SUPPLEMENTARY INFORMATION

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1 { OI: 1.138/n31 Srifie n nlyze APs on week 1 s of iet High-ft iet BrU High-ft iet BrU 4 High-ft iet BrU 6 High-ft iet BrU Lin - Lin - : C34 + : C C45 1 C S C4- Aipoyte preursors (APs) C4+ Count 3 1 C C31 C9 C4 PgfRα-HB-GFP All single ells All single ells GFP GFP -.3 ±.1% 7.8 ±.% GFP - GFP ± 6.3 ±.5%.5% 1.6 ±.% 7. ±.1% C9 C C ±.1% 1. ± 1.9% C9 GFP +.8 ±.4%.4 ± 1.% 3.8 ± 1.% 54.6 ± 1.3% 86. ± 1.7% 1.8 ±.6% C9 PgfRα-HB-GFP mie GFP + ell prolifertion 1wk vs 4 ** 3 Perent BrU Positive Cells 1 VWAT Supplementry Figure 1 Chrteriztion of -inue ipoyte hyperplsi. (A) Experimentl sheme for BrU time ourse in Figure B. (B) Representtive flow ytometry ot plots initing sequentil gting use to ientify AP popultions, inluing C4 + ipoyte progenitors (C45 -, C31 -, C34 +, C9 +, S1 +, C4 + ), C4 - preipoytes (C45 -, C31 -, C34 +, C9 +, S1 +, C4 - ), n totl APs (C45 -, C31 -, C34 +, C9 +, S1 + ) (C) Flow ytometry plots of SVF from VWAT of PgfRα-HB-GFP mie showing tht the mjority of GFP + ells re C9 + ; C34 +, of whih 97.1 ± 1.5% re lso S1 +. (n = 3 mie) () Quntifition of BrU inorportion into GFP + ells from VWAT of mle PgfRα-HB-GFP mie uring 1 week of or feeing n BrU tretment. (n = 5 mie for n 7 mie for.) Signifine etween the inite groups in () ws lulte using two-tile stuent s t-test. Ext p-vlues re liste in Supplementry Tle 1. Error rs or vlues represent men ± s.e.m. ** (p<.1). : high-ft iet, : stnr iet, BrU: romoeoxyuriine Mmilln Pulishers Limite. All rights reserve

2 n BrU Pulse Chse s of iet BrU Anlyze ipoyte nulei y flow ytometry Perent BrU Positive Nulei Aipoyte Nulei n week BrU pulse VWAT SWAT 8 weeks of iet Supplementry Figure Aipoyte preursor prolifertion uring week of feeing oes not le to ipogenesis. (A) Shemti n epiting the seon week BrU pulse-hse in Supplementry Figure B. (B) BrU inorportion into ipoyte nulei fter pulse-hse from the seon week of feeing. (n = 5 for ll groups). Error rs or vlues represent men ± s.e.m. : high-ft iet, : stnr iet, BrU: romoeoxyuriine Mmilln Pulishers Limite. All rights reserve

3 pakt (T38) AKT pakt (S473) AKT Lin - : C34 + : C9 + : S-1 + Lin - : C34 + : C9 + : S µg/ul Ins.1 µg/ul Ins Wort y 3 of iet 1 µg/ul Ins.1 µg/ul Ins Wort 6 k 6 k 6 k 6 k ritrry units pakt T38/ AKT 3 ns e pakt1 (S473) ß tin y 3 of iet ritrry units pakt S473/ AKT 6 ns 4 y 3 of iet S1-negtive ells S1-positive ells 6 k 4 k no A no A nti-phospho-t38-pe nti-phospho-s473-pe Supplementry Figure 3 High-ft iet oes not inue AKT phosphoryltion in whole ft. (A) Western lot nlysis for phosphorylte AKT t the inite sites n totl AKT, in lystes from whole VWAT on y 3 of iet. Eh lne represents one niml. Unroppe lots re shown in Supplementry Figure 7I-L. (B) Quntifition of pakt western lots in (A), normlize to totl AKT. (C-) VWAT ws trete with the inite onentrtions of insulin prior to tissue igestion, n APs were isolte in the presene of wortmnnin n phosphtse inhiitors to limit further hnges in pthwy tivtion (see methos). APs were then fixe n nlyze y flow ytometry for levels of phosphorylte AKT t threonine 38 (C) or serine 473 (). The sme AP ell isoltion proeure ws use in the experiments in Figure 4B-F n 5B-C. (E) Western lot for phosphorylte AKT1 (S473) with lystes from APs enrihe from SVF vi S-1 e pull own (S1-positive) n remining unselete ells (S1-negtive) (see methos) fter 3 ys of or. Eh lne represents poole ells from mie. Unroppe lots re shown in Supplementry Figure 7M-N. Signifine etween the inite groups in (B) ws lulte using two-tile stuent s t-test. Error rs represent men ± s.e.m. : high-ft iet, : stnr iet Mmilln Pulishers Limite. All rights reserve

