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1 DOI: 1.13/n7 Reltive Pprg mrna Time (weeks) Interspulr Inguinl Epididyml Reltive undne Lipid droplet size ( m ) >5 weeks 1 week weeks 3 weeks weeks weeks 9 weeks Reltive undne Time (weeks) smll (-7 m ) medium (7-1 m ) lrge (1-5 m ) very lrge (>5 m ) Figure S1 Britening of inguinl dipose tissue during 1-week old stimultion t C is reversile within 5 weeks t 3 C. ) Young dult (-9 weeks) mle C57BL/ mie were old-stimulted for 7 dys t C nd susequently returned to regulr housing temperture of 3 C. Gene expression of the generl dipoyte mrker Pprg in different dipose tissue depots during old nd susequent wrm dpttion s in Fig. 1 is lrgely onstnt during temperture dpttion. Men ± s.e.m. of n = nimls. Soure dt is provided in SI tle 5. -) Quntifition of the ltered lipid droplet distriution of HE-stined prffin setions of inguinl ft during temperture dpttion s in Fig. 1. ) The sizes of the lipid droplets were determined y utomted imge nlysis nd lssified into size ins. Smll lipid droplets re typil of multiloulr rown/rite dipoytes forming during old stimultion, medium sized re typil of white dipoytes in younger mie with lrge lipid droplets developing t inresing ge. Men of n = nimls (> ojets per niml). Soure dt is provided in SI tle 5. ) Reltive undne of smll, medium nd lrge lipid droplets over temperture dpttion time ourse s in Fig. 1. The old-indued inrese in rown/rite dipoyte-typil smll lipid droplets is ompletely reversed fter 5 weeks. Men ± s.e.m. of n = nimls (> ojets per niml). Soure dt is provided in SI tle Mmilln Pulishers Limited. All rights reserved.

2 frt site promoter HBEGF- pa pgk neo-kn pgk pa 3 UTR promoter CreER T pa pgk neo-kn pgk pa 3 UTR Trnsgeni Cold-stimulted Control Trnsgeni Cold-stimulted Control Interspulr Epipidyml Tuulin Tuulin Inguinl Skeletl musle Tuulin Tuulin Figure S Bteril rtifiil onstruts (BACs) for novel trnsgeni mouse strins nd hrteristion of Up1-GFP strin. -) Sheme depiting the BAC onstruts used for genertion of the Up1-GFP () nd Up1- CreER () mouse lines. HBEGF: hepring-inding epiderml growth ftor-like growth ftor, : enhned green fluoresent protein, pa: ovine polya sequene, pgk: phosphoglyerte kinse promoter, neo-kn: neomyin-knmyin resistne ssette, pgk pa: phosphoglyerte kinse polya sequene, UTR: untrnslted region, CreER T : seond genertion of tmoxifen-induile Cre reominse estrogen reeptor fusion gene. ) Chrteristion of the Up1-GFP mouse strin. Adult mle Up1-GFP trnsgeni mie nd wildtype littermtes were kept t 3 C or oldstimulted for 7 dys t C. Tissues were lysed nd nlysed y Western Blotting for, nd tuulin. Control: diluted lyste of Up1- GFP mie. Unropped dt is depited in supplementry Fig. 7-f Mmilln Pulishers Limited. All rights reserved.

3 ROSA NEO - STOP Cre-medited reomintion tdrfp loxp site loxp 7 site unreomined lous primer reomined lous primer ROSA tdrfp % of totl ells 3 1 drenls interspulr Reomined ells rin olon epididyml hert inguinl intestine kidney liver - tmoxifen tmoxifen lung pnres skeletl musle spleen thymus % of totl ells 3 1 interspulr Reomined ells inguinl epididyml - old old e f g Reomined ells d % of totl ells 3 1 Reomined ells 1 3 Tmoxifen diet feeding (dys) % of totl ells Tmoxifen gvge 3 Tmoxifen how 1 Interspulr Inguinl Epididyml Reltive Up1 mrna Interspulr Inguinl Epididyml Reltive Rfp mrna Interspulr Inguinl Epididyml h % of totl ells 3 1 Interspulr Reomined ells Inguinl Epididyml Tmoxifen during old Tmoxifen prior to old i Interspulr weight (mg) old old Figure S3 Chrteristion of the Up1-CreER mouse strin. ) Sheme depiting genomi modifition of ROSA-tdRFP mie y Cre reominse nd primer inding sites used for quntifition of reomintion. ) Perentge of ells tht underwent genomi reomintion in vrious tissues of Up1-CreER x ROSA-tdRFP nimls with or without tmoxifen indution. Men s.e.m. of n = 5 nimls, P <.1. ) Perentge of ells tht underwent genomi reomintion in dipose tissues of Up1- CreER x ROSA-tdRFP nimls fter tmoxifen indution with or without 1 week old stimultion. Men s.e.m. of n = 5 nimls for unstimulted nd n = nimls for stimulted, P <.1. d) Cre-medited genomi reomintion in interspulr during tmoxifen dministrtion y diet for up to 3 dys nd sturtion urve fit. Men s.e.m. of n = nimls (dy 9), n = 3 nimls (dy 13), n = nimls (dys 1, 3, nd 3) nd n = 5 nimls (dy 1). Soure dt is provided in SI tle 5. e) Comprison of the Cre-medited genomi reomintion rte in different dipose tissues fter different routes of tmoxifen dministrtion. Young dult mie were either gvged with doses of. mg tmoxifen on 5 onseutive dys or fed tmoxifen how diet for 1 dys. Men s.e.m. of n = nimls for epididyml on tmoxifen how nd n = 3 nimls for ll others, not signifint. Soure dt is provided in SI tle 5. f-g) Rfp nd Up1 expression in seprted nd dipoyte frtions of dipose tissues of old-stimulted tmoxifen-treted Up1-CreER x ROSA-tdRFP mie. Men s.e.m. of n = 7 extrts (derived from pooled dipose tissues of mie eh), P <.1. h) Comprison of the Cre-medited genomi reomintion rte in different dipose tissues with either onurrent or former tmoxifen dministrtion. Young dult mie were either gvged with doses of.5 mg tmoxifen on 5 onseutive dys nd old-stimulted weeks lter or dult mie without prior tmoxifen dministrtion were oldstimulted in prllel with the other group while reeiving these doses of tmoxifen. Animls were srified diretly fter old stimultion. Men s.e.m. of n = 7 nimls for inguinl nd n = nimls for interspulr nd epididyml, P <.1. i) Tissue weight of interspulr in young dult mie with or without one week old stimultion. Men s.e.m. of n = 5 nimls Mmilln Pulishers Limited. All rights reserved.

