Histone Deacetylases Promote ER Stress Induced ...

Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 DOI: 10.1159/000489367 © 2018 The Author(s) www.karger.com/cpb online:April April30,30, 2018 Published online: 2018 Published by S. Karger AG, Basel and Biochemistry Published www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium Accepted: February 08, 2018

This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (http://www.karger.com/Services/OpenAccessLicense). Usage and distribution IRUFRPPHUFLDOSXUSRVHVDVZHOODVDQ\GLVWULEXWLRQRIPRGLÀHGPDWHULDOUHTXLUHVZULWWHQSHUPLVVLRQ

Original Paper

Histone Deacetylases Promote ER Stress Induced Epithelial Mesenchymal Transition in Human Lung Epithelial Cells Daishun Liu Honglan Zhu Guichuan Huang

Ling Gong

Shenglan Pu

Yang Wu

Wei Zhang

'HSDUWPHQWRI5HVSLUDWRU\0HGLFLQH7KH)LUVW3HRSOH·V+RVSLWDORI=XQ\L7KH7KLUG$IÀOLDWHG+RVSLWDO of Zunyi Medical College, Institute of Respiratory Diseases in Zunyi, Zunyi, Guizhou, China

Key Words Histone deacetylase • Endoplasmic reticulum stress • Epithelial–mesenchymal transition • 3XOPRQDU\ÀEURVLV Abstract Background/Aims: Epithelial to mesenchymal transition (EMT) is a crucial process involved LQSXOPRQDU\ÀEURVLV7KLVVWXG\DLPHGWRH[SORUHWKHUROHRIKLVWRQHGHDFHW\ODVHV+'$&V  and endoplasmic reticulum (ER) stress in EMT in human lung epithelial cells. Methods: Human lung adenocarcinoma A549 cells were treated with bleomycin and tunicamycin to induce EMT. 7KHSUROLIHUDWLRQRI$FHOOVZDVGHWHFWHGE\077DVVD\7KHH[SUHVVLRQRI+'$&VDQG(07 PDUNHUVZDVGHWHFWHGE\3&5DQG:HVWHUQEORWDQDO\VLV7KHVHFUHWLRQRI7*)DŽDQGFROODJHQ ,ZDVH[DPLQHGE\(/,6$Results: A549 cells switched from a cobblestone-like appearance WR DQ HORQJDWHG ÀEUREODVW OLNH DSSHDUDQFH DIWHU H[SRVXUH WR WXQLFDP\FLQ RU EOHRP\FLQ DFFRPSDQLHG E\ LQFUHDVHG H[SUHVVLRQ RI 1FDGKHULQ į60$ DQG &ROODJHQ , 0HDQZKLOH *53ZDVXSUHJXODWHGLQ$FHOOVH[SRVHGWRWXQLFDP\FLQRUEOHRP\FLQ7KHVHFKDQJHV induced by tunicamycin or bleomycin could be abrogated by 4-PBA. Moreover, tunicamycin DQGEOHRP\FLQSURPRWHGWKHH[SUHVVLRQRI+'$&DQG+'$&DQG+'$&VLQKLELWRU6$+$ abrogated the morphological and biochemical changes in A549 cells. 4-PBA and SAHA LQKLELWHGWKHXSUHJXODWLRQRISXOPRQDU\ÀEURVLVIDFWRUV7*)DŽDQG,/DQGWKHDFWLYDWLRQ of Smad pathway induced by tunicamycin or bleomycin. Conclusions: :HSURYLGHWKHÀUVW evidence that tunicamycin and bleomycin induce ER stress and EMT in lung epithelial cells via the upregulation of HDACs. HDACs inhibitor could inhibit ER stress induced upregulation RI SXOPRQDU\ ÀEURVLV IDFWRUV DQG WKH DFWLYDWLRQ RI 6PDG SDWKZD\ +'$&V LQKLELWRUV DUH SURPLVLQJDJHQWVIRUWKHWKHUDS\RISXOPRQDU\ÀEURVLV © 2018 The Author(s) Published by S. Karger AG, Basel

Introduction

†‹‘’ƒ–Š‹…’—Ž‘ƒ”›ϐ‹„”‘•‹•ȋ  Ȍ‹•ƒ…Š”‘‹…’”‘‰”‡••‹˜‡Ž—‰†‹•‡ƒ•‡‘ˆ—‘™ ‡–‹‘Ž‘‰›Ǥ…‡–Š‡’ƒ–‹‡–•ƒ”‡†‹ƒ‰‘•‡†ƒ•  ǡ–Š‡‹”‡†‹ƒ•—”˜‹˜ƒŽ‹•‘Ž›͵ǦͶ›‡ƒ”•ǡƒ† Daishun Liu

Department of Respiratory Medicine, The First People’s Hospital of Zunyi, Zunyi, Guizhou (China) E-Mail ldslwtg@126.com

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

–Š‡‘”–ƒŽ‹–›”ƒ–‡‹•Š‹‰Š‡”–Šƒƒ›…ƒ…‡”•ȏͳǡʹȐǤ–’”‡•‡––Š‡‡–‹‘Ž‘‰›ƒ†’ƒ–Š‘‰‡‡•‹• ‘ˆ  ‹••–‹ŽŽ‘–…Ž‡ƒ”ǡƒ†–Š‡‡ˆϐ‹…ƒ…›ˆ‘”–Š‡–”‡ƒ–‡–‘ˆ  ‹•Ž‘™ȏ͵ȐǤŠ‡”‡ˆ‘”‡ǡˆ—”–Š‡” —†‡”•–ƒ†‹‰ ‘ˆ ‘Ž‡…—Žƒ” ‡…Šƒ‹• ‘ˆ   ™‹ŽŽ „‡ ‹’‘”–ƒ– –‘ †‡˜‡Ž‘’ ‘˜‡Ž ƒ† ‡ˆˆ‡…–‹˜‡•–”ƒ–‡‰‹‡•ˆ‘”–Š‡’”‡˜‡–‹‘ƒ†–Š‡”ƒ’›‘ˆ  Ǥ Š‡ ’ƒ–Š‘‰‡‹… ’”‘…‡•• ‘ˆ   ‹˜‘Ž˜‡• –Š‡ †‹•–—”„‡† „ƒŽƒ…‡ ‘ˆ –Š‡ ƒ’‘’–‘•‹• ƒ† ’”‘Ž‹ˆ‡”ƒ–‹‘ ‘ˆ ϐ‹„”‘„Žƒ•– ƒ• ™‡ŽŽ ƒ• –Š‡ ‘˜‡”’”‘†—…–‹‘ ‘ˆ ‡š–”ƒ…‡ŽŽ—Žƒ” ƒ–”‹š ȋȌ …‘’‘‡–• •—…Š ƒ• …‘ŽŽƒ‰‡ ȏͶȐǤ ›‘ϐ‹„”‘„Žƒ•– ‹• ƒ ‹’‘”–ƒ– •‘—”…‡ ˆ‘” –Š‡ •›–Š‡•‹• ‘ˆ …‘ŽŽƒ‰‡ǡ ƒ† ‡’‹–Š‡Ž‹ƒŽǦ–‘Ǧ‡•‡…Š›ƒŽ –”ƒ•‹–‹‘ ȋȌ ’Žƒ›• ‹’‘”–ƒ– ”‘Ž‡ ‹ –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ ›‘ϐ‹„”‘„Žƒ•– ȏͷǡ ͸ȐǤ  ”‡ˆ‡”• –‘ –Š‡ ’Š‡‘‡‘ –Šƒ– ‡’‹–Š‡Ž‹ƒŽ …‡ŽŽ• •–‹—Žƒ–‡† „› …‡”–ƒ‹ •–‹—Ž‹ ‘„–ƒ‹ ‡•‡…Š›ƒŽ …‡ŽŽ ’Š‡‘–›’‡• ƒ† ’Š›•‹‘Ž‘‰‹…ƒŽ …Šƒ”ƒ…–‡”‹•–‹…••—…Šƒ•–Š‡…Šƒ‰‡‘ˆ…‡ŽŽ‘”’Š‘Ž‘‰›ˆ”‘ϐŽƒ–‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•‹–‘•’‹†Ž‡Ǧ •Šƒ’‡†ϐ‹„”‘„Žƒ•–•ǡ”‡†—…‡†‡š’”‡••‹‘‘ˆǦ…ƒ†Š‡”‹ƒ†‹…”‡ƒ•‡†‡š’”‡••‹‘‘ˆǦ…ƒ†Š‡”‹ǡ ȽǦǡ‘ŽŽƒ‰‡ ƒ†‘ŽŽƒ‰‡ ȏ͹ȐǤ‘™‡˜‡”ǡ–Š‡”‘Ž‡‘ˆ‹  ‹•—…Ž‡ƒ”Ǥ ‡…‡– •–—†‹‡• •—‰‰‡•– –Šƒ– •‡˜‡”ƒŽ ˆƒ…–‘”• •—…Š ƒ• •‘‹‰ǡ …Š”‘‹… ƒ•’‹”ƒ–‹‘ ƒ† ˜‹”ƒŽ‹ˆ‡…–‹‘…‘—Ž†’‘–‡–‹ƒŽŽ›”‡•—Ž–‹‡†‘’Žƒ•‹…”‡–‹…—Ž—•–”‡••ȋ•–”‡••Ȍ‘ˆ–›’‡ ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ƒ†—Ž–‹ƒ–‡Ž›Ž‡ƒ†•–‘–Š‡ƒ’‘’–‘•‹•ƒ†‘ˆ–Š‡•‡…‡ŽŽ•ȏͺǦ ͳͲȐǤŠ‡•–‹—Žƒ–‹‘„›–—‹…ƒ›…‹ǡ–Šƒ’•‹‰ƒ”‰‹ƒ†„Ž‡‘›…‹…‘—Ž†‹†—…‡•–”‡••ƒ† ’”‘‘–‡‘ˆ ƒ†”‡ƒŽ–—„—Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡ„—–ͶǦ’Š‡›ŽǦ„—–›”‹…ƒ…‹†ȋͶǦȌ ‹Š‹„‹–‡†–Š‡ƒ…–‹˜ƒ–‹‘‘ˆ•–”‡••ƒ†–Š‡‹†—…–‹‘‘ˆȏͳͳǦͳͶȐǤ ‹•–‘‡ †‡ƒ…‡–›Žƒ•‡• ȋ•Ȍ ’ƒ”–‹…‹’ƒ–‡ ‹ –Š‡ ”‡‰—Žƒ–‹‘ ‘ˆ †›ƒ‹… ‡“—‹Ž‹„”‹— •–ƒ–‡ ‘ˆ Š‹•–‘‡ ‘” ‘ǦŠ‹•–‘‡ ƒ…‡–›Žƒ–‹‘Ȁ†‡ƒ…‡–›Žƒ–‹‘ ƒ† –Š‡‹” †‡ƒ…‡–›Žƒ•‡ ƒ…–‹˜‹–› …‘—Ž†„‡‹Š‹„‹–‡†„›Š‹•–‘‡†‡ƒ…‡–›Žƒ•‡‹Š‹„‹–‘”ȋ‹ȌǤ‡…‡–•–—†‹‡•Šƒ˜‡•Š‘™–Šƒ– •ƒ”‡‹˜‘Ž˜‡†‹–Š‡ˆ‘”ƒ–‹‘‘ˆƒ†–Š‡‹†—…–‹‘‘ˆ•–”‡••ȏͳͷǦʹͲȐǤ‘™‡˜‡”ǡ ™Š‡–Š‡”•ƒˆˆ‡…–•–”‡••ƒ†‘ˆŽ—‰‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•†—”‹‰–Š‡†‡˜‡Ž‘’‡–‘ˆ ’—Ž‘ƒ”›ϐ‹„”‘•‹•‡‡†•–‘„‡‡š’Ž‘”‡†Ǥ –Š‹••–—†›ǡ™‡‡’Ž‘›‡†–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹–‘‹†—…‡•–”‡••‹Š—ƒ Ž—‰‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ƒ†—•‡†‹–‘‹˜‡•–‹‰ƒ–‡–Š‡”‘Ž‡‘ˆ•‹–Š‡‘†—Žƒ–‹‘‘ˆ •–”‡••ƒ†‘ˆŠ—ƒŽ—‰‡’‹–Š‡Ž‹—ƒ†‹–Š‡’ƒ–Š‘‰‡‡•‹•‘ˆ’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ Materials and Methods Cell culture and treatment —ƒŽ—‰ƒ†‡‘…ƒ”…‹‘ƒͷͶͻ…‡ŽŽ•™‡”‡’—”…Šƒ•‡†ˆ”‘‡”‹…ƒ›’‡—Ž–—”‡‘ŽŽ‡…–‹‘ȋ ǦǦͳͺͷȌǡƒ†…—Ž–—”‡†‹ ͳ͸ͶͲ‡†‹—•—’’Ž‡‡–‡†™‹–ŠͳͲΨˆ‡–ƒŽ„‘˜‹‡•‡”—ȋ Ȍƒ– ͵͹Ǐ‹ͷΨʹ‹…—„ƒ–‘”Ǥ‡ŽŽ•™‡”‡…—Ž–—”‡†ˆ‘”ʹͶŠǡ•‡”—•–ƒ”˜‡†ˆ‘”ʹͶŠǡ–Š‡–”‡ƒ–‡†™‹–ŠͲǤͷɊ‰ȀŽ –—‹…ƒ›…‹‘”„Ž‡‘›…‹ǡ‘”†‹ˆˆ‡”‡–…‘…‡–”ƒ–‹‘•‘ˆͶǦ‘”‹•—„‡”‘›Žƒ‹Ž‹†‡Š›†”‘šƒ‹…ƒ…‹† ȋȌˆ‘”†‹ˆˆ‡”‡–’‡”‹‘†Ǥ MTT assay ͷͶͻ…‡ŽŽ•™‡”‡•‡‡†‡†‹–‘ͻ͸Ǧ™‡ŽŽ’Žƒ–‡•ƒ–ƒ†‡•‹–›‘ˆͷǡͲͲͲ…‡ŽŽ•Ȁ™‡ŽŽǡ…—Ž–—”‡†‹ƒŠ—‹†‹ϐ‹‡† …Šƒ„‡”ƒ–͵͹Ǐ‘˜‡”‹‰Š–ƒ†–Š‡–”‡ƒ–‡†™‹–Š–—‹…ƒ›…‹‘”„Ž‡‘›…‹Ǥƒ…Š†ƒ›ˆ‘”•‹š…‘•‡…—–‹˜‡†ƒ›•ǡ ˜‹ƒ„Ž‡…‡ŽŽ•™‡”‡‡˜ƒŽ—ƒ–‡†™‹–Šƒ••ƒ›‹–ȋ‹‰ƒǡȌƒ……‘”†‹‰–‘–Š‡ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘•Ǥ ʹͲɊȋͷ‰ȀȌ•‘Ž—–‹‘™ƒ•ƒ††‡†–‘‡ƒ…Š™‡ŽŽƒ†–Š‡’Žƒ–‡•™‡”‡‹…—„ƒ–‡†ƒ–͵͹Ǐˆ‘”ͶŠǡ–Š‡ ͳͷͲɊ™ƒ•ƒ††‡†–‘‡ƒ…Š™‡ŽŽƒ†–Š‡’Žƒ–‡•™‡”‡‹…—„ƒ–‡†ƒ–”‘‘–‡’‡”ƒ–—”‡ˆ‘”ͳͲ‹ǤŠ‡ ƒ„•‘”’–‹‘˜ƒŽ—‡‘ˆ‡˜‡”›™‡ŽŽȋȌ™ƒ•”‡ƒ†ƒ–ͶͻͲ—•‹‰ƒ‹…”‘’Žƒ–‡”‡ƒ†‡”ȋͺͲͲǡ‹‘Ǧ‡ǡȌǤ RT-PCR ‘–ƒŽ™ƒ•‡š–”ƒ…–‡†ˆ”‘ͷͶͻ…‡ŽŽ•„›—•‹‰•‹’Ž—•ȋƒƒ”ƒȌƒ……‘”†‹‰–‘–Š‡‹•–”—…–‹‘•ǡ ƒ†–Š‡‹–‡‰”‹–›‘ˆ–‘–ƒŽ™ƒ•…‘ϐ‹”‡†„›•’‡…–”‘’Š‘–‘‡–‡”Ǥ‘–ƒŽ™ƒ•”‡˜‡”•‡Ž›–”ƒ•…”‹„‡† ‹–‘ … —•‹‰ ”‡˜‡”•‡ –”ƒ•…”‹’–‹‘ ‹– ȋƒƒ”ƒȌǡ ƒ†  ™ƒ• ’‡”ˆ‘”‡† ™‹–Š … ƒ• –Š‡ –‡’Žƒ–‡ —†‡” –Š‡ …‘†‹–‹‘•ǣ ͻͶǏ ˆ‘” ͳ ‹Ǣ ͻͶǏ ˆ‘” ͵Ͳ •‡… Ǧ ͷʹǏ ˆ‘” ͵Ͳ •‡… Ǧ ͹ʹǏ ˆ‘” ͳ ‹ǡ ͵Ͳ …›…Ž‡•Ǣ ͹ʹǏ ˆ‘” ͳͲ ‹Ǥ ŽŽ ’”‹‡”• ™‡”‡ ’—”…Šƒ•‡† ˆ”‘ ‹ˆ‡ ‡…Š‘Ž‘‰‹‡• ȋȌ ƒ† –Š‡ •‡“—‡…‡• ™‡”‡ ƒ• ˆ‘ŽŽ‘™•ǣ ͹ͺ ˆ‘”™ƒ”† ͷǯǦ   Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ   

