S1 SUPPLEMENTARY FIGURE LEGENDS Figure S1 ...

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SLCO4A1. PRDM16. ADRB2. ELOVL3. DIO2. PGC1ACPT1B. 0. 5. 10. 15. Re la tive m. RNA. E xp re ssio n vehicle. Rosi. GQ-16 average ± SEM; *p
SUPPLEMENTARY FIGURE LEGENDS Figure S1. GQ-16 is a selective and partial agonist of PPARγ. (A) Competition of unlabeled GQ16 and rosiglitazone for PPARγLBD-bound [3H]rosiglitazone. (B) U-937 pro-monocytes, transiently cotransfected with different full-length nuclear receptors and the luciferase reporter gene under the control of DR-1TKLuc, were incubated with vehicle (DMSO), bezafibrate (100 µM), rosiglitazone (10 µM), 9-cis-retinoic (10 µM) or GQ-16 (30 µM). Figure S2. GQ-16 Induces a PPARγ conformational ensemble that is suboptimal for coactivator recruitment. In vitro interaction between PPARγ and GST-SRC1(381-882) in the presence of either the TZD troglitazone or GQ-16. Figure S3. Effects of GQ-16 on activity and body composition. (A) Ambulatory activity of mice treated with rosiglitazone (4 mg/kg/d) or GQ-16 (20 mg/kg/d) (n = 4 per group). (B) Body composition analysis of mice treated as in (A) for 7 days. All data are means ± s.e.m. **p < 0.01. Figure S4. Effects of PPARγ ligands on gene expression in adipose tissues. Gene expression from epididymal, brown, or inguinal adipose tissue isolated from mice treated with vehicle (DMSO, n = 5), rosiglitazone (4 mg/kg/d, n = 6), or GQ-16 (20 mg/kg/d, n = 6) for 7 days. Figure S5. Deuterium incorporation levels into peptidic fragments of PPARγ in the absence or presence of ligands rosiglitazone and GQ-16. Numbers are listed as the percentage of deuterium incorporation at specified time points. Figure S6. Hydration of H12 during the MD simulation. PPARγ-RSG complex (black), PPARγGQ-16 (orange). Figure S7. Position and orientation of the full agonist rosiglitazone compared to select PPARγ partial agonists. (A-E) The full agonist rosiglitazone (A, PDB 2PRG) binds perpendicular to Helix 3 (in foreground, Helix 12 is at extreme left), while GQ-16 (B), PA-082 (C, PDB 2FVJ), MRL-24 (D, PDB 2Q5P), and nTZDpa (E, 2Q5S) bind in a north-south orientation, parallel to Helix 3.

S1

Supplementary Table 1. Components of standard chow and high fat diets obtained from the University of Campinas animal facility.

 

Standard  chow  (g)  

High  fat  diet  (g)  

Casein  

202  

200  

Sucrose  

100  

100  

Cornstarch  

397  

115.5  

130.5  

132  

-­‐  

312  

Soybean  oil  

70  

40  

Cellulose  

50  

50  

Mineral  Mix  American  

35  

35  

Vitamin  mix  AIN-­‐93  

10  

10  

L-­‐cystine  

3  

3  

Choline  

2.5  

2.5  

Dextrinated  starch   Lard  

     Institute  of  Nutrition  (AIN)-­‐93  

S2

Supplementary Table 2. Crystallographic Statistics Data Analysis Space group

P212121

Unit cell (Å)

83.69, 84.39, 96.98

Resolution (Å)

42.19 – 2.10

Rmerge

0.056(0.468)

Mean (I/σI)

12.4(2.5)

Completeness

98.3(97.4)

Unique reflections

39972(5691)

Multiplicity

3.5(3.4)

Refinement Statistics Resolution (Å)

42.19 – 2.10

Rwork/Rfree

20.77/24.60

Bond Angles RMS (°)

1.317

Bond Lengths RMS (Å)

0.010

2

Average Protein B (Å ) 2

Average Ligand B (Å )

49.86 48.78

Ramachandran Analysis: Most Favored (%)

97.9

Additionally Allowed (%)

2.1

Disallowed (%)

0.0

S3

FIGURE S1 A

B 10

110

* 8

Rosiglitazone

90

Fold activation

% Bound [ 3H]Rosiglitazone

100 GQ-16

80 70 60 50

6

*

*

*

4

*

40

2

30 20

DMSO

10 0 -12

-4

Bezafibrate 10 M

-11

-10

-9 -8 -7 -6 log [competitor] M

-5

-4

-3

-5

Rosiglitazone 10 M 9-cis-retinoic acid 10-5 M GQ-16 10

-4,5

0

+

+

+ +

+

+ +

+ +

+ +

M Empty vector

S4

+

+

+ PPARγ

+ PPARα

+ PPARδ

+ RXRα

FIGURE S2

S5

S6

G

Q

-1

6

i

e os

Fat mass (g)

35

R

6

i

20 cl

-1

25

hi

Q

30

ve

G

e os

0

cl

Lean mass (g)

5000

R

6

10000

hi

-1

i

e

os

cl

15000

ve

Q

R

hi

G

ve

X ambulatory (counts/day)

FIGURE S3 15

10

**

5

0

D IO C 2 O C X2 O X C 5B PT C 1B O C X8 O U XB5 C B FACOP1 C B X2 PGOXP4 CC1 8B PTB FAH 1B C BR SL IDE P4 C UCA O P PR 4 A 1 D H1R CM SAL ID1E6 DC A EL ROB4 PRO A2 VL 1 PGDM 3 PGC 16 1 EL CA1B O V A L3 D R B 2 D IO PG 2 C 1A

Relative mRNA Expression

Relative mRNA Expression

Relative mRNA Expression 15

5

4

3

1

20

15

10

5

0 ** * **

GQ-16 3

1 **

H C R SL IDE C A O PR 4 A D 1 M A 16 D EL RB O 2 VL 3 D I PG O2 C C 1A PT 1B

C O X 5 C B O C X2 O X 8 U B C FA P1 B PG P4 C 1B

10

5

inguinal WAT

vehicle 4

vehicle

Rosi

Rosi

Relative mRNA Expression

C O X5 C B O C X2 O X C 8B PT FA 1B B P U 4 C P1 H C R SL IDE C A O PR 4 A D 1 M PG 16 C EL 1B O V A L3 D R B 2 D IO PG 2 C 1A

FIGURE S4 epiWAT

Rosi

vehicle

GQ-16

5

0

BAT average ± SEM; *p