Surface plasmon resonance ( SPR ) analysis of the Nbr1 - Ams1 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using the GST Capture Kit . Ams1 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through the chip surface .

O O O O O O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

Surface plasmon resonance (SPR) analysis of the Nbr1-Ams1 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ams1 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


SPR analysis of the Nbr1 - Ape4 interaction . GST - tagged ZZ1 - L1 fragment of Nbr1 purified from E . coli was captured on a CM5 sensor chip using the GST Capture Kit . Ape4 purified from S . pombe ( two - fold serial dilutions from 1000 nM to 62 . 5 nM ) was flowed through the chip surface .

O O O O B-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O

SPR analysis of the Nbr1-Ape4 interaction. GST-tagged ZZ1-L1 fragment of Nbr1 purified from E. coli was captured on a CM5 sensor chip using the GST Capture Kit. Ape4 purified from S. pombe (two-fold serial dilutions from 1000 nM to 62.5 nM) was flowed through the chip surface.


The effects of individually mutating the first two residues of Ams1 on its Nbr1 - binding ability . Nbr1 - Ams1 interactions were examined by the Pil1 co - tethering assay . Scale bar , 3 μm .

O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

The effects of individually mutating the first two residues of Ams1 on its Nbr1-binding ability. Nbr1-Ams1 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


The effects of individually mutating the third residue and other residues in the U - shaped loop of Ape4 on its Nbr1 - binding ability . Nbr1 - Ape4 interactions were examined by the Pil1 co - tethering assay . Scale bar , 3 μm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O

The effects of individually mutating the third residue and other residues in the U-shaped loop of Ape4 on its Nbr1-binding ability. Nbr1-Ape4 interactions were examined by the Pil1 co-tethering assay. Scale bar, 3 μm.


( A - C ) . Mice body weight gain ( A ) , the lean mass and fat mass ( B ) and gWAT index ( C ) . At 8 weeks of age , male C57BL / 6 mice were switched to HFD . After 12 weeks of HFD feeding , mice were divided into three groups receiving non - exercise , endurance exercise or resistance exercise for 14 days . ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C). Mice body weight gain (A), the lean mass and fat mass (B) and gWAT index (C). At 8 weeks of age, male C57BL/6 mice were switched to HFD. After 12 weeks of HFD feeding, mice were divided into three groups receiving non-exercise, endurance exercise or resistance exercise for 14 days. (n = 8 per group).


( D ) . Relative changes in metabolites in response to resistance exercise . Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non - exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) . Shades of yellow and blue represent fold increase and fold decrease of a metabolite , respectively ( see color scale ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D). Relative changes in metabolites in response to resistance exercise. Heat maps show changes of metabolites in the serums from mice receiving resistance exercise or non-exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group). Shades of yellow and blue represent fold increase and fold decrease of a metabolite, respectively (see color scale).


( G ) . Two tailed Pearson ' s correlation coefficient analysis of plasma AKG level and body mass index ( BMI ) in Chinese adults ( 10 males and 35 females ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G). Two tailed Pearson's correlation coefficient analysis of plasma AKG level and body mass index (BMI) in Chinese adults (10 males and 35 females).


( H ) . Serum AKG concentration - time profile obtained before and after 40 - min resistant exercise . At 8 weeks of age , male C57BL / 6 mice were switched to HFD and continuously fed with HFD for 12 weeks . At 20 weeks of age , mice received resistance exercise for 40 min . Another group of chow fed male C57BL / 6 mice ( 10 weeks ) received resistance exercise for 40 min . The serum AKG level were tested at 0 , 1 , 2 , 4 and 6 hrs after exercise ( n = 8 - 10 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H). Serum AKG concentration-time profile obtained before and after 40-min resistant exercise. At 8 weeks of age, male C57BL/6 mice were switched to HFD and continuously fed with HFD for 12 weeks. At 20 weeks of age, mice received resistance exercise for 40 min. Another group of chow fed male C57BL/6 mice (10 weeks) received resistance exercise for 40 min. The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after exercise (n = 8-10 per group).


