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1 X p -lu c ct ivi ty doi:.8/nture8 S CsA - THA + DAPI Merge FSK THA TUN Supplementry Figure : A. Ad-Xp luc ctivity in primry heptocytes exposed to FSK, THA, or TUN s indicted. Luciferse ctivity normlized to Ad- RSV βgl ctivity. Averge from three independent experiments shown (P <.). B. Immunocytochemicl nlysis of endogenous nd α stining in primry heptocytes exposed to THA. Pre-tretment with CsA indicted. Scle r; µm.
2 doi:.8/nture8 S (gi6) Coverge: 6.% K.SSSVPPYLR.D R.NVGSDIAVLR.R K.EAQDTSDGIIQK.N R.NSGSELQVYYASPR.S K.AEPQPLSPASSSYSVSSPR.S G S T WT F F CBD F F F F GST pull-down 6 HA/ Input HA/ IP IgG % input - + HA/ Coomssie stining Supplementry Figure : A. Top, α peptide sequences recovered from immunoprecipittes of endogenous prepred from HEK9T cells. Percent coverge indicted. Bottom, immunolot showing recovery of HA-tgged α from IPs of endogenous prepred from HEK9T cells. B. Top, immunolot of HA-tgged α recovered from HEK9T lystes following pull-down ssy with recominnt GST- polypeptide frgments (F-F6). Amino cid endpoints for ech frgment indicted. Bottom, Coomssie stined gel showing GST- peptide frgments employed in GST pull down ssys.
3 doi:.8/nture8 S TCL Cyto M / N LDM PM HDM KDEL DAPI Merge IB: nti- IB: nti-kdel EEA DAPI Merge IB: nti-eea IB: nti-tgn6 TGN6 DAPI Merge IB: nti-cyto. C Cyto. C DAPI Merge IB: nti-tuulin IB: nti- c Trypsin (min) d KDEL DAPI Merge GRP9 Supplementry Figure : is peripherlly ssocited with the endoplsmic reticulum. A. Immunolots of sucellulr frctions prepred from CRL-89 slivry glnd cells. protein mounts in cytoplsmic (cyto), mitochondril nd nucler (M/N), high or low density microsoml (HDM, LDM) nd plsm memrne (PM) frctions reltive to totl cell lyste (TCL) shown. Co-frctiontion with ER (KDEL), endosoml (EEA), Golgi (TGN6), mitochondril (cyto C), nucler (), or cytoplsmic (tuulin) mrkers shown. B. Immunocytochemicl studies of CRL slivry glnd cells showing reltive colocliztion of with sucellulr mrkers in A. Scle rs: µm. C. Immunolot showing reltive protese sensitivity of nd ER-resident GRP9 proteins in ER-contining HDM frctions incuted with trypsin. D. Immunocytochemicl nlysis of cultured mouse primry heptocytes stined with nti- or ER specific nti-kdel ntiserum. Scle r: µm.
4 doi:.8/nture8 S X p- lu c c tiv it y Insulin- Insulin+ THA TUN lu y Xp- c ctivit 7 6 SIK SIKi THA TUN c lu y Xp- c ctivit N/ (S7A) (S7A) N THA TUN d Ad li Fsted Xp-luc Supplementry Figure : A. Effect of insulin (nm) on Xp-luc reporter ctivity in heptocytes. Cells were exposed to TUN, THA, or vehicle for 8 hours, followed y incution with insulin for hours. (P <.; P <.; n=). B. Effect of Adenovirl SIK over-expression or RNAi-medited knockdown on Xp-luc reporter ctivity in primry heptocytes exposed to THA or TUN s indicted. (P <.; P <.; n=). C. Ad-Xp luc reporter ctivity in heptocytes expressing ctive N nd phosphoryltion-defective, ctive Ser7Al. Exposure to THA or TUN indicted. (P <.; P <.; n=). D. Live imging nlysis of d liitum fed nd 6 hour fsted mice expressing denovirlly encoded Xp-luciferse reporter.
