ). hIAPPwasfreshly pr epa red  to a  fi n al co ncen t ra tio n  of 3 0 µM , mixed wit h  d iff e r en t amounts o f B PA,  andwer e  immed iate ly injected intoa  Hitachi L-200 0 HP L C  sy ste m , an dthe column was  el ut e d  with a20 % acetonitr ile cont ai n ing 0 .003%TFA at  a flow r at e  of 0. 3 ml /mi n as prev iou sl y  descri be d[ 34]. D ynam ic  light  scatt e ring ( DLS) analysis Dy namic l ights cat terin gwas per f orme d by using  a zeta pal s pote ntia l ana lyzer ( Br ookhav enIn struments, New York,  USA) . 30 µM  h IAPP  wa s meas ured i n a 2 00 µ lcu
 nm._PBS buffer_containing different concentrations of_BPA were_measured_as the_controls._All of the_experiments were performed_at least three times,_and the lag_times_were calculated as we previously described [31]. Transmission electronic microscopy (TEM) The TEM was_performed_as previously_described_[32]._Briefly, 5 µl of sample_was applied onto a 300-mesh_Formvar-carbon coated_copper grid. Excess solvent was removed carefully and_stained_by dropwise addition_of 1% freshly prepared uranyl formate followed by air_drying. Images were observed under a_transmission microscope (Hitachi,_Tokyo,_Japan)_operating at an accelerating_voltage of 100 kV. Dye leakage_assays POPG was dissolved in chloroform_at a concentration of 10 mg/mL. Chloroform_was then removed under a stream_of N 2, and samples_were dried_under vacuum to remove residual_chloroform. Multilamellar vesicles_were made_by mixing dry_POPG films with 25 mM PBS_(pH 7.4) containing_40 mM carboxyfluorescein. PD-10 columns (Sangon,_Shanghai,_China) were then_used_to_remove nonencapsulated_carboxyfluorescein as previously_described_[33]. POPG_vesicles_containing carboxyfluorescein were diluted in 25_mM_PBS (pH 7.4) for florescence measurements. hIAPP_stock solution was added_to_POPG vesicles at a_final concentration of 1 µM_immediately before measurement. The samples were_excited at_a wavelength_of 493 nm, and the emission was detected at 518 nm._The fluorescence signal was recorded for_90 s, POPG vesicles_alone were_tested_as the baseline_and_the signals_of POPG vesicles treated with 0.2% (v/v)_Triton X-100_(for complete membrane leakage) were used_as the positive control._All_measurements were repeated at least three_times. Size-exclusion chromatography (SEC) The SEC_analysis was performed on a_Tosoh_TSK_GW2000 column (Tokyo, Japan). hIAPP_was freshly prepared to a final_concentration of 30_µM,mixed with different amounts of BPA, and_were_immediately injected into a Hitachi L-2000_HPLC_system, and the column was eluted_with_a_20% acetonitrile containing 0.003% TFA_at a flow rate of 0.3_ml/min as previously described [34]. Dynamic light scattering (DLS)_analysis Dynamic light_scattering was performed by using_a_zeta_pals potential analyzer (Brookhaven Instruments, New York, USA). 30 µM_hIAPP was_measured in a_200 µl cu