4 Perent BrU Positive Cells 6 4 kcl/ grm BW week SWAT Veh Wort Veh Wort Totl foo intke 4 ys **** **** Akt fl/fl PgfRα-re; Akt fl/fl PgfRα-re Akt fl/fl PgfRα-re; Akt fl/fl e Perent BrU Positive Cells Akt fl/fl PgfRα-re; Akt fl/fl AP ells S V PgfRα-re; Akt fl/fl Akt fl/fl Lin+ ells S V 1wk vs SWAT PgfRα-re Akt flox full length n Exons 4 n 5 remove Grms Boy weight 6 weeks 3 1 Akt fl/fl PgfRα-re; Akt fl/fl Supplementry Figure 4 eletion of AKT in the ipoyte linege oes not ffet oy weight or foo intke. (A) Quntifition of BrU inorportion into SWAT APs fter 1 week of or with ily injetion of wortmnnin (Wort), or vehile (Veh) (n = 5 mie for eh group). (B) PCR nlysis of NA isolte from AP ells from the VWAT n SWAT epots of femle mie show suessful exision of the floxe exons 4 n 5 from Akt gene, while Lin + ells (C45 + loo ells n C31 + enothelil ells) ontin the full-length Akt gene. Eh lne represents ells isolte from the inite epot of iniviul mie. (C) Boy weight of Akt fx/fl (n = 9 mie) n PgfRα-re; Akt fl/fl (n = 1 mie) t 6 weeks of ge. () Quntifition of foo intke normlize to oy weight in the inite groups of mie on or uring the first 4 ys of feeing. (n = 3 mie for Akt fl/fl n PgfRα-re; Akt fl/fl, n = 4 mie for PgfRα-re, n = mie for Akt fl/fl, n n = 3 mie for PgfRα-re; Akt fl/fl. (E) BrU inorportion into SWAT APs of the inite groups uring the first week of feeing ompre to ontrols. (n = 7 mie for Akt fl/fl, n = 4 mie for PgfRαre; Akt fl/fl, n = 14 mie for PgfRα-re; Akt fl/fl, n = 8 mie for Akt fl/fl, n = 5 mie for wil-type n PgfRα-re ). Signifine in () ws lulte using one-wy ANOVA with Tukey s test for multiple omprisons. Ext p-vlues re liste in Supplementry Tle 1. Sttistis soure t for 4 n e foun in Supplementry Tle. Error rs represent men ± s.e.m. ** (p<.1), **** (P<.1) Mmilln Pulishers Limite. All rights reserve

5 Foo intke (kcl) per week o/o; Akt -/- o/o Foo intke in kcl ** *** o/o o/o; Akt -/- *** s of ge Perentge of totl Foo intke (kcl/grm BW) per week Foo intke Normlize to oy weight * * o/o o/o; Akt -/ s of ge VWAT o/o * o/o; Akt -/ Aipoyte imeter (µm) Supplementry Figure 5 AKT is require for the tivtion of ipoyte preursors t the onset of oesity in o/o mie. (A-B) ly intke of in rw kilolories (kcl) (A) or kcl normlize to oy weight (B) for the inite groups of young mie fter wening t 3 weeks of ge. 3 enotes foo intke etween the ges of 3 n 4 weeks. (n = 4 for wil-type, n = 3 for o/o, n = 7-8 for o/o; Akt -/- ) (C-) H&E-stine setions of VWAT (C) n orresponing ipoyte size mesurements () for the inite groups. (n = 7 for o/o; Akt -/-, n = 4 for o/o). Signifine etween inite groups in (A-B) ws lulte using two-tile stuent s t-test. Signifine in () ws lulte using two-wy ANOVA with Bonferroni s test for multiple omprisons. Sle rs in (C) re 5μm. * or (P<.5), ** or (p<.1), *** or (P<.1), **** or (P<.1). For (A-B), * inites signifine of o/o over wil-type, inites signifine of o/o; Akt -/- over wil-type, n inites signifine of o/o; Akt -/- over o/o. Ext p-vlues re liste in Supplementry Tle 1. Sttistis soure t for 5A-B n e foun in Supplementry Tle. Error rs represent men ± s.e.m Mmilln Pulishers Limite. All rights reserve