 Ǧ͵ǯǢ

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

ʹ ˆ‘”™ƒ”† ͷǯǦ 



  

Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ    Ǧ͵ǯǢ ͸ ˆ‘”™ƒ”† ͷǯǦ     

Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ

Ǧ͵ǯǢ Ǧ…ƒ†Š‡”‹ ˆ‘”™ƒ”† ͷǯǦ

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 Ǧ͵ǯǢ Ǧ…ƒ†Š‡”‹ ˆ‘”™ƒ”† ͷǯǦ 



Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ     Ǧ͵ǯǢ ȽǦ ˆ‘”™ƒ”† ͷǯǦ       Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ



 

 Ǧ͵ǯǢ •ƒ†ʹ ˆ‘”™ƒ”† ͷǯǦ

   Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ      Ǧ͵ǯǢ •ƒ†͵ ˆ‘”™ƒ”† ͷǯǦ

  

Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ    

   Ǧ͵ǯǢ  ǦȾͳ ˆ‘”™ƒ”† ͷǯǦ



 Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ

    Ǧ͵ǯǢ Ǧ͵ʹ ˆ‘”™ƒ”† ͷǯǦ  

Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ 

Ǧ͵ǯǢ ǦͳȾ ˆ‘”™ƒ”† ͷǯǦ  Ǧ͵ǯ ”‡˜‡”•‡ ͷǯǦ  

Ǧ͵ǯǢ ȾǦƒ…–‹ ˆ‘”™ƒ”† ͷǯǦ    Ǧ͵ǯ”‡˜‡”•‡ͷǯǦ

 

Ǧ͵ǯǤ ’”‘†—…–•™‡”‡”—„›ʹΨ‰‡Ž‡Ž‡…–”‘’Š‘”‡•‹•ƒ†•–ƒ‹‡†™‹–Š‡–Š‹†‹—„”‘‹†‡Ǥ ‘”“—ƒ–‹–ƒ–‹‘ ‘ˆ  Ž‡˜‡Ž•ǡ ”‡ƒŽǦ–‹‡  ™ƒ• ’‡”ˆ‘”‡† ™‹–Š ȾǦƒ…–‹ ƒ• ‹–‡”ƒŽ …‘–”‘Ž ƒ†  Ž‡˜‡Ž• ™‡”‡ …ƒŽ…—Žƒ–‡† „› –Š‡ ʹǦȟȟ– ‡–Š‘†Ǥ ƒ…Š – ˜ƒŽ—‡ —•‡† ˆ‘” –Š‡ …ƒŽ…—Žƒ–‹‘• ™ƒ• –Š‡ ‡ƒ ‘ˆ –Š‡ –”‹’Ž‹…ƒ–‡ ‡š’‡”‹‡–•Ǥ Western blot analysis ‘–ƒŽ’”‘–‡‹•™‡”‡‹•‘Žƒ–‡†ˆ”‘ͷͶͻ…‡ŽŽ•ƒ†“—ƒ–‹–ƒ–‡†„›„‹…‹…Š‘‹…ƒ…‹†ȋȌ‡–Š‘†ǤͷͲ ρ‰ ’”‘–‡‹• ™‡”‡ Ž‘ƒ†‡† ‘–‘ ͳʹΨ Ȃ  ˆ‘” –Š‡ •‡’ƒ”ƒ–‹‘ ƒ† –”ƒ•ˆ‡””‡† –‘   ‡„”ƒ‡•Ǥ Š‡‡„”ƒ‡•™‡”‡–Š‡‹…—„ƒ–‡†™‹–Š•’‡…‹ϐ‹…ƒ–‹„‘†›ˆ‘” ͹ͺǡȽǦǡʹǡ͸‘”ȾǦƒ…–‹ ȋ‡ŽŽ‹‰ƒŽ‹‰ǡƒ˜‡”•ǡǡȌƒ–Ͷ͑‘˜‡”‹‰Š–ǤŠ‡‡„”ƒ‡•™‡”‡™ƒ•Š‡†ƒ†‹…—„ƒ–‡†™‹–Š …‘Œ—‰ƒ–‡†•‡…‘†ƒ”›ƒ–‹„‘†‹‡•ˆ‘”ʹŠƒ–”‘‘–‡’‡”ƒ–—”‡ǤŠ‡‡„”ƒ‡•™‡”‡‡š’‘•‡†—•‹‰‹– ƒ†–Š‡•‹‰ƒŽ•™‡”‡ƒƒŽ›œ‡†™‹–Š—ƒ–‹–›‘‡ͶǤ͸ʹ•‘ˆ–™ƒ”‡ǤŠ‡‡š’‡”‹‡–•™‡”‡”‡’‡ƒ–‡†–Š”‡‡–‹‡•Ǥ ELISA Š‡•—’‡”ƒ–ƒ–•™‡”‡…‘ŽŽ‡…–‡†ˆ”‘ͷͶͻ…‡ŽŽ•ƒ† ǦȾͳƒ†…‘ŽŽƒ‰‡ …‘–‡–•‹–Š‡•—’‡”ƒ–ƒ–• ™‡”‡‡ƒ•—”‡†—•‹‰‹–•ȋƬ•›•–‡•ǡ‹‡ƒ’‘Ž‹•ǡǡȌˆ‘ŽŽ‘™‹‰–Š‡ƒ—ˆƒ…–—”‡”ǯ•’”‘–‘…‘Ž•Ǥ Statistical analysis ƒ–ƒ ™‡”‡ ‡š’”‡••‡† ƒ• ‡ƒά ƒ† ƒƒŽ›œ‡† „›  ʹʹǤͲ •–ƒ–‹•–‹…ƒŽ •‘ˆ–™ƒ”‡Ǥ ‘’ƒ”‹•‘• „‡–™‡‡ –™‘ ‰”‘—’• ™‡”‡ ’‡”ˆ‘”‡† —•‹‰  ǡ ƒ† …‘’ƒ”‹•‘• ƒ‘‰ —Ž–‹’Ž‡ ‰”‘—’• ™‡”‡ ’‡”ˆ‘”‡†—•‹‰‘‡Ǧ™ƒ›ˆ‘ŽŽ‘™‡†„›—‡›ǯ•’‘•–ǦŠ‘…–‡•–ǤδͲǤͲͷ™ƒ•…‘•‹†‡”‡†•–ƒ–‹•–‹…ƒŽŽ› •‹‰‹ϐ‹…ƒ–Ǥ