5 doi:.8/nture8 S Fsted Refed Xp-luc Fsted Refed Fsted Refed (p) Supplementry Figure : A. Live imging nlysis showing effect of IP TUN dministrtion on Xp- luc ctivity in 8 hour fsted compred to hour refed mice. B. Immunolot of, α, nd proteins in liver extrcts from fsted nd refed mice. S6 h Xp-luc h X p - l uc cti vi ty 7 6 Supplementry Figure 6: Live imging (left) nd quntittive nlysis (right, P< h h.; P <.; n=) of Xp- luc ctivity in mice or hours following IP dministrtion of tunicmycin or DMSO vehicle.
6 mrna levels doi:.8/nture8 S7 USi Xp-luc i X p -luc ctivity USi i THA TUN c USi i Xp d USi i TUN: (p) Xp Supplementry Figure 7: A. nd B. Effect of denovirlly encoded α RNAi on Xp-luc reporter ctivity in liver (A) nd primry heptocytes (B, P <.; P<.; n=) exposed to THA or TUN s indicted. C. Q-PCR nlysis of heptic Xp nd mrna mounts in control (USi) nd i expressing mice. D. Immunolot showing full-length nd proteolyticlly cleved (p) α s well s XBP proteins in heptic extrcts from mice depleted of α y RNAi-medited knockdown (i) reltive to controls following IP dministrtion of TUN or DMSO vehicle s indicted. 6
7 doi:.8/nture8 S8 TUN: HA/ TUN: USi i p S9 Supplementry Figure 8: Immunolots showing heptic protein mounts in mice injected with denoviruses encoding HA-tgged (A) or RNAi (B) s shown in figure (pnels C nd D). Intrperitonel (IP) dministrtion of TUN or control vehicle indicted. 7
8 doi:.8/nture8 S9 N N N FSK: c Reltive ChI P s ig nl (G6Pse) IP IgG % input 8 6 USi i p FSK- FSK+ (p) CRE - luc ctivity 8 N /N (S7A) (S7A)/N FSK- FSK+ S Supplementry Figure 9: α disrupts : dependent trnscription. A. Immunolot showing recovery of from IPs of prepred from nucler extrcts of primry heptocytes expressing denovirl N or green fluorescent protein () control. B. Effect of N expression on Ad-CRE luc ctivity in primry heptocytes co-expressing either wild-type or phosphoryltion-defective, ctive Ser7Al. Exposure to FSK indicted. (P <.; P <.; n=). C. ChIP ssy of primry heptocytes showing no effect of over-expression or RNAi medited depletion (i) on occupncy of phospho(ser) in cells exposed to FSK or DMSO vehicle (P <.; n=). USi, cells expressing with denovirlly encoded unspecific RNAi. 8
9 doi:.8/nture8 p S h: m: h : m : RRKKKEYVKCLENR RRKKKEYVKCLENR RKKKKEYMLGLEAR RKKKKEYMLGLEAR 7 VAVLE VAVLE LKA AL LKA AL Co n IgG R/ A WT IP R/ A WT Input R/A WT HA/ c T r ov y A F6 ec er..8. WT R/ A d X p- lu c c tiv it y WT R/A e CR E - l uc c t ivi ty 8 FSK- FSK+ WT R/A Supplementry Figure : Chrcteriztion of interction-defective mutnt protein. A. Alignment of nd α sequences in the ZIP domins, showing conservtion t Arg in nd Arg7 in humn α. B nd C. Co-immunoprecipittion ssy (B) nd densitometric nlysis (C) showing recovery of wild-type nd Arg7Al (R/A) mutnt α proteins from IPs of. D. nd E. Trnsient ssy of HEK9T cells showing reltive effects of wild-type α nd R/A mutnt α on Xp-luc (D) or CRE-luc (E) reporter ctivity in cells exposed to TUN or FSK s indicted (P<.; n=). 9
10 doi:.8/nture8 S X p -l u c c ti vi t y THA TUN C R E -l uc c t ivi ty N /N FSK- FSK+ S Supplementry Figure : ntgonizes α ctivity. A. nd B. Effect of denovirl over-expression on Xp-luc (A) nd CRE-luc (B) reporter ctivities in primry heptocytes exposed to ER stress ctivtors (TUN, THA; pnel A) or FSK (pnel B). Effect of in cells co-expressing N (B) indicted. (P<.; n=).