6 SWAT P1 P3 P5 8 SWAT Akt -/- LipiTOX Isoletin IB4 Perentge of totl Akt -/- P1 ] P3 P Lipi roplet imeter (µm) 3 Ft mss 6 weeks Ft mss (grms) 1 Akt fl/fl PgfRα-re Akt fl/fl Supplementry Figure 6 AKT is not require for the norml evelopment of white ipose tissue. (A-B) Confol imges (A) of eveloping wil-type n Akt -/- SWAT stine with LipiTOX ( lipi stin) n Isoletin IB4 (n enothelil ell stin) n (B) orresponing lipi roplet size quntifition se on LipiTOX stining (n = 3 mie for ll groups exept n = 4 mie for Akt -/- P5). Eh group inlues mie from -3 ifferent litters. (C) Eho MRI quntifition of ft mss from the inite groups of mie t 6 weeks of ge. (n = 13 mie for Akt fl/fl n n = 14 mie for PgfRα-re; Akt fl/fl ). Sle rs in (A) re 5μm. Error rs represent men ± s.e.m. PX inites postntl y X Mmilln Pulishers Limite. All rights reserve

7 5A short exposure memrne ut 5A long exposure memrne ut Akt1 Atin 6 k Akt memrne ut 5 k 4 k Akt1 Atin 6 k Akt memrne ut 5 k 4 k 3 k 3 k 5B top pnel, short exposure pakt 5 k memrne ut 5B top pnel, long exposure 16 k 11 k 8 k 6 k 5 k memrne ut e 5B top pnel Akt Atin Atin 4 k 4 k f g h 5B ottom pnel, short exposure 5B ottom pnel, long exposure 5B ottom pnel 6 k 16 k 11 k 8 k 6 k pakt1 memrne ut 5 k memrne ut Akt1 Atin Atin 4 k 4 k Supplementry Figure 7 Unroppe western lots. (A-B) Western lots from the experiment in Figure 5A. (C-E) Western lots from the experiment in Figure 5B top pnel. (F-H) Western lots from the experiment in Figure 5B ottom pnel. (I-J) Western lots from the experiment in Supplementry Figure 3A top pnel. (K-L) Western lots from the experiment in Supplementry Figure 3A ottom pnel. (M-N) Western lots from the experiment in Supplementry Figure 3E Mmilln Pulishers Limite. All rights reserve

8 i j Supplementry 3A top pnel Supplementry 3A top pnel 6 k 5 k 4 k pakt (T38) 6 k 5 k 4 k Akt kgroun n k Supplementry 3A ottom pnel l Supplementry 3A ottom pnel 6 k 5 k 4 k 6 k pakt (S473) 5 k 4 k Akt kgroun n m n Supplementry 3E ottom pnel, short exposure 16 k 11 k 8 k Supplementry 3E ottom pnel, long exposure 16 k 11 k 8 k memrne ut Atin 6 k 5 k 4 k pakt1 memrne ut Atin 6 k 5 k 4 k 3 k 3 k Supplementry Figure 7 ontinue Mmilln Pulishers Limite. All rights reserve