Results

Tunicamycin and bleomycin induce EMT of A549 cells ‘”†‡”–‘ϐ‹†–Š‡‘’–‹ƒŽ…‘…‡–”ƒ–‹‘ƒ†–‹‡ˆ‘”–Š‡–”‡ƒ–‡–‘ˆͶͷͻ…‡ŽŽ•„› –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ™‹–Š‘—– …ƒ—•‹‰ ‡š…‡••‹˜‡ …‡ŽŽ †‡ƒ–Šǡ †‹ˆˆ‡”‡– …‘…‡–”ƒ–‹‘• ‘ˆ –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ȋͲǦͳͲǤͲ Ɋ‰ȀŽȌ ™‡”‡ —•‡† –‘ –”‡ƒ– ͷͶͻ …‡ŽŽ• ˆ‘” ͳʹ Šǡ ʹͶ ŠǡͶͺŠƒ†͹ʹŠǡƒ†…‡ŽŽ˜‹ƒ„‹Ž‹–›™ƒ•†‡–‡…–‡†„›ƒ••ƒ›Ǥ‡ˆ‘—†–Šƒ––—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹Š‹„‹–‡† –Š‡ ˜‹ƒ„‹Ž‹–› ‘ˆ ͷͶͻ …‡ŽŽ• ‹ ƒ †‘•‡ ƒ† –‹‡ †‡’‡†‡– ƒ‡” ȋ†ƒ–ƒ ‘– •Š‘™ȌǤ Š‡”‡ˆ‘”‡ǡ ͷͶͻ …‡ŽŽ• ™‡”‡ –”‡ƒ–‡† ™‹–Š –Š”‡‡ †‹ˆˆ‡”‡– …‘…‡–”ƒ–‹‘• ‘ˆ–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ȋͲǤͷǡͳǤͲǡ͵ǤͲɊ‰ȀŽȌˆ‘”ͶͺŠǡƒ†™‡ˆ‘—†…‡ŽŽ•‡Ž‘‰ƒ–‡† ˆ”‘–Š‡…‘„„Ž‡•–‘‡•Šƒ’‡ȋ ‹‰Ǥͳ”‡†ƒ””‘™ȌǤ‡…ƒ—•‡ƒ›…‡ŽŽ•†‹‡†ƒˆ–‡”–”‡ƒ–‡– ™‹–ŠͳǤͲƒ†͵ǤͲɊ‰ȀŽ–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ǡͲǤͷɊ‰ȀŽ–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ ™‡”‡•‡Ž‡…–‡†–‘–”‡ƒ–ͷͶͻ…‡ŽŽ•ˆ‘”ʹͶŠǡͶͺŠƒ†͹ʹŠǡƒ†™‡ˆ‘—†…‡ŽŽ•‡Ž‘‰ƒ–‡†ˆ”‘ –Š‡…‘„„Ž‡•–‘‡•Šƒ’‡ƒ–Ͷͺƒ†͹ʹŠƒˆ–‡”–”‡ƒ–‡–ȋ ‹‰Ǥͳ”‡†ƒ””‘™ȌǤŠ‡…Šƒ‰‡•‹ ͷͶͻ…‡ŽŽ‘”’Š‘Ž‘‰›‹†‹…ƒ–‡†–Šƒ–‡•‡…Š›ƒŽ…Šƒ‰‡‘……—””‡†ƒˆ–‡”–—‹…ƒ›…‹ƒ† „Ž‡‘›…‹–”‡ƒ–‡–Ǥ   ‘ ˆ—”–Š‡” ‡š’Ž‘”‡ ™Š‡–Š‡” –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡  ‘ˆ ͷͶͲ …‡ŽŽ•ǡ ™‡†‡–‡…–‡†–Š‡‡š’”‡••‹‘‘ˆǦ”‡Žƒ–‡†‘Ž‡…—Ž‡•‹ͷͶͻ…‡ŽŽ•ƒˆ–‡”–—‹…ƒ›…‹ƒ† „Ž‡‘›…‹–”‡ƒ–‡–Ǥ‡ˆ‘—†–Šƒ–‡š’”‡••‹‘‘ˆ‡•‡…Š›ƒŽƒ”‡”Ǧ…ƒ†Š‡”‹

1823

Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

Fig. 1. —‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡  ‹ ͷͶͻ …‡ŽŽ•Ǥ ǣ Šƒ•‡ …‘Ǧ –”ƒ•– ‹ƒ‰‡• ‘ˆ ͷͶͻ …‡ŽŽ• –”‡ƒ–‡† ™‹–Š ǡ  ‘” ˜‡Š‹…Ž‡ …‘–”‘Ž ˆ‘” Ͷͺ ŠǤ ǣ Šƒ•‡ …‘–”ƒ•– ‹ƒ‰‡• ‘ˆ ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–ŠͲǤͷ Ɋ‰ȀŽ‘”ˆ‘”ͲǡʹͶǡ Ͷͺƒ†͹ʹŠǤƒ‰‹ϐ‹…ƒ–‹‘ǣ šʹͲͲǤǡǣǦƒƒŽ›•‹• ‘ˆ Ǧ…ƒ†Š‡”‹ǡ ȽǦ ƒ† Ǧ…ƒ†Š‡”‹  Ž‡˜‡Ž• ‹ ͷͶͻ …‡ŽŽ• –”‡ƒ–‡† ™‹–Š  ȋͲǤͷ Ɋ‰ȀŽȌǡ  ȋͲǤͷ Ɋ‰ȀŽȌ ‘” ˜‡Š‹…Ž‡ …‘–”‘Ž ȋȌ ˆ‘” Ͷͺ ŠǤ ǡ ǣ ‡Ǧ •–‡”„Ž‘–ƒƒŽ›•‹•‘ˆȽǦ ‹ ͷͶͻ …‡ŽŽ• –”‡ƒ–‡† ™‹–Š  ȋͲǤͷ Ɋ‰ȀŽȌǡ  ȋͲǤͷ Ɋ‰ȀŽȌ ‘” ˜‡Š‹…Ž‡ …‘–”‘Ž ȋȌ ˆ‘” Ͷͺ ŠǤ ǣ   ƒƒŽ›•‹• ‘ˆ …‘ŽŽƒ‰‡  Ž‡˜‡Ž ‹–Š‡•—’‡”ƒ–ƒ–‘ˆͷͶͻ …‡ŽŽ•–”‡ƒ–‡†™‹–ŠȋͲǤͷɊ‰ȀŽȌǡȋͲǤͷɊ‰ȀŽȌǡ‘”…‘–”‘Žȋ—–”‡ƒ–‡†Ȍˆ‘”ͶͺŠǤŽŽ†ƒ–ƒ™‡”‡ ˆ”‘͵•‡’ƒ”ƒ–‡‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡȗȗ’δͲǤͲͳ˜•Ǥ…‘–”‘ŽǤǣ–—‹…ƒ›…‹Ǣǣ„Ž‡‘›…‹Ǥ

‹…”‡ƒ•‡†ƒˆ–‡”–—‹…ƒ›…‹–”‡ƒ–‡–ǡƒ†’”‘–‡‹‡š’”‡••‹‘‘ˆȽǦ‹…”‡ƒ•‡† ƒˆ–‡”–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹–”‡ƒ–‡–ǡ™Š‹Ž‡‡š’”‡••‹‘‘ˆ‡’‹–Š‡Ž‹ƒŽ…‡ŽŽƒ”‡” Ǧ…ƒ†Š‡”‹†‡…”‡ƒ•‡†ƒˆ–‡”–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹–”‡ƒ–‡– ȋ ‹‰Ǥͳǡǡǡ ȌǤ  ƒ••ƒ›•Š‘™‡†–Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹…”‡ƒ•‡†–Š‡•‡…”‡–‹‘‘ˆ…‘ŽŽƒ‰‡ „›ͷͶͻ …‡ŽŽ• ȋ ‹‰Ǥ ͳ ȌǤ ‘ŽŽ‡…–‹˜‡Ž›ǡ –Š‡•‡ †ƒ–ƒ ’”‘˜‹†‡ ‡˜‹†‡…‡ –Šƒ– –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡‹ͷͶͻ…‡ŽŽ•Ǥ Tunicamycin and bleomycin induce ER stress in A549 cells ‘‹˜‡•–‹‰ƒ–‡™Š‡–Š‡”–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹†—…‡•–”‡••‹ͷͶͻ…‡ŽŽ•ǡ–Š‡ ‡š’”‡••‹‘‘ˆ…Šƒ’‡”‘‡ ͹ͺ™ƒ•†‡–‡…–‡†„›ǦǤ‡ˆ‘—†–Šƒ––”‡ƒ–‡– ™‹–ŠͷͶͻ…‡ŽŽ•„›–—‹…ƒ›…‹‘”„Ž‡‘›…‹ƒ–†‹ˆˆ‡”‡–…‘…‡–”ƒ–‹‘ƒ††‹ˆˆ‡”‡––‹‡ ’‡”‹‘†ƒŽŽŽ‡†–‘‹…”‡ƒ•‡†‡š’”‡••‹‘‘ˆ ͹ͺ…‘’ƒ”‡†–‘…‘–”‘Žȋ ‹‰ǤʹǡǡǡȌǤ   ‡š– ™‡ ‡š’Ž‘”‡† ™Š‡–Š‡”  •–”‡•• ‹Š‹„‹–‘” ͶǦ …‘—Ž† ‹Š‹„‹– –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡†  •–”‡•• ‹ ͷͶͻ …‡ŽŽ•Ǥ  ͷͶͻ …‡ŽŽ• ™‡”‡ –”‡ƒ–‡† ™‹–Š –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹ …‘„‹ƒ–‹‘ ™‹–Š Ͳǡ ͲǤͷǡ ‘” ͳǤͲ  ͶǦǡ Ǧ ƒƒŽ›•‹• •Š‘™‡† –Šƒ– ͶǦ”‡†—…‡† ͹ͺ‡š’”‡••‹‘‹†—…‡†„›–—‹…ƒ›…‹‘”„Ž‡‘›…‹‹ƒ†‘•‡Ǧ †‡’‡†‡– ƒ‡” ȋ ‹‰Ǥ ʹǡ ǡ ǡ ȌǤ ͷͶͻ …‡ŽŽ• ™‡”‡ –Š‡ –”‡ƒ–‡† ™‹–Š –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹ …‘„‹ƒ–‹‘ ™‹–Š ͳǤͲ  ͶǦǤ ‡•–‡” „Ž‘– ƒƒŽ›•‹• •Š‘™‡† –Šƒ– ͶǦ ”‡†—…‡† ͹ͺ’”‘–‡‹‡š’”‡••‹‘‹†—…‡†„›–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ǡ„—––Š‡”‡™ƒ•‘ •‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡‹ ͹ͺ’”‘–‡‹‡š’”‡••‹‘„‡–™‡‡…‘–”‘Ž‰”‘—’ƒ†ͶǦ–”‡ƒ–‡† ‰”‘—’ȋ ‹‰Ǥʹ ǡǡǡȌǤƒ‡–‘‰‡–Š‡”ǡ–Š‡•‡”‡•—Ž–••—‰‰‡•––Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ ‹†—…‡•–”‡••‹ͷͶͻ…‡ŽŽ•Ǥ ER stress mediates EMT in A549 cells ‹…‡™‡•Š‘™‡†–Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‘–‘Ž›‹†—…‡„—–ƒŽ•‘‹†—…‡ •–”‡••‹ͷͶͻ…‡ŽŽ•ǡ™‡™‘†‡”‡†™Š‡–Š‡”–Š‡”‡‹•ƒ…‘””‡Žƒ–‹‘„‡–™‡‡•–”‡••ƒ† Ǥ‡—•‡†•–”‡••‹Š‹„‹–‘”ͶǦ–‘–”‡ƒ–ͷͶͻ…‡ŽŽ•–‘‰‡–Š‡”™‹–Š–—‹…ƒ›…‹‘”