( I ) . Serum AKG levels after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1 day or resistance exercise for 40 min . ( n = 8 - 10 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I). Serum AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1 day or resistance exercise for 40 min.(n = 8-10 per group).


( J ) . Muscles AKG levels after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into two groups receiving either non - exercise or resistance exercise for 40 min ( n = 6 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J). Muscles AKG levels after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into two groups receiving either non-exercise or resistance exercise for 40 min (n = 6 per group).


Muscle ICDHm ( K ) , enzyme activity after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Muscle ICDHm (K), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


GDH ( L ) , enzyme activity after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GDH (L), enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


α - KGDH ( M ) enzyme activity after exercise . Male C57BL / 6 mice ( 10 weeks ) fed with normal chow were divided into three groups receiving non - exercise , running wheel free access for 1day or resistance exercise for 40 min ( n = 8 - 9 per group ) .

α-KGDH (M) enzyme activity after exercise. Male C57BL/6 mice (10 weeks) fed with normal chow were divided into three groups receiving non-exercise, running wheel free access for 1day or resistance exercise for 40 min (n = 8-9 per group).


( A ) . Serum AKG concentration - time profile obtained from male C57BL / 6 mice ( 10 weeks ) fed with normal chow before or after AKG gavage ( 10 mg / kg ) . The serum AKG level were tested at 0 , 1 , 2 , 4 and 6 hrs after gavage ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after AKG gavage (10 mg/kg). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after gavage (n = 8 per group).


( B - D ) . Water intake ( B ) , cumulative food intake ( C ) and body weight gain ( D ) of male C57BL / 6 mice . At 12 weeks of age , mice were switched to HFD and received tap water or water supplemented with 2 % AKG for 11 weeks ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B-D). Water intake (B), cumulative food intake (C) and body weight gain (D) of male C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).


Fat and lean mass ( E ) of body composition from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fat and lean mass (E) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).


representative images ( F ) of body composition from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O

representative images (F) of body composition from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( G - I ) . Weight index of BAT ( G ) , gWAT ( H ) and iWAT ( I ) from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-I). Weight index of BAT (G), gWAT (H) and iWAT (I) from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( J ) . Serum AKG level of male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O

(J). Serum AKG level of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( K - L ) . Representative images ( K ) and quantification ( L ) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(K-L). Representative images (K) and quantification (L) of gWAT and iWAT HE staining from male mice after 11 weeks of AKG supplementation (n = 9 per group).


( M - O ) . Water intake ( M ) , cumulative food intake ( N ) and body weight gain ( O ) of female C57BL / 6 mice . At 12 weeks of age , mice were switched to HFD and received tap water or water supplemented with 2 % AKG for 11 weeks ( n = 9 per group ) .

(M-O). Water intake (M), cumulative food intake (N) and body weight gain (O) of female C57BL/6 mice. At 12 weeks of age, mice were switched to HFD and received tap water or water supplemented with 2% AKG for 11 weeks (n = 9 per group).


( P - Q ) . Fat and lean mass index ( P ) and representative image ( Q ) of body composition from female mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

(P-Q). Fat and lean mass index (P) and representative image (Q) of body composition from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( R - T ) . Weight index of BAT ( R ) , gWAT ( S ) and iWAT ( T ) from female mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

(R-T). Weight index of BAT (R), gWAT (S) and iWAT (T) from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( U - V ) . Representative images ( U ) and quantification ( V ) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

(U-V). Representative images (U) and quantification (V) of gWAT and iWAT HE staining from female mice after 11 weeks of AKG supplementation (n = 9 per group).


( A - B ) . Oxygen consumption in male C57BL / 6 mice after 11 weeks of AKG supplementation ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B). Oxygen consumption in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).


( C ) . Body temperature of male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O

(C). Body temperature of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( D - E ) . Representative images ( D ) and quantification ( E ) of BAT thermogenesis induced by 6 hr cold exposure at 4 ℃ in male mice supplemented with AKG for 11 weeks ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-E). Representative images (D) and quantification (E) of BAT thermogenesis induced by 6 hr cold exposure at 4℃ in male mice supplemented with AKG for 11 weeks (n = 9 per group).