4 Up1-CreER Up1-GFP ROSA-tdRFP Primry ntiody DAPI GFP RFP Alex 7 () Figure S The expression of memrne-ound nd solule RFP in rown dipoytes of Up1-Trer trnsgeni nimls is highly effiient. Adult mle Up1-Trer mie were old-stimulted for 7 dys t C. The interspulr ws ut into semi-thin ryosetions, immunohistohemilly stined for (Alex7-oupled seondry ntiody) nd ounterstined with DAPI. The lower three rows depit imges of from littermte nimls tht were either Up1-GFP or ROSA-tdRFP negtive (rows nd 3) or stining ontrols without primry ntiody (row ). Size r = 1 µm Mmilln Pulishers Limited. All rights reserved.

5 Temperture ( C) 1 5 Tmoxifen indution Time (weeks) Reltive Cide mrna Reltive Cox71 mrna Unstimulted ontrol weeks post stimultion Interspulr Inguinl Epididyml Interspulr Inguinl Epididyml Figure S5 Cold-indued inrese in rite-typil gene expression in inguinl dipose tissue is reversed during whitening. ) Environmentl temperture nd tmoxifen indution during rite dipoyte tring study. Young dult (-9 weeks) mle Up1-CreER x ROSA-tdRFP mie were fed tmoxifen diet nd old-stimulted for 7 dys t C eginning 3 dys fter the diet hnge. After the old stimultion the nimls were returned to regulr housing temperture of 3 C on tmoxifen-free diet. -) Former rite dipoytes lose the rown dipoyte-typil gene expression during whitening. Young dult mle Up1-CreER x ROSA-tdRFP mie were oldstimulted on tmoxifen diet nd returned to regulr housing temperture on tmoxifen-free diet susequently s depited in (). Gene expression of Cide nd Rfp in the nd dipoyte frtions of different dipose tissues ws determined weeks fter the old stimultion (Up1 nd Rfp levels re depited in Fig. f,g). The unstimulted ontrol nimls were fed tmoxifen diet simultneously ut were mintined t 3 C onstntly. Men s.e.m. of n = extrts for ontrol nd n = 7 extrts for post stimultion smples (derived from pooled dipose tissues of mie eh). not signifint Mmilln Pulishers Limited. All rights reserved.

6 FSC-A d RFP AAD AAD Figure S Gting strtegy for dipoyte sortings y flow ytometry. Representtive lots for the seprtion of RFP nd RFP - dipoytes from inguinl dipose tissue. Adult mle Up1-CreER x ROSA-tdRFP mie were old-stimulted for 7 dys t C nd dipose tissues were seprted into nd dipoyte frtions y entrifugtion. ) Stter plot of inguinl dipose tissue with gte for dipoyte popultion. ) Histogrm of the gted popultion depiting RFP nd RFP - popultions. -d) Stter plots of the seleted RFP () nd RFP - (d) popultions with gting for 7-AAD negtive (live) dipoytes onstituting the sorted ojets Mmilln Pulishers Limited. All rights reserved.

7 ing epi 7 musle d ing epi 7 musle e 7 7 ing epi musle f ing epi musle Figure S7 Unropped Western lot dt. -) Unropped primry dt of the Western Blots depited in Fig. 1f. ) FABP, ) GAPDH nd. -f) Unropped primry dt of the Western Blots depited in supplementry Fig.. ) short exposure, d) Tuulin, e) long exposure, f). ing: inguinl dipose tissue, epi: epididyml dipose tissue Mmilln Pulishers Limited. All rights reserved.

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