1824

Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

„Ž‡‘›…‹ǡ ƒ† †‡–‡…–‡† …‡ŽŽ ‘”’Š‘Ž‘‰‹…ƒŽ …Šƒ‰‡• ƒ† –Š‡ ‡š’”‡••‹‘ ‘ˆ  ”‡Žƒ–‡† ‘Ž‡…—Ž‡•Ǥ ‘’ƒ”‡† –‘ –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ –”‡ƒ–‡† ‰”‘—’ǡ ƒ††‹–‹‘ƒŽ –”‡ƒ–‡– ™‹–Š †‹ˆˆ‡”‡– …‘…‡–”ƒ–‹‘• ȋͲǤͷǡ ͳǤͲ Ȍ ‘ˆ ͶǦ Ž‡† –‘ ”‡†—…‡† —„‡” ‘ˆ …‡ŽŽ• ™‹–Š ‡Ž‘‰ƒ–‡† ϐ‹„”‘„Žƒ•– …‡ŽŽ ‘”’Š‘Ž‘‰›ǡ ™Š‹Ž‡ ͶǦ –”‡ƒ–‡† ‰”‘—’ •Š‘™‡† ‘ …Šƒ‰‡• ‹ …‡ŽŽ ‘”’Š‘Ž‘‰› ȋ ‹‰Ǥ ͵ǡ Ȍǡ ‹†‹…ƒ–‹‰ –Šƒ– ͶǦ •—’’”‡••‡† –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹†—…‡†‡•‡…Š›ƒŽ…Šƒ‰‡‹ͷͶͻ…‡ŽŽ•Ǥ‘’ƒ”‡†–‘–—‹…ƒ›…‹‘”„Ž‡‘›…‹–”‡ƒ–‡† ‰”‘—’ǡͳǤͲͶǦ†‡…”‡ƒ•‡†‡š’”‡••‹‘‘ˆ‡•‡…Š›ƒŽƒ”‡”•Ǧ…ƒ†Š‡”‹ƒ† ȽǦ ȋ’ δͲǤͲͷȌ ȋ ‹‰Ǥ ͵ǡ ǡ ǡ ȌǤ ͶǦ ƒŽ•‘ †‡…”‡ƒ•‡† ’”‘–‡‹ ‡š’”‡••‹‘ ‘ˆ ȽǦ ȋ’ δͲǤͲͷȌǡ„—––Š‡”‡™ƒ•‘•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡‹ȽǦ’”‘–‡‹‡š’”‡••‹‘„‡–™‡‡…‘–”‘Ž ‰”‘—’ƒ†ͶǦ–”‡ƒ–‡†‰”‘—’ȋ ‹‰Ǥ͵ ǡǡ ǡ ȌǤ —”–Š‡”‘”‡ǡͶǦ‹Š‹„‹–‡†–—‹…ƒ›…‹ ƒ†„Ž‡‘›…‹‹†—…‡†•‡…”‡–‹‘‘ˆ…‘ŽŽƒ‰‡ „›ͷͶͻ…‡ŽŽ•ȋ ‹‰Ǥ͵ȌǤŠ‡•‡”‡•—Ž–••—‰‰‡•– –Šƒ–•–”‡••‡†‹ƒ–‡•‘ˆͷͶͻ…‡ŽŽ•Ǥ

Fig. 2.—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹†—…‡•–”‡••‹ͷͶͻ…‡ŽŽ•ǤͷͶͻ…‡ŽŽ•™‡”‡’Žƒ–‡†‹͸Ǧ™‡ŽŽ’Žƒ–‡• ‘‡†ƒ›’”‹‘”–‘–”‡ƒ–‡–™‹–Š‘”ǡƒ†…‡ŽŽ•™‡”‡‰”‘™–Šƒ””‡•–‡†‹•‡”—Ǧˆ”‡‡‡†‹—ˆ‘”ʹͶŠǤ ǡǣǦƒƒŽ›•‹•‘ˆ ͹ͺ‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Šǡ‘”…‘–”‘Žˆ‘”ͶͺŠǤǡǣǦ ƒƒŽ›•‹•‘ˆ ͹ͺŽ‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–ŠͲǤͷɊ‰ȀŽ‘”ˆ‘”ͲǡʹͶǡͶͺƒ†͹ʹŠǤǡ ǡ ǡ

ǣǦƒƒŽ›•‹•‘ˆ ͹ͺ‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–Ǧ ‡–™‹–ŠͶǦǤ ǡǡǡǣ‡•–‡”„Ž‘–ƒƒŽ›•‹•‘ˆ ͹ͺ’”‘–‡‹Ž‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘” ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠͶǦǤŽŽ†ƒ–ƒ™‡”‡ˆ”‘͵•‡’ƒ”ƒ–‡‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡ ȗȗ’δͲǤͲͳ˜•Ǥ…‘–”‘ŽǤ͓’δͲǤͲͷǡ͓͓’δͲǤͲͷ˜•Ǥ‘”Ǥǣ–—‹…ƒ›…‹Ǣǣ„Ž‡‘›…‹Ǥ

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Fig. 3.•–”‡••‹†—…‡•‹ͷͶͻ…‡ŽŽ•ǤͷͶͻ…‡ŽŽ•™‡”‡’Žƒ–‡†ƒ–ͳέͳͲ͸…‡ŽŽ•Ȁ™‡ŽŽ‹͸Ǧ™‡ŽŽ’Žƒ–‡•‘‡ †ƒ›’”‹‘”–‘–”‡ƒ–‡–™‹–Š‘”ǡ–Š‡–Š‡…‡ŽŽ•™‡”‡‰”‘™–Šƒ””‡•–‡†‹•‡”—Ǧˆ”‡‡‡†‹—ˆ‘”ʹͶ ŠǤǡǣŠƒ•‡…‘–”ƒ•–‹ƒ‰‡•‘ˆͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡– ™‹–ŠͶǦǤǡǡǡ ǣǦƒƒŽ›•‹•‘ˆǦ…ƒ†Š‡”‹ƒ†ȽǦŽ‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š ȋͲǤͷɊ‰ȀŽȌ‘”ȋͲǤͷɊ‰ȀŽȌˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠͶǦȋͲǤͷȌǤ ǡǡ ǡ ǣ‡Ǧ •–‡”„Ž‘–ƒƒŽ›•‹•‘ˆȽǦ‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡– ™‹–ŠͶǦǤǣ ƒƒŽ›•‹•‘ˆ…‘ŽŽƒ‰‡ Ž‡˜‡Ž‹–Š‡•—’‡”ƒ–ƒ–‘ˆͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–ŠȋͲǤͷɊ‰Ȁ ŽȌ‘”ȋͲǤͷɊ‰ȀŽȌˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠͶǦȋͲǤͷȌǤŽŽ†ƒ–ƒ™‡”‡ˆ”‘͵ •‡’ƒ”ƒ–‡‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡȗȗ’δͲǤͲͳ˜•Ǥ…‘–”‘ŽǤ͓’δͲǤͲͷǡ͓͓’δͲǤͲͷ˜•Ǥ‘”Ǥǣ–—‹…ƒ›…‹Ǣ ǣ„Ž‡‘›…‹Ǥ

Tunicamycin and bleomycin upregulate HDACs in A549 cells ‡š–™‡‡š’Ž‘”‡†™Š‡–Š‡”ƒ…–‹˜ƒ–‹‘‹•‹˜‘Ž˜‡†‹–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ ‹†—…‡†  •–”‡•• ‹ ͷͶͻ …‡ŽŽ•Ǥ Ǧ ƒƒŽ›•‹• •Š‘™‡† –Šƒ– ʹ ƒ† ͸  Ž‡˜‡Ž•‹…”‡ƒ•‡†ƒˆ–‡”„Ž‡‘›…‹–”‡ƒ–‡–ǡ™Š‹Ž‡‘Ž›ʹŽ‡˜‡Ž•‹…”‡ƒ•‡†ƒˆ–‡” –—‹…ƒ›…‹ –”‡ƒ–‡– ‹ ͷͶͻ …‡ŽŽ• ȋ ‹‰Ǥ Ͷǡ ȌǤ ‹‹Žƒ”Ž›ǡ ‡•–‡” „Ž‘– ƒƒŽ›•‹• •Š‘™‡† –Šƒ–ʹƒ†͸’”‘–‡‹Ž‡˜‡Ž•‹…”‡ƒ•‡†ƒˆ–‡”–—‹…ƒ›…‹ƒ†„Ž‡‘›…‹–”‡ƒ–‡– ‹ͷͶͻ…‡ŽŽ•ȋ ‹‰ǤͶǡȌǤ Š‡•‡†ƒ–ƒ‹†‹…ƒ–‡–Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹—’”‡‰—Žƒ–‡ •‹ͷͶͻ…‡ŽŽ•Ǥ

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Tunicamycin and bleomycin induce ER stress by upregulating HDACs ‘ ”‡˜‡ƒŽ –Š‡ ƒ••‘…‹ƒ–‹‘ „‡–™‡‡ • ƒ†  •–”‡•• ‹ ͷͶͻ …‡ŽŽ• ‹†—…‡† „› –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ǡ ™‡ †‡–‡…–‡† –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹†—…‡†  •–”‡•• ‹ ͷͶͻ …‡ŽŽ• –”‡ƒ–‡† „› ’ƒǦ ‹Š‹„‹–‘” Ǥ ‘’ƒ”‡† ™‹–Š –—‹…ƒ›…‹ ‰”‘—’ ‘” „Ž‡‘›…‹‰”‘—’ǡƒ††‹–‹‘ƒŽ–”‡ƒ–‡–™‹–Š‘”ͶǦ†‡…”‡ƒ•‡†ʹƒ†͸ ‡š’”‡••‹‘ȋ ‹‰Ǥͷǡǡǡ ȌǤ —”–Š‡”‘”‡ǡ‹Š‹„‹–‡†–—‹…ƒ›…‹‘”„Ž‡‘›…‹ ‹†—…‡†—’”‡‰—Žƒ–‹‘‘ˆ ͹ͺŽ‡˜‡Ž‹ͷͶͻ…‡ŽŽ•ȋ ‹‰Ǥͷǡǡ ǡȌǤ‹‹Žƒ”Ž›ǡ‡•–‡” „Ž‘– ƒƒŽ›•‹• •Š‘™‡† –Šƒ–  ‘” ͶǦ †‡…”‡ƒ•‡† –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹†—…‡† —’”‡‰—Žƒ–‹‘‘ˆʹƒ†͸’”‘–‡‹Ž‡˜‡Ž•ƒ†–Š‡”‡™ƒ•‘•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡‹ ʹƒ†͸’”‘–‡‹‡š’”‡••‹‘„‡–™‡‡…‘–”‘Ž‰”‘—’ƒ†ͶǦ‘”–”‡ƒ–‡† ‰”‘—’ ȋ ‹‰Ǥ ͷ ǡ ǡ ǡ ȌǤ —”–Š‡”‘”‡ǡ  ‹Š‹„‹–‡† –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹†—…‡† —’”‡‰—Žƒ–‹‘‘ˆ ͹ͺ’”‘–‡‹Ž‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•„—––Š‡”‡™ƒ•‘•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡‹

͹ͺ’”‘–‡‹‡š’”‡••‹‘„‡–™‡‡…‘–”‘Ž‰”‘—’ƒ†–”‡ƒ–‡†‰”‘—’ȋ ‹‰ǤͷǡǡǡȌǤ ‘ŽŽ‡…–‹˜‡Ž›ǡ–Š‡•‡”‡•—Ž–•‹†‹…ƒ–‡–Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹ƒ›‹†—…‡•–”‡••‹ ͷͶͻ…‡ŽŽ•˜‹ƒ–Š‡—’”‡‰—Žƒ–‹‘‘ˆʹƒ†͸‡š’”‡••‹‘Ǥ HDACs inhibitor inhibits tunicamycin or bleomycin induced EMT ‡š– ™‡ ‡šƒ‹‡† –Š‡ ‡ˆˆ‡…– ‘ˆ  ‘ –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹†—…‡†  ‘ˆ ͷͶͻ …‡ŽŽ•Ǥ ‘’ƒ”‡† ™‹–Š –—‹…ƒ›…‹ ‰”‘—’ ‘” „Ž‡‘›…‹ ‰”‘—’ǡ ƒ††‹–‹‘ƒŽ –”‡ƒ–‡– ™‹–ŠȋͲǤ͵ƒ†ͳǤͲɊȌ‘”–”‡ƒ–‡–™‹–ŠȋͲǤ͵ɊȌƒŽ‘‡’”‘‘–‡†‡Ž‘‰ƒ–‡† ϐ‹„”‘„Žƒ•–‹… ‘”’Š‘Ž‘‰› …Šƒ‰‡ ‘ˆ …‡ŽŽ• ȋ ‹‰Ǥ ͸ǡ ȌǤ ‡ƒ™Š‹Ž‡ǡ ͲǤ͵ Ɋ  ‹Š‹„‹–‡† –—‹…ƒ›…‹‘”„Ž‡‘›…‹‹†—…‡†—’”‡‰—Žƒ–‹‘‘ˆ‡•‡…Š›ƒŽƒ”‡”•ȽǦƒ†ƒŽ•‘ ‹Š‹„‹–‡†–—‹…ƒ›…‹‹†—…‡†—’”‡‰—Žƒ–‹‘‘ˆ‡•‡…Š›ƒŽƒ”‡”Ǧ…ƒ†Š‡”‹ƒ– Ž‡˜‡Ž• ‹ ͷͶͻ …‡ŽŽ• ȋ ‹‰Ǥ ͸ǡ ǡ ǡ ȌǤ ‹‹Žƒ”Ž›ǡ ‡•–‡” „Ž‘– ƒƒŽ›•‹• •Š‘™‡† –Šƒ– 