11 doi:.8/nture8 S TUN: (p) TUN: USi - + i - + (p) Supplementry Figure : Immunolots showing effect of denovirl α over-expression (A) or RNAi-medited depletion (B) on α protein mounts in fsted mice, s depicted in figure (pnels C nd D). Amounts of dephosphorylted ctive shown. Mice were nlyzed hours fter IP TUN or vehicle dministrtion.
12 Blood glucose (m g/ dl) doi:.8/nture8 S Len o/o Xp-luc CRE-luc Len o/o (p) p Xp-S Xp-U pjnk 6 Len o/ o d/d m R NA levels Len G6Pse PEPCK o/o d/d Supplementry Figure : Top left, live imging nlysis of Ad-Xp luc nd JNK JNK peif lph eif lph Ad-CRE luc ctivities in oese o/o mice reltive to len controls. Top right, immunolot of heptic extrcts from wild-type nd o/o mice showing mounts of full length nd cleved α (p) s well s other ER stress mrkers (Xp, phospho JNK, EIFlph (phospho nd totl)). Reltive mounts of nd lso shown. Bottom left, circulting glucose concentrtions (left) nd mrna mounts for gluconeogenic genes (right) in o/o, nd d/d mice reltive to len controls (P<.; P <.; n=8).
13 doi:.8/nture8 S Len N d/d N (p) Len N DIO N (p) S Supplementry Figure : Immunolots showing α (p), dephosphorylted, nd protein mounts in heptic extrcts from control () nd N over-expressing d/d (A) or high ft diet fed (B; DIO) mice reltive to len nimls under fsted conditions. Metolic effects of N over-expression shown in figure (Pnels B nd C).
14 Blood glucose (m g/ dl) Blood glucose (m g/ dl ) doi:.8/nture8 S Len N d/d N pjnk JNK pirs (Ser7) IRS pakt (Thr8) Akt Len N DIO N pjnk JNK pirs (Ser7) IRS pakt (Thr8) Akt c 7 6 Len/ Len/N d/d/ d/d/n 6 9 Time fter glucose (min) d Len/ Len/N DIO/ DIO/N 6 9 Time fter glucose (min) Supplementry Figure : Effect of heptic N expression on insulin signling nd glucose tolernce. A. nd B. Immunolot nlysis of liver extrcts from d/d (A) or high ft diet fed (B; DIO) mice reltive to len controls. Effect of denovirl N or control indicted. C. nd D. Glucose tolernce testing of len compred to d/d (C) or high ft diet fed (D) oese mice expressing denovirlly encoded N or control protein. Mice were injected IP with glucose ( g/kg ody weight), nd circulting lood glucose concentrtions were monitored s shown (P <.; P <.; n=).
15 doi:.8/nture8 Time fter glucose (min) Time fter glucose (min) S6 Fsting ER stress Oesity Gluconeogenesis Gluconeogenesis Gluconeogenesis ER qulity control ER qulity control ER qulity control NUCLEUS NUCLEUS NUCLEUS Supplementry Figure 6: Cross-tlk etween ER stress nd fsting pthwys t the level of trnscriptionl coctivtor. ER stress nd camp pthwys trigger dephosphoryltion nd nucler trnsloction of. ER stress stimultes the expression of ER qulity control genes y promoting the ssocition of with α, while fsting signls increse gluconeogenic gene expression y incresing the : interction. As consequence of its ility to ind to, α down-regultes the gluconeogenic progrm y interfering with the : interction. The cross-tlk etween these pthwys ppers criticl for glucose homeostsis ecuse reductions in heptic α protein mounts in oesity promote reciprocl increses in gluconeogenic gene expression nd in heptic glucose output.
* * * * * liver kidney ileum. Supplementary Fig.S1
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