The mRNA expression of thermogenic genes ( F ) in BAT of male mice after 11 weeks of AKG supplementation ( n = 3 - 6 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The mRNA expression of thermogenic genes (F) in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).


Immunoblots and quantification ( G ) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation ( n = 3 - 6 per group ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

Immunoblots and quantification (G) of UCP1 protein in BAT of male mice after 11 weeks of AKG supplementation (n = 3-6 per group).


( H - I ) . DAB staining ( H ) and quantification ( I ) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(H-I). DAB staining (H) and quantification (I) of UCP1 in BAT of male mice supplemented with AKG for 11 weeks (n = 9 per group).


( J ) . Serum levels of NEFA in male mice supplemented with AKG for 11 weeks ( n = 9 per group ) .

(J). Serum levels of NEFA in male mice supplemented with AKG for 11 weeks (n = 9 per group).


( K ) . The mRNA expression of CD137 , CD40 , TBX1 , TMEM26 , CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks ( n = 8 per group ) .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(K). The mRNA expression of CD137, CD40, TBX1, TMEM26, CITED1 and slc27a1 in iWAT of male mice supplemented with AKG for 11 weeks (n = 8 per group).


( L - M ) . Respiratory exchange ratio ( RER ) in male C57BL / 6 mice after 11 weeks of AKG supplementation ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(L-M). Respiratory exchange ratio (RER) in male C57BL/6 mice after 11 weeks of AKG supplementation (n = 8 per group).


( N - O ) . Immunoblots ( N ) and quantification ( O ) of p - HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation ( n = 3 per group ) .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male mice after 11 weeks of AKG supplementation (n = 3 per group).


( P - Q ) . Representative images ( P ) and quantification ( Q ) of p - HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation ( n = 9 per group ) .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O

(P-Q). Representative images (P) and quantification (Q) of p-HSL DAB staining in gWAT and iWAT of male mice after 11 weeks of AKG supplementation (n = 9 per group).


( R ) . The mRNA expression of PPARγ , FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks ( n = 6 per group ) .

O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

(R). The mRNA expression of PPARγ, FASN and ACC in the gWAT and iWAT from male mice supplemented with AKG for 11 weeks (n = 6 per group).


( S - V ) . Serum levels of E ( S ) , NE ( T ) , T3 ( U ) , and T4 ( V ) in male mice supplemented with AKG for 11 weeks ( n = 8 - 9 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(S-V). Serum levels of E (S), NE (T), T3 (U), and T4 (V) in male mice supplemented with AKG for 11 weeks (n = 8-9 per group).


( A ) . Serum AKG concentration - time profile obtained from male C57BL / 6 mice ( 10 weeks ) fed with normal chow before or after i . p AKG ( 10 mg / kg body weight ) . The serum AKG level were tested at 0 , 1 , 2 , 4 and 6 hrs after injection ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A). Serum AKG concentration-time profile obtained from male C57BL/6 mice (10 weeks) fed with normal chow before or after i.p AKG (10 mg/kg body weight). The serum AKG level were tested at 0, 1, 2, 4 and 6 hrs after injection (n = 8 per group).


( B - C ) . Representative images ( B ) and quantification ( C ) of BAT thermogenesis after 6 hrs cold exposure at 4 ℃ . Male C57BL / 6 mice ( 10 weeks ) were i . p . injected with 10 mg / kg AKG or saline and immediately exposed to cold stress at 4 ℃ ( n = 8 per group ) .

(B-C). Representative images (B) and quantification (C) of BAT thermogenesis after 6 hrs cold exposure at 4℃. Male C57BL/6 mice (10 weeks) were i.p. injected with 10 mg/kg AKG or saline and immediately exposed to cold stress at 4℃ (n = 8 per group).


( D ) . Immunoblots and quantification of p - HSL and ATGL in the gWAT of male C57BL / 6 mice ( 10 weeks ) 3 hrs after i . p . injection of 10 mg / kg AKG or saline ( n = 3 per group ) .

O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D). Immunoblots and quantification of p-HSL and ATGL in the gWAT of male C57BL/6 mice (10 weeks) 3 hrs after i.p. injection of 10 mg/kg AKG or saline (n = 3 per group).


( E ) . Serum E