Fig. 4.—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹†—…‡–Š‡‡š’”‡••‹‘‘ˆʹƒ†͸‹ͷͶͻ…‡ŽŽ•ǤͷͶͻ…‡ŽŽ• ™‡”‡’Žƒ–‡†ƒ–ͳέͳͲ͸…‡ŽŽ•Ȁ™‡ŽŽ‹͸Ǧ™‡ŽŽ’Žƒ–‡•‘‡†ƒ›’”‹‘”–‘–”‡ƒ–‡–™‹–Š‘”ǡƒ†–Š‡…‡ŽŽ• ™‡”‡‰”‘™–Šƒ””‡•–‡†‹•‡”—Ǧˆ”‡‡‡†‹—ˆ‘”ʹͶŠǤŠ‡ͷͶͻ…‡ŽŽ•™‡”‡‡š’‘•‡†–‘ȋͲǤͷɊ‰ȀŽȌǡ ȋͲǤͷɊ‰ȀŽȌ‘”ȋ…‘–”‘ŽȌˆ‘”ͶͺŠǤǡǣǦƒƒŽ›•‹•‘ˆʹƒ†͸Ž‡˜‡Ž•‹ ͷͶͻ…‡ŽŽ•Ǥǡǣ‡•–‡”„Ž‘–ƒƒŽ›•‹•‘ˆʹƒ†͸’”‘–‡‹Ž‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•ǤŽŽ†ƒ–ƒ™‡”‡ˆ”‘ ͵•‡’ƒ”ƒ–‡‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡȗȗ’δͲǤͲͳ˜•Ǥ…‘–”‘ŽǤǣ–—‹…ƒ›…‹Ǣǣ„Ž‡‘›…‹Ǥ

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Fig. 5. —‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡  •–”‡•• „› —’”‡‰—Žƒ–‹‰ •Ǥ ͷͶͻ …‡ŽŽ• ™‡”‡ ’Žƒ–‡† ‹ ͸Ǧ™‡ŽŽ’Žƒ–‡•ƒ†‰”‘™–Šƒ””‡•–‡†‹•‡”—Ǧˆ”‡‡‡†‹—ˆ‘”ʹͶŠǤǡǡǡ ǣǦƒƒŽ›•‹•‘ˆʹƒ† ͸‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–Š‘” ͶǦǤǡǡ ǡǣǦƒƒŽ›•‹•‘ˆ ͹ͺ‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘” ™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–Š‘”ͶǦǤ ǡ ǡǡǣ‡•–‡”„Ž‘–ƒƒŽ›•‹•‘ˆʹƒ†͸’”‘–‡‹ Ž‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–Š‘”ͶǦǤǡ ǡǡǣ‡•–‡”„Ž‘–ƒƒŽ›•‹•‘ˆ ͹ͺ’”‘–‡‹Ž‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘” ™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–Š‘”ͶǦǤŽŽ†ƒ–ƒ™‡”‡ˆ”‘͵•‡’ƒ”ƒ–‡‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡȗȗ’δͲǤͲͳ ˜•Ǥ…‘–”‘ŽǤ͓’δͲǤͲͷǡ͓͓’δͲǤͲͷ˜•Ǥ‘”Ǥǣ–—‹…ƒ›…‹Ǣǣ„Ž‡‘›…‹Ǥ

†‡…”‡ƒ•‡† –—‹…ƒ›…‹ ‘” „Ž‡‘›…‹ ‹†—…‡† —’”‡‰—Žƒ–‹‘ ‘ˆ ȽǦ ƒ– ’”‘–‡‹ Ž‡˜‡Ž „—– –Š‡”‡™ƒ•‘•‹‰‹ϐ‹…ƒ–†‹ˆˆ‡”‡…‡‹ȽǦ’”‘–‡‹‡š’”‡••‹‘„‡–™‡‡…‘–”‘Ž‰”‘—’ƒ† –”‡ƒ–‡†‰”‘—’ȋ ‹‰Ǥ͸ ǡǡ ǡ ȌǤ ƒ††‹–‹‘ǡ‹Š‹„‹–‡†–—‹…ƒ›…‹‘”„Ž‡‘›…‹

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

Fig. 6. ‹Š‹„‹–•–—‹…ƒ›…‹‘”„Ž‡‘›…‹‹†—…‡†ǤͷͶͻ…‡ŽŽ•™‡”‡’Žƒ–‡†ƒ–ͳέͳͲ͸…‡ŽŽ•Ȁ™‡ŽŽ ‹͸Ǧ™‡ŽŽ’Žƒ–‡•‘‡†ƒ›’”‹‘”–‘–”‡ƒ–‡–™‹–Š‘”ǡƒ†…‡ŽŽ•™‡”‡‰”‘™–Šƒ””‡•–‡†‹•‡”—Ǧˆ”‡‡ ‡†‹—ˆ‘”ʹͶŠǤǡǣŠƒ•‡…‘–”ƒ•–‹ƒ‰‡•‘ˆͷͶͻ…‡ŽŽ•‹…—„ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘” ™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠǤǡǡǡ ǣǦƒƒŽ›•‹•‘ˆǦ…ƒ†Š‡”‹ƒ†ȽǦŽ‡˜‡Ž•‹ͷͶͻ …‡ŽŽ•–”‡ƒ–‡†™‹–ŠȋͲǤͷɊ‰ȀŽȌ‘”ȋͲǤͷɊ‰ȀŽȌˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠȋͲǤ͵ ɊȌǤ ǡǡ ǡ ǣ‡•–‡”„Ž‘–ƒƒŽ›•‹•‘ˆȽǦ‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—– ’”‡–”‡ƒ–‡–™‹–ŠǤǣ ƒƒŽ›•‹•‘ˆ…‘ŽŽƒ‰‡ Ž‡˜‡Ž‹–Š‡•—’‡”ƒ–ƒ–‘ˆͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š ȋͲǤͷɊ‰ȀŽȌ‘”ȋͲǤͷɊ‰ȀŽȌˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠȋͲǤ͵ȌǤŽŽ†ƒ–ƒ ™‡”‡ˆ”‘͵•‡’ƒ”ƒ–‡‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡȗȗ’δͲǤͲͳ˜•Ǥ…‘–”‘ŽǤ͓’δͲǤͲͷǡ͓͓’δͲǤͲͷ˜•Ǥ‘”Ǥǣ –—‹…ƒ›…‹Ǣǣ„Ž‡‘›…‹Ǥ

‹†—…‡†•‡…”‡–‹‘‘ˆ‘ŽŽƒ‰‡ „›ͷͶͻ…‡ŽŽ•ȋ ‹‰Ǥ͸ȌǤƒ‡–‘‰‡–Š‡”ǡ–Š‡•‡”‡•—Ž–••—‰‰‡•– –Šƒ–•‡†‹ƒ–‡–—‹…ƒ›…‹‘”„Ž‡‘›…‹‹†—…‡†‘ˆͷͶͻ…‡ŽŽ•Ǥ •‹Š‹„‹–‘”‹Š‹„‹–••–”‡••‹†—…‡†—’”‡‰—Žƒ–‹‘‘ˆ’—Ž‘ƒ”›ϔ‹„”‘•‹•ˆƒ…–‘”•ƒ† the activation of Smad pathway ‘ ‡š’Ž‘”‡ ™Š‡–Š‡”  •–”‡•• ƒˆˆ‡…–• –Š‡ ‡š’”‡••‹‘ ‘ˆ ϐ‹„”‘•‹•Ǧ”‡Žƒ–‡† ˆƒ…–‘”•ǡ ™‡ †‡–‡…–‡†–Š‡‡š’”‡••‹‘‘ˆ”‡Ž‡˜ƒ–ˆƒ…–‘”•ƒ–ƒ†’”‘–‡‹Ž‡˜‡Ž•„›Ǧƒ† Ǥ

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

Fi

7

Fig. 7. ‹Š‹„‹–•–Š‡—’”‡‰—Žƒ–‹‘‘ˆ’—Ž‘ƒ”›ϐ‹„”‘•‹•ˆƒ…–‘”•ƒ†–Š‡ƒ…–‹˜ƒ–‹‘‘ˆƒ†’ƒ–Š™ƒ›Ǥ ͷͶͻ …‡ŽŽ• ™‡”‡ ’Žƒ–‡† ‹ ͸Ǧ™‡ŽŽ ’Žƒ–‡• ƒ† ‰”‘™–Š ƒ””‡•–‡† ‹ •‡”—Ǧˆ”‡‡ ‡†‹— ˆ‘” ʹͶ ŠǤ ǡ ǡ ǡ ǣ ǦƒƒŽ›•‹•‘ˆ ǦȾͳǡ Ǧ͵ʹƒ† ǦͳȾ‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡†™‹–Š‘”ˆ‘”ʹͶŠ™‹–Š‘” ™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–Š‘”ͶǦǤǣ ƒƒŽ›•‹•‘ˆ ǦȾͳŽ‡˜‡Ž•‹–Š‡•—’‡”ƒ–ƒ–‘ˆͷͶͻ …‡ŽŽ•–”‡ƒ–‡†™‹–ŠȋͲǤͷɊ‰ȀŽȌ‘”ȋͲǤͷɊ‰ȀŽȌˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–ŠȋͲǤ͵ ɊȌ‘”ͶǦȋͳǤͲȌǤ ǡ ǡǡ ǣǦƒƒŽ›•‹•‘ˆƒ†ʹƒ†ƒ†͵Ž‡˜‡Ž•‹ͷͶͻ…‡ŽŽ•–”‡ƒ–‡† ™‹–Š‘”ˆ‘”ͶͺŠ™‹–Š‘”™‹–Š‘—–’”‡–”‡ƒ–‡–™‹–Š‘”ͶǦǤŽŽ†ƒ–ƒ™‡”‡ˆ”‘͵•‡’ƒ”ƒ–‡ ‡š’‡”‹‡–•Ǥȗ’δͲǤͲͷǡȗȗ’δͲǤͲͳǡ˜•Ǥ…‘–”‘ŽǢ͓’δͲǤͲͷǡ͓͓’δͲǤͲͷ˜•Ǥ‘”Ǥǣ–—‹…ƒ›…‹Ǣǣ „Ž‡‘›…‹Ǥ

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

Š‡”‡•—Ž–••Š‘™‡†–Šƒ––—‹…ƒ›…‹‹…”‡ƒ•‡† ǦȾͳƒ† Ǧ͵ʹ‡š’”‡••‹‘Ž‡˜‡Ž• ƒ•™‡ŽŽƒ•–Š‡•‡…”‡–‹‘‘ˆ ǦȾͳ’”‘–‡‹‹ͷͶͻ…‡ŽŽ•ǡ™Š‹Ž‡„Ž‡‘›…‹‹…”‡ƒ•‡† ǦȾͳǡ Ǧ͵ʹƒ† ǦͳȾ‡š’”‡••‹‘Ž‡˜‡Ž•ƒ•™‡ŽŽƒ•–Š‡•‡…”‡–‹‘‘ˆ ǦȾͳ’”‘–‡‹‹ͷͶͻ …‡ŽŽ•ȋ ‹‰Ǥ͹ǡǡǡǡȌǤ‘™‡˜‡”ǡ–Š‡…Šƒ‰‡•‹†—…‡†„›–—‹…ƒ›…‹‘”„Ž‡‘›…‹…‘—Ž† „‡ƒ–ƒ‰‘‹œ‡†„›ͶǦ‘”ȋ ‹‰Ǥ͹ǡǡǡǡȌǤ ‡š–ǡ ™‡ ‡šƒ‹‡† –Š‡ ‡ˆˆ‡…–• ‘ˆ  ‹Š‹„‹–‘” ƒ† • ‹Š‹„‹–‘” ‘  ǦȾͳȀƒ† ’ƒ–Š™ƒ›‹ͷͶͻ…‡ŽŽ•ǤǦƒƒŽ›•‹••Š‘™‡†–Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹…”‡ƒ•‡† –Š‡‡š’”‡••‹‘‘ˆƒ†ʹ„—–Šƒ†‘•‹‰‹ϐ‹…ƒ–‡ˆˆ‡…–•‘–Š‡‡š’”‡••‹‘‘ˆƒ†͵ ǡ…‘’ƒ”‡†–‘…‘–”‘Ž‰”‘—’ȋ ‹‰Ǥ͹ ǡ ǡǡ ȌǤ‹Š‹„‹–‡†ƒ†͵‡š’”‡••‹‘ ƒ†–Š‡—’”‡‰—Žƒ–‹‘‘ˆƒ†ʹŽ‡˜‡Ž•‹†—…‡†„›–—‹…ƒ›…‹‘”„Ž‡‘›…‹ǡ„—–ͶǦ ‘Ž›‹Š‹„‹–‡†–Š‡—’”‡‰—Žƒ–‹‘‘ˆƒ†ʹŽ‡˜‡Ž‹†—…‡†„›„Ž‡‘›…‹ȋ ‹‰Ǥ͹ ǡ ǡǡ ȌǤ •—ƒ”›ǡ–Š‡•‡”‡•—Ž–•‹†‹…ƒ–‡–Šƒ–•‡†‹ƒ–‡–Š‡—’”‡‰—Žƒ–‹‘‘ˆ’—Ž‘ƒ”› ϐ‹„”‘•‹•ˆƒ…–‘”•ƒ†–Š‡ƒ…–‹˜ƒ–‹‘‘ˆƒ†’ƒ–Š™ƒ›ƒˆ–‡”•–”‡••Ǥ Discussion

—Ž‘ƒ”› ϐ‹„”‘•‹• ‹• ƒ …‘’Ž‡š ’ƒ–Š‘Ž‘‰‹…ƒŽ ’”‘…‡••ǡ ƒ† ’—Ž‘ƒ”›  ‹• ‘‡ ‘ˆ –Š‡‹’‘”–ƒ–‡…Šƒ‹••—†‡”Ž›‹‰‹–•’ƒ–Š‘‰‡‡•‹•Ǥ—‰›‘ϐ‹„”‘„Žƒ•–…‡ŽŽ•ƒ”‡ƒ‹Ž› †‡”‹˜‡†ˆ”‘‘ˆƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•Ǥ‘™‡˜‡”ǡ…—””‡–—†‡”•–ƒ†‹‰‘ˆ’—Ž‘ƒ”› ”‡ƒ‹•Ž‹‹–‡†Ǥ‡…‡–•–—†‹‡••Š‘™‡†–Šƒ–‹–Š‡’ƒ–Š‘‰‡‡•‹•‘ˆ’—Ž‘ƒ”›ϐ‹„”‘•‹•ǡ …‡”–ƒ‹ ‰‡‡–‹… ƒ† ‡˜‹”‘‡–ƒŽ ˆƒ…–‘”• •—…Š ƒ• •‘‹‰ǡ …Š”‘‹… ƒ•’‹”ƒ–‹‘ǡ ˜‹”ƒŽ ‹ˆ‡…–‹‘ǡ—…‹ͷ‰‡‡’”‘‘–‡”’‘Ž›‘”’Š‹•ǡ–‡Ž‘‡”ƒ•‡‰‡‡—–ƒ–‹‘ǡƒ† —–ƒ–‹‘•ƒ›•–‹—Žƒ–‡•–”‡••‹ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡŽ‡ƒ†‹‰–‘–Š‡‘……—””‡…‡‘ˆ ȏͻǦͳͳȐǤ ‘—•‡Ž—‰ϐ‹„”‘•‹•ǡ„Ž‡‘›…‹‹†—…‡†•–”‡••ǡ™Š‹…Š–Š‡‡†‹ƒ–‡†–Š‡ ’”‘‰”‡••‹‘‘ˆȏͳ͸ȐǤ ƒ‰”‡‡‡–™‹–Š’”‡˜‹‘—•ϐ‹†‹‰•ǡ‹–Š‹••–—†›™‡†‡‘•–”ƒ–‡† –Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹†—…‡†•–”‡••‹ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡƒ†•–”‡•• …ƒ—•‡†‹ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•Ǥ •ƒ”‡…”—…‹ƒŽŽ›‹’Ž‹…ƒ–‡†‹ƒ›’Š›•‹‘Ž‘‰‹…ƒŽƒ†’ƒ–Š‘Ž‘‰‹…ƒŽ’”‘…‡••‡•ǡƒ† • ‹Š‹„‹–‘” …‘—Ž† „‡‡ϐ‹– …ƒ…‡” ƒ† ϐ‹„”‘–‹… †‹•‡ƒ•‡•Ǥ  …‡”–ƒ‹ –—‘” …‡ŽŽ•ǡ • Ž‘…ƒŽ‹œ‡†‹ƒˆˆ‡…––Š‡‘……—””‡…‡‘ˆ•–”‡••ȏʹͳǦʹ͵ȐǤ –Š‹••–—†›ǡ™‡’”‘˜‹†‡†–Š‡ϐ‹”•– ‡˜‹†‡…‡–Šƒ––—‹…ƒ›…‹ƒ†„Ž‡‘›…‹‹†—…‡†•–”‡••‹ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•˜‹ƒ –Š‡—’”‡‰—Žƒ–‹‘‘ˆʹȀ͸ǡ™Š‹…Š‡†‹ƒ–‡–Š‡ˆ‘”ƒ–‹‘‘ˆǤ‘™‡˜‡”ǡ™Š‡–Š‡”‘–Š‡” –›’‡•‘ˆ•ƒ”‡‹˜‘Ž˜‡†‹•–”‡••‹†—…‡†‡‡†•–‘„‡ˆ—”–Š‡”‡š’Ž‘”‡†Ǥ ˜‡”› ”‡…‡–•–—†›•—‰‰‡•–‡†–Šƒ–…Žƒ•• •ȋͳǡʹǡ͵ƒ†ͺȌ’Žƒ›ƒƒŒ‘””‘Ž‡–‘”‡‰—Žƒ–‡ ‘ˆŠ—ƒ”‡ƒŽ‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡ™Š‹Ž‡…Žƒ•• ƒ•ȋͶƒ†ͷȌƒ”‡—Ž‹‡Ž›–‘„‡ ‹˜‘Ž˜‡† ȏʹͶȐǤ  ƒ††‹–‹‘ǡ  ‹Š‹„‹–‘” ͻ͵ͻ ‹Š‹„‹–‡†  ‘ˆ Š—ƒ ”‡ƒŽ ’”‘š‹ƒŽ –—„—Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡƒ†”‡†—…‡†–Š‡ƒ……——Žƒ–‹‘‘ˆȽǦƒ†‹ϐŽƒƒ–‘”›ƒ†’”‘Ǧ ϐ‹„”‘–‹……›–‘‹‡•ǤŠ‡‡…Šƒ‹•ƒ›„‡†—‡–‘–Š‡‹Š‹„‹–‹‘‘ˆƒ†Ǧ‹†‡’‡†‡– ǦȾ •‹‰ƒŽ‹‰ „›  ‹Š‹„‹–‘” ƒ† ‹˜‘Ž˜‡  ƒ†  ͵Ȁ ’ƒ–Š™ƒ›• ȏʹͷȐǤ ’ƒ”– ˆ”‘ „‡‹‰ƒ•–”‡••‹Š‹„‹–‘”ǡͶƒŽ•‘ƒ…–•ƒ•ƒ‹Š‹„‹–‘”ȋ…Žƒ•• ƒ† Ȍ™Š‡—•‡† ƒ–Š‹‰Š‡”†‘•‡•Ǥ –Š‹••–—†›™‡ˆ‘—†–Šƒ–Ͷ†‹†‘–…ƒ—•‡•‹‰‹ϐ‹…ƒ–…Šƒ‰‡•‹–Š‡ Ž‡˜‡Ž• ‘ˆ  ʹ ƒ† ͸ǡ ’‡”Šƒ’• „‡…ƒ—•‡ ™‡ —•‡† Ž‘™ †‘•‡ ‘ˆ ͶǤ Š‡•‡ †ƒ–ƒ ’”‘˜‹†‡ •–”‘‰•—’’‘”––Šƒ–•‹Š‹„‹–‘”‹Š‹„‹–••–”‡••‹†—…‡†ƒ†–Š‡—’”‡‰—Žƒ–‹‘‘ˆ ’—Ž‘ƒ”›ϐ‹„”‘•‹•ˆƒ…–‘”•Ǥ Š‡•‹‰ƒŽ‹‰‡…Šƒ‹••‹˜‘Ž˜‡†‹•–”‡••‹†—…‡†ƒ”‡…‘’Ž‡šǤ•–”‡•• ‹†—…‡†‹ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•–Š”‘—‰Šƒ†ʹƒ†”…’ƒ–Š™ƒ›•ȏͳͶȐǤ –Š‹••–—†› ™‡ˆ‘—†–Šƒ–•–”‡••‹…”‡ƒ•‡†ƒ†ʹ„—–‘–ƒ†͵Ž‡˜‡Ž•ǡ„—–•‹Š‹„‹–‘”  ‹Š‹„‹–‡† –Š‡ ‡š’”‡••‹‘ ‘ˆ ƒ†ʹ ƒ† ƒ†͵ •Ǥ Š—•ǡ ƒ†ʹ ’ƒ–Š™ƒ› ƒ› „‡ ‹˜‘Ž˜‡† ‹  •–”‡•• ƒ†  ‹ ƒŽ˜‡‘Žƒ” ‡’‹–Š‡Ž‹ƒŽ …‡ŽŽ• ‹†—…‡† „› –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹Ǥ ‘™‡˜‡”ǡ ƒ ”‡…‡– •–—†› •Š‘™‡† –Šƒ–  ǦȾ ‡†‹ƒ–‡†  ‹ ”‡–‹ƒŽ ’‹‰‡– ‡’‹–Š‡Ž‹— –Š”‘—‰Š …‘’Ž‡š ‹–‡”’Žƒ› „‡–™‡‡ ƒ† ’ƒ–Š™ƒ›ǡ –ǡ ’͵ͺǡ ͳȀʹ ’ƒ–Š™ƒ›• ƒ† ƒ‰‰‡†Ȁ‘–…Š ’ƒ–Š™ƒ› ȏʹ͸ȐǤ  ƒ††‹–‹‘ǡ ‹ǦͳͶͷ ™ƒ• ”‡…‡–Ž› ”‡’‘”–‡† –‘ ‘†—Žƒ–‡  ǦȾͳ Ȁƒ†͵ •‹‰ƒŽ‹‰ –‘ ”‡‰—Žƒ–‡ ‡†‘–Š‡Ž‹ƒŽǦ–‘Ǧ‡•‡…Š›ƒŽ –”ƒ•‹–‹‘

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

Fig. 8.”‘’‘•‡†‡…Šƒ‹••„›™Š‹…Š –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ …ƒ—•‡ ’—ŽǦ ‘ƒ”›ϐ‹„”‘•‹•Ǥ—‹…ƒ›…‹ƒ†„Ž‡‘Ǧ ›…‹—’”‡‰—Žƒ–‡ʹȀ͸ǡ™Š‹…Š–Š‡ ‹†—…‡  •–”‡•• ‹ ƒŽ˜‡‘Žƒ” ‡’‹–Š‡Ž‹ƒŽ …‡ŽŽ•Ǥ‡š–ǡ•–”‡••ƒ…–‹˜ƒ–‡•ƒ†ʹȀ͵ ’ƒ–Š™ƒ› ƒ† ’”‘‘–‡•  ‘ˆ ƒŽ˜‡‘Ǧ Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǤŠ‡ƒ……——Žƒ–‹‘‘ˆ ȽǦƒ†–Š‡”‡Ž‡ƒ•‡‘ˆ‹ϐŽƒƒ–‘”› ƒ†’”‘Ǧϐ‹„”‘–‹……›–‘‹‡•…‘–”‹„—–‡–‘ ’—Ž‘ƒ”› ϐ‹„”‘•‹•Ǥ • ‹Š‹„‹–‘”• •—…Š ƒ•  ‘”  •–”‡•• ‹Š‹„‹–‘”• •—…Š ƒ• ͶǦ ‹Š‹„‹– –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹‹†—…‡†•–”‡••ǡƒ†„Ž‘… –Š‡ ƒ…–‹˜ƒ–‹‘ ‘ˆ ƒ† ’ƒ–Š™ƒ› ƒ† –‘’”‡˜‡–’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ

ȏʹ͹ȐǤ —”–Š‡”•–—†‹‡•ƒ”‡‡…‡••ƒ”›–‘‹˜‡•–‹‰ƒ–‡–Š‡…”‘••–ƒŽ‘ˆƒ†’ƒ–Š™ƒ›™‹–Š‘–Š‡” ’ƒ–Š™ƒ›•‹‡†‹ƒ–‹‰‘ˆƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•‡š’‘•‡†–‘•–”‡••Ǥ —”‹‰ ’—Ž‘ƒ”› ϐ‹„”‘•‹•ǡ –Š‡ ‡š’”‡••‹‘ ‘ˆ ’”‘Ǧϐ‹„”‘–‹… ˆƒ…–‘”• ƒ† ƒ–‹Ǧϐ‹„”‘–‹… ˆƒ…–‘”•‹•—’”‡‰—Žƒ–‡†ƒ††‘™”‡‰—Žƒ–‡†ǡ”‡•’‡…–‹˜‡Ž›Ǥ”‘Ǧϐ‹„”‘–‹…ˆƒ…–‘”••—…Šƒ• ǦȾͳǡ ǦͳȾǡ ǦȽǡ Ǧͳ͹ǡ Ǧʹ͹ǡ Ǧͳ͵ƒ† Ǧ͵ʹ™‡”‡Š‹‰ŠŽ›‡š’”‡••‡†‹’—Ž‘ƒ”›ϐ‹„”‘•‹•ȏʹͺǡ ʹͻȐǤ•–”‡••…ƒ’”‘‘–‡–Š‡‡š’”‡••‹‘ƒ†•‡…”‡–‹‘‘ˆ ǦȾͳ‹Ž—‰‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡ ƒ†’Žƒ›•ƒ‹’‘”–ƒ–”‘Ž‡‹–Š‡’”‘‰”‡••‹‘‘ˆ’—Ž‘ƒ”›ϐ‹„”‘•‹•ȏ͵ͲȐǤ –Š‹••–—†›™‡ ˆ‘—† –Šƒ– –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡†  •–”‡•• „› —’”‡‰—Žƒ–‹‰ •ǡ ™Š‹…Š –Š‡ ’”‘‘–‡† –Š‡ ‡š’”‡••‹‘ ‘ˆ  ǦȾͳǡ Ǧ͵ʹ ƒ† ǦͳȾǤ Š‡•‡ ’”‘Ǧϐ‹„”‘–‹… ˆƒ…–‘”• ƒ› …‘–”‹„—–‡–‘’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ Conclusion

‡ ’”‘˜‹†‡ –Š‡ ϐ‹”•– ‡˜‹†‡…‡ –Šƒ– –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ ‹†—…‡  •–”‡•• ˜‹ƒ —’”‡‰—Žƒ–‹‰ • ‹ ƒŽ˜‡‘Žƒ” ‡’‹–Š‡Ž‹ƒŽ …‡ŽŽ•Ǥ  •–”‡•• –Š‡ ƒ…–‹˜ƒ–‡• ƒ†ʹȀ͵ ’ƒ–Š™ƒ›ƒ†’”‘‘–‡•‘ˆƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•Ǥ‘•‡“—‡–Ž›ǡ–Š‡ƒ……——Žƒ–‹‘‘ˆ ȽǦƒ†–Š‡”‡Ž‡ƒ•‡‘ˆ‹ϐŽƒƒ–‘”›ƒ†’”‘Ǧϐ‹„”‘–‹……›–‘‹‡•™‹ŽŽŽ‡ƒ†–‘’—Ž‘ƒ”› ϐ‹„”‘•‹•Ǥ ‘™‡˜‡”ǡ • ‹Š‹„‹–‘” ‘”  •–”‡•• ‹Š‹„‹–‘” …‘—Ž† ‹Š‹„‹–  •–”‡•• ‹†—…‡† „› –—‹…ƒ›…‹ ƒ† „Ž‡‘›…‹ǡ ƒ† „Ž‘… –Š‡ ƒ…–‹˜ƒ–‹‘ ‘ˆ ƒ† ’ƒ–Š™ƒ› ƒ† ǡ –Š—• ’”‡˜‡–‹‰’—Ž‘ƒ”›ϐ‹„”‘•‹•ȋ ‹‰ǤͺȌǤŠ‡•‡”‡•—Ž–••—‰‰‡•––Šƒ– •’”‘‘–‡•–”‡•• ‹†—…‡†‹Š—ƒƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǡƒ†•‹Š‹„‹–‘”•ƒ”‡’”‘‹•‹‰ƒ‰‡–• ˆ‘”–Š‡–Š‡”ƒ’›‘ˆ’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ Acknowledgements

Š‹• •–—†› ™ƒ• •—’’‘”–‡† „› ‰”ƒ–• ˆ”‘ —‹œŠ‘— ”‘˜‹…‡ ‡ƒŽ–Š ‡’ƒ”–‡– ȋ‘Ǥ ‰œ™ŒŒʹͲͳͶǦͳǦͲ͸ͶȌǡ —‹œŠ‘— ”‘˜‹…‡ ‡ƒŽ–Š ‡’ƒ”–‡– ȋ‘Ǥ ‰œ™ŒŒʹͲͳͷǦͳǦͲͳ͹Ȍ ƒ† …‹‡…‡ ƒ† ‡…Š‘Ž‘‰› ‰‡…› ‘ˆ —‹œŠ‘— ”‘˜‹…‡ ȋ‘Ǥ „”ƒ…Š ‘ˆ —‹œŠ‘— –ƒŽ‡– –‡ƒ ʹͲͳͷǦͶͲͳͺȌǤ Disclosure Statement

‘…‘ϐŽ‹…–‘ˆ‹–‡”‡•–•‡š‹•–•Ǥ

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Physiol Biochem 2018;46:1821-1834 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000489367 and Biochemistry Published online: April 30, 2018 www.karger.com/cpb Liu et al.: HDACs Promotes EMT of Lung Epithelium

References ͳ

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ƒ‰Š— ǡ ”‡—†‡„‡”‰‡”ǡƒ‰ǡ—”–‹• ǡ’ƒ†ƒǡƒ›‡• ǡ‹ŽŽ‡”› ǡ‘’‡ʹ†ǡ‡ŽŽ‡‰”‹‹ǣ ‹‰Š’”‡˜ƒŽ‡…‡‘ˆƒ„‘”ƒŽƒ…‹†‰ƒ•–”‘Ǧ‘‡•‘’Šƒ‰‡ƒŽ”‡ϐŽ—š‹‹†‹‘’ƒ–Š‹…’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ—”‡•’‹”  ʹͲͲ͸Ǣʹ͹ǣͳ͵͸ǦͳͶʹǤ ƒŽ›•›ǡ‹†Ǧ—œƒˆƒ ǡ‘–‡ŽŽƒǡ••‡”ǣ …‹†‡…‡ƒ†’”‡˜ƒŽ‡…‡‘ˆ‹†‹‘’ƒ–Š‹…’—Ž‘ƒ”›ϐ‹„”‘•‹•ǣ ”‡˜‹‡™‘ˆ–Š‡Ž‹–‡”ƒ–—”‡Ǥ—”‡•’‹”‡˜ʹͲͳʹǢʹͳǣ͵ͷͷǦ͵͸ͳǤ –‘‹‘—ǡƒ”‰ƒ”‹–‘’‘—Ž‘• ǡ‹ƒˆƒƒ•ǣŠƒ”ƒ…‘Ž‘‰‹…ƒŽ–”‡ƒ–‡–‘ˆ‹†‹‘’ƒ–Š‹…’—Ž‘ƒ”› ϐ‹„”‘•‹•ǣˆ”‘–Š‡’ƒ•––‘–Š‡ˆ—–—”‡Ǥ—”‡•’‹”‡˜ʹͲͳ͵ǢʹʹǣʹͺͳǦʹͻͳǤ ‘††ǡ—œ‹ƒ ǡ–ƒƒ•ǣ‘Ž‡…—Žƒ”ƒ†…‡ŽŽ—Žƒ”‡…Šƒ‹••‘ˆ’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ ‹„”‘‰‡‡•‹• ‹••—‡‡’ƒ‹”ʹͲͳʹǢͷǣͳͳǤ Š‘ǣ—””‡–‘…‡’–ˆ‘”–Š‡ƒ–Š‘‰‡‡•‹•‘ˆ †‹‘’ƒ–Š‹…—Ž‘ƒ”› ‹„”‘•‹•ǤŽ‹‡•—Ž‘‘Ž ʹͲͳ͵ǢͳǣͳͲͲͺǤ ‘Ž–‡”• ǡ‘ŽŽƒ”†ǡ ‘‡•ǣƒ–Š‘‰‡‡•‹•‘ˆ‹†‹‘’ƒ–Š‹…’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ—‡˜ƒ–Š‘Ž ʹͲͳͶǢͻǣͳͷ͹Ǧͳ͹ͻǤ ƒ‰‡ǡ‘”‘ǣƒ†‹–‡”•–‹–‹ƒŽŽ—‰†‹•‡ƒ•‡ǣƒ›•–‡”‹‘—•”‡Žƒ–‹‘•Š‹’Ǥ—””’‹—Ž‡† ʹͲͳʹǢͳͺǣͷͳ͹Ǧͷʹ͵Ǥ Ž‹’‹‘ǡ ‘‡•ǡ‹ƒ‘ǡƒ‰ ǡ—Žƒ”‹ǡ”‡‡—ƒ”ǡƒ—‡”Ǧ ‡•‡ǡƒ”†ǡƒǡ ‹ǣ ’‹–Š‡Ž‹ƒŽ–‘‡•‡…Š›ƒŽ–”ƒ•‹–‹‘‹†—…‡†„›„Ž‡‘›…‹‘” ȋ„ͳȌȀ ‹—”‹‡‹†—…‡†’Ž—”‹’‘–‡– •–‡…‡ŽŽǦ†‡”‹˜‡†ƒŽ˜‡‘Žƒ”›’‡ ǦŽ‹‡…‡ŽŽ•Ǥ‹ˆˆ‡”‡–‹ƒ–‹‘ʹͲͳͳǢͺʹǣͺͻǦͻͺǤ ƒ”ǡ‡‡ ǣ‡…‡–ƒ†˜ƒ…‡•‹‹†‹‘’ƒ–Š‹…’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ—„‡”…‡•’‹”‹•ȋ‡‘—ŽȌʹͲͳ͵Ǣ͹ͶǣͳǦ͸Ǥ ƒ™•‘ǡ”‘••‘ ǡ‘Ž‘•—Š‹ǡ‘Ž†ƒ ǡŠ‡‰ǡƒ‡ǡŽƒ…™‡ŽŽǡ—ǡƒ”‹ǡƒ”‡ ǡ‹ŽŽ‡”

ǡ‘›† ǡŽƒ…™‡ŽŽǣ†‘’Žƒ•‹…”‡–‹…—Ž—•–”‡••‹ƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•‹•’”‘‹‡– ‹  ǣƒ••‘…‹ƒ–‹‘™‹–ŠƒŽ–‡”‡†•—”ˆƒ…–ƒ–’”‘–‡‹’”‘…‡••‹‰ƒ†Š‡”’‡•˜‹”—•‹ˆ‡…–‹‘Ǥ Š›•‹‘Ž—‰ ‡ŽŽ‘ŽŠ›•‹‘ŽʹͲͲͺǢʹͻͶǣͳͳͳͻǦͳͳʹ͸Ǥ ”‘’•‹ ǡƒ™•‘ǡŽƒ…™‡ŽŽǣ‹‰Š–’Žƒ…‡ǡ”‹‰Š––‹‡ǣ–Š‡‡˜‘Ž˜‹‰”‘Ž‡‘ˆŠ‡”’‡•˜‹”—•‹ˆ‡…–‹‘ƒ•ƒ Dz•‡…‘†Š‹–dz‹‹†‹‘’ƒ–Š‹…’—Ž‘ƒ”›ϐ‹„”‘•‹•Ǥ Š›•‹‘Ž—‰‡ŽŽ‘ŽŠ›•‹‘ŽʹͲͳʹǢ͵ͲʹǣͶͶͳǦͶͶͶǤ ƒ••‡”‹ǡŠ‘–žǡŠƒ”ƒǡ—•–‹ǣŠ‡…Š‡‹…ƒŽ…Šƒ’‡”‘‡ͶǦ’Š‡›Ž„—–›”ƒ–‡‹Š‹„‹–•ƒ†‹’‘‰‡‡•‹• „›‘†—Žƒ–‹‰–Š‡—ˆ‘Ž†‡†’”‘–‡‹”‡•’‘•‡Ǥ ‹’‹†‡•ʹͲͲͻǢͷͲǣʹͶͺ͸ǦʹͷͲͳǤ Š‘‰ǡŠ‘—ǡǡ‹‘‘ǡ‹—ǡƒˆƒŽ˜‹ǡ”‹•Šƒ˜‡‹ǡ—„‘—”†ǡ‡ƒ‹‘ǡ‹ŽŽ‹•ǡ ‹ ǡ†—‘‹•ǡ”ƒ†ƒŽŽǡ‡‡”• ǡ‘”‘ǣ‘Ž‡‘ˆ‡†‘’Žƒ•‹…”‡–‹…—Ž—•–”‡••‹‡’‹–Š‡Ž‹ƒŽǦ ‡•‡…Š›ƒŽ–”ƒ•‹–‹‘‘ˆƒŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•ǣ‡ˆˆ‡…–•‘ˆ‹•ˆ‘Ž†‡†•—”ˆƒ…–ƒ–’”‘–‡‹Ǥ ‡•’‹”‡ŽŽ ‘Ž‹‘ŽʹͲͳͳǢͶͷǣͶͻͺǦͷͲͻǤ ƒŒ‘”‡ǡŠ‡‰ǡ‡‰”›•‡ǡ‘œ ǡ„†‘Ž”ƒ•—Ž‹ƒǡƒ™•‘ǡŽƒ…™‡ŽŽǣŽ˜‡‘Žƒ”‡’‹–Š‡Ž‹ƒŽ …‡ŽŽ•—†‡”‰‘‡’‹–Š‡Ž‹ƒŽǦ–‘Ǧ‡•‡…Š›ƒŽ–”ƒ•‹–‹‘‹”‡•’‘•‡–‘‡†‘’Žƒ•‹…”‡–‹…—Ž—•–”‡••Ǥ ‹‘Ž Š‡ʹͲͳͳǢʹͺ͸ǣ͵Ͳͻ͹ʹǦ͵ͲͻͺͲǤ ‘‘ǡ‹ǡŠ‘ǡ ƒ‰ ǡ‡‡ǣ†‘’Žƒ•‹…”‡–‹…—Ž—•–”‡••‹†—…‡•‡’‹–Š‡Ž‹ƒŽǦ‡•‡…Š›ƒŽ –”ƒ•‹–‹‘–Š”‘—‰Šƒ—–‘’Šƒ‰›˜‹ƒƒ…–‹˜ƒ–‹‘‘ˆ…Ǧ”…‹ƒ•‡Ǥ‡’Š”‘š’‡’Š”‘ŽʹͲͳͶǢͳʹ͸ǣͳʹ͹ǦͳͶͲǤ Šƒ‘ǡ‹ǡƒ‘ ǡŠ‡ǡƒǡŠƒ‰ǡ—ǣŠ‡›Ž„—–›”‹…ƒ…‹†‹Š‹„‹–•‡’‹–Š‡Ž‹ƒŽǦ ‡•‡…Š›ƒŽ–”ƒ•‹–‹‘†—”‹‰„Ž‡‘›…‹Ǧ‹†—…‡†Ž—‰ϐ‹„”‘•‹•Ǥ‘š‹…‘Ž‡––ʹͲͳͷǢʹ͵ʹǣʹͳ͵ǦʹʹͲǤ ƒŠƒŽ‹ǡƒ”…ƒ”ǡŠ‹ƒ‹›ƒǣŠ‡‡‡”‰‹‰”‘Ž‡‘ˆŠ‹•–‘‡†‡ƒ…‡–›Žƒ•‡•ȋ•Ȍ‹”‡‰—Žƒ–‹‘Ǥ ‡–Š‘†•œ›‘ŽʹͲͳͳǢͶͻͲǣͳͷͻǦͳ͹ͶǤ Š‘ ǡ‘‘ǡƒ” ǡ ǡƒ‰ ǡ‹ǡ‡‡ǡƒ‰ ǡ‡‡ǣˆˆ‡…–•‘ˆŠ‹•–‘‡†‡ƒ…‡–›Žƒ•‡ ‹Š‹„‹–‘”‘‡š–”ƒ…‡ŽŽ—Žƒ”ƒ–”‹š’”‘†—…–‹‘‹Š—ƒƒ•ƒŽ’‘Ž›’‘”‰ƒ…—Ž–—”‡•Ǥ Š‹‘ŽŽŽ‡”‰› ʹͲͳ͵Ǣʹ͹ǣͳͺǦʹ͵Ǥ —‘ ǡŠ—ƒ‰ǡ‹†‡•ǡƒ…Š‡œ ǡŠƒǡŠ‹–‡ǡƒ•› ǣ”•‡‹…–”‹‘š‹†‡‹Š‹„‹–•–”ƒ•ˆ‘”‹‰ ‰”‘™–Šˆƒ…–‘”ǦȾͳǦ‹†—…‡†ϐ‹„”‘„Žƒ•––‘›‘ϐ‹„”‘„Žƒ•–†‹ˆˆ‡”‡–‹ƒ–‹‘in vitroƒ†„Ž‡‘›…‹‹†—…‡†Ž—‰ ϐ‹„”‘•‹•in vivoǤ‡•’‹”‡•ʹͲͳͶǢͳͷǣͷͳǤ ‹ƒ‘ǡŠ‡ǡ‹—ǡ—‘ǡ‡ǡ‹—ǣ”‹…Š‘•–ƒ–‹ǡƒŠ‹•–‘‡†‡ƒ…‡–›Žƒ•‡‹Š‹„‹–‘”ǡ•—’’”‡••‡• ’”‘Ž‹ˆ‡”ƒ–‹‘ƒ†‡’‹–Š‡Ž‹ƒŽȂ‡•‡…Š›ƒŽ–”ƒ•‹–‹‘‹”‡–‹ƒŽ’‹‰‡–‡’‹–Š‡Ž‹—…‡ŽŽ•Ǥ ‡ŽŽ‘Ž‡† ʹͲͳͶǢͳͺǣ͸Ͷ͸Ǧ͸ͷͷǤ ƒŠƒŽ‹ǡƒ”…ƒ”ǡ ƒ‰ǡ‹ŽŽ‹ƒ•ǡ‘‘‡ ǡ‘ϐ‹Ž‘ ǡŠ‹ƒ‹›ƒǣ…–‹˜ƒ–‹‘‘ˆ–Š‡—ˆ‘Ž†‡† ’”‘–‡‹”‡•’‘•‡…‘–”‹„—–‡•–‘™ƒ”†–Š‡ƒ–‹–—‘”ƒ…–‹˜‹–›‘ˆ˜‘”‹ƒ•–ƒ–Ǥ‡‘’Žƒ•‹ƒʹͲͳͲǢͳʹǣͺͲǦͺ͸Ǥ

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ʹͺ ʹͻ

͵Ͳ

ƒ‘ǡƒŽŽ—”‹ǡ‘ŽŠ‡ǡƒ‰ǡ ‹•—•ǡŠ‡ ǡƒǡ—…Ž‡›ǡƒŽ—•—ǡ‘‘–Šƒƒ†ƒ•™ƒ›ǡ ‘•Š‹ ǡ–ƒ†ŒƒǡŠƒŽŽƒǣ”‡ƒ–‡–™‹–Š’ƒ‘„‹‘•–ƒ–‹†—…‡• Ž—…‘•‡Ǧ‡‰—Žƒ–‡†”‘–‡‹͹ͺƒ…‡–›Žƒ–‹‘ƒ† ‡†‘’Žƒ•‹…”‡–‹…—Ž—•–”‡••‹„”‡ƒ•–…ƒ…‡”…‡ŽŽ•Ǥ‘Žƒ…‡”Š‡”ʹͲͳͲǢͻǣͻͶʹǦͻͷʹǤ ƒŠƒŽ‹ǡƒ”…ƒ”ǡ”ƒ„Š—ǡ‡–‘ǡŠ‹ƒ‹›ƒǣŽƒ•• Š‹•–‘‡†‡ƒ…‡–›Žƒ•‡•Ž‘…ƒŽ‹œ‡–‘–Š‡‡†‘’Žƒ•‹… ”‡–‹…—Ž—ƒ†‘†—Žƒ–‡–Š‡—ˆ‘Ž†‡†’”‘–‡‹”‡•’‘•‡Ǥ  ʹͲͳʹǢʹ͸ǣʹͶ͵͹ǦʹͶͶͷǤ Š‘‹ǡ‡‡ ǡ—”œǡƒ•‡ ǡ›—ǡ‹ ǡ‹ǡ ‹ǡ‹ƒ‘ǡ ‡‘‰ǣŽƒ•• • •’‡…‹ϐ‹…ƒŽŽ›”‡‰—Žƒ–‡Ǧ…ƒ†Š‡”‹‡š’”‡••‹‘‹Š—ƒ”‡ƒŽ‡’‹–Š‡Ž‹ƒŽ…‡ŽŽ•Ǥ ‡ŽŽ‘Ž‡†ʹͲͳ͸ǢʹͲǣʹʹͺͻǦ ʹʹͻͺǤ ƒ‰ǡ‡‡ǡ‹ ǡ‹ǡ‹ǡ‹ǡƒ‰ǡ ƒ‰ǡ‡‘ǣŠ‡”ƒ’‡—–‹…ƒ…–‹˜‹–›‘ˆ–Š‡ Š‹•–‘‡†‡ƒ…‡–›Žƒ•‡‹Š‹„‹–‘”ͻ͵ͻ‘”‡ƒŽϐ‹„”‘•‹•Ǥ – —‘’Šƒ”ƒ…‘ŽʹͲͳ͹ǢͶʹǣʹͷǦ͵ͳǤ Š‡ǡ‹ƒ‘ǡƒ‰ǡ—‘ǡ‡ǡ‹—ǣŠ‡…‘’Ž‡š‹–‡”’Žƒ›„‡–™‡‡ͳȀʹǡ ȾȀƒ†ǡƒ†

ƒ‰‰‡†Ȁ‘–…Š•‹‰ƒŽ‹‰’ƒ–Š™ƒ›•‹–Š‡”‡‰—Žƒ–‹‘‘ˆ‡’‹–Š‡Ž‹ƒŽǦ‡•‡…Š›ƒŽ–”ƒ•‹–‹‘‹”‡–‹ƒŽ’‹‰‡– ‡’‹–Š‡Ž‹—…‡ŽŽ•Ǥ‘‡ʹͲͳͶǢͻǣ‡ͻ͸͵͸ͷǤ ‹ƒ‰ǡŠƒ‰ǡƒ‰ǡ‹ǣͳ‘†—Žƒ–‡• ǦȾͳǦ †—…‡† †‘–Š‡Ž‹ƒŽǦ–‘Ǧ‡•‡…Š›ƒŽ”ƒ•‹–‹‘–Š”‘—‰Š‘™”‡‰—Žƒ–‹‘‘ˆ‹ǦͳͶͷǤ‡ŽŽ Š›•‹‘Ž‹‘…Š‡ʹͲͳ͹ǢͶʹǣ͵ͷ͹Ǧ͵͹ʹǤ ‘‰ǡŠƒ‰ǡ‡ǡ— ǡ‹ǡ—ǡƒ‰ǡ‹ǡƒ‘ ǣ›‡”‰›‘ˆ Ǧʹ͹ƒ† ǦȽ‹”‡‰—Žƒ–‹‰ ͳͲ‡š’”‡••‹‘‹Ž—‰ϐ‹„”‘„Žƒ•–•Ǥ ‡•’‹”‡ŽŽ‘Ž‹‘ŽʹͲͳ͵ǢͶͺǣͷͳͺǦͷ͵ͲǤ ‹ǡŠ‡ǡŠƒ‰ǡƒ‰ǡ‘‰ǡ—ǡ‡ǡ‹ǡƒ‘ ǣ ƒ†–•‹‰ƒŽ‹‰’ƒ–Š™ƒ›•”‡‰—Žƒ–‹‰  ǦȽǦ‹†—…‡† Ǧ͵ʹ‡š’”‡••‹‘‹Š—ƒŽ—‰ϐ‹„”‘„Žƒ•–•ǣ‹’Ž‹…ƒ–‹‘•‹ƒ‹”™ƒ›‹ϐŽƒƒ–‹‘Ǥ —‘Ž‘‰›ʹͲͳͷǢͳͶͶǣʹͺʹǦʹͻͲǤ ƒ‹–”ƒǡƒ‘ǡ ƒ”…‹ƒǣ—–ƒ–•—”ˆƒ…–ƒ–ʹ’”‘–‡‹•ƒ••‘…‹ƒ–‡†™‹–Šˆƒ‹Ž‹ƒŽ’—Ž‘ƒ”›ϐ‹„”‘•‹• ƒ†Ž—‰…ƒ…‡”‹†—…‡ ǦȾͳ•‡…”‡–‹‘Ǥ”‘…ƒ–Ž…ƒ†…‹ʹͲͳʹǢͳͲͻǣʹͳͲ͸ͶǦʹͳͲ͸ͻǤ

1834