9 Supplementry Tle 1 Ext p-vlues Figure 1 C Bseline VWAT vs VWAT.68 VWAT vs VWAT.41 VWAT vs SWAT.39 Figure B 1 week vs p <.1 multipliity-juste p-vlue C VWAT 1 week vs for C4- p =. VWAT 1 week vs for C4+ p =.9 VWAT 1 hour C4- vs C4+ p =.54 VWAT 1 hour C4- vs C4+ p =.4 VWAT 1 hour vs for C4- p =.1 VWAT 1 hour vs for C4+ p <.1 SWAT 1 hour C4- vs C4+ p =.51 SWAT 1 hour C4- vs C4+ p =. F vs.39 Figure 3 B VWAT vs for 7 weeks iet p =.7 multipliity-juste p-vlue C week 1 vs week 7 p =.74 E vs p =.8 Figure 4 A vs y 1 p =.7 vs y p =.63 vs y 3 p =.4 vs y 4 p =.15 C pakt T38 vs p =.13 pakt S473 vs p =.6 Figure 5 C pakt vs p =.18 Veh vs Wort p <.1 E PgfRα-Cre; Akt fl/fl vs Akt fl/fl p =.51 multipliity-juste p-vlue PgfRα-Cre; Akt fl/fl vs PgfRα-Cre p =.5 multipliity-juste p-vlue PgfRα-Cre; Akt fl/fl vs p =. multipliity-juste p-vlue F vs p =.11 vs PgfRα-Cre; Akt fl/fl p =. Figure 6 A vs p =.45 Akt1 vs p =.1 Akt vs p =. vs Akt p =.1 Akt1 vs Akt p <.1 o/o vs Akt-/-;o/o week 3 p <.1 multipliity-juste p-vlue o/o vs Akt-/-;o/o week 4 p <.1 multipliity-juste p-vlue o/o vs Akt-/-;o/o week 5 p <.1 multipliity-juste p-vlue o/o vs Akt-/-;o/o week 6 p <.1 multipliity-juste p-vlue E o/o vs Akt-/-;o/o week 3 p <.1 multipliity-juste p-vlue o/o vs Akt-/-;o/o week 4 p <.1 multipliity-juste p-vlue o/o vs Akt-/-;o/o week 5 p <.1 multipliity-juste p-vlue o/o vs Akt-/-;o/o week 6 p <.1 multipliity-juste p-vlue F vs o/o p =.1 Akt-/-; o/o vs o/o p =.11 G vs Akt-/-; o/o p =.31 vs o/o p <.1 Akt-/-; o/o vs o/o p <.1 Supplementry Figure 1 vs p =.89 Supplementry Figure 4 Akt fl/fl vs PgfRα-Cre p <.1 multipliity-juste p-vlue Akt fl/fl vs Akt fl/fl p =.15 multipliity-juste p-vlue Akt fl/fl vs PgfRα-Cre; Akt fl/fl p =.55 multipliity-juste p-vlue PgfRα-Cre; Akt fl/fl vs PgfRα-Cre p <.1 multipliity-juste p-vlue PgfRα-Cre; Akt fl/fl vs Akt fl/fl p =.1 multipliity-juste p-vlue PgfRα-Cre; Akt fl/fl vs PgfRα-Cre; Akt fl/fl p =.35 multipliity-juste p-vlue Supplementry Figure 5 A week 4 Akt-/-; o/o vs p =.63 week 4 o/o vs p =.1 week 4 o/o vs Akt-/-; o/o p =.1 week 5 o/o vs p =.7 week 5 Akt-/-; o/o vs p =. week 6 o/o vs p =.3 week 6 Akt-/-; o/o vs p =.4 B week 4 Akt-/-; o/o vs p =.18 week 4 o/o vs p =.151 week 5 Akt-/-; o/o vs p =.5 week 5 o/o vs p =.3 week 5 o/o vs Akt-/-; o/o p =.134 week 6 Akt-/-; o/o vs p <.1 week 6 o/o vs p =.14 week 6 o/o vs Akt-/-; o/o p =.7-3µm o/o vs Akt-/-;o/o p =.396 multipliity-juste p-vlue 3-4µm o/o vs Akt-/-;o/o p =. multipliity-juste p-vlue 4-5µm o/o vs Akt-/-;o/o p <.1 multipliity-juste p-vlue 5-6µm o/o vs Akt-/-;o/o p =.11 multipliity-juste p-vlue 6-7µm o/o vs Akt-/-;o/o p =.36 multipliity-juste p-vlue 9-1µm o/o vs Akt-/-;o/o p <.1 multipliity-juste p-vlue 1-11µm o/o vs Akt-/-;o/o p <.1 multipliity-juste p-vlue 11-1µm o/o vs Akt-/-;o/o p <.1 multipliity-juste p-vlue 1-13µm o/o vs Akt-/-;o/o p =.6 multipliity-juste p-vlue Supplementry Tle 1 Ext p-vlues. Ext p-vlues for sttistil tests tht show signifine Mmilln Pulishers Limite. All rights reserve

10 Supplementry Tle Vlues for iniviul t points with n<5 Figure B VWAT AP VWAT AP SWAT AP SWAT AP Figure 5C Figure 6 Figure 6E pakt/akt pakt1/akt Boy weight Akt-/-;o/o o/o Ft mss o/o; Akt-/- o/o Supplementry Figure 4 Akt fl/fl Foo Intke PgfRαrere PgfRα- Akt fl/fl Akt fl/fl PgfRαre; Akt fl/fl Supplementry Figure 5A Foo intke in kcl o/o week week week Foo intke in kcl o/o; Akt-/- week week week Supplementry Figure 5B Foo intke in kcl normlize to BW o/o week week week Foo intke in kcl normlize to BW o/o; Akt-/- week week week Supplementry Tle Iniviul t points. Iniviul t points re reporte for experiments with ny group ontining less thn 5 mie, n results not plotte s iniviul t points in the figures Mmilln Pulishers Limite. All rights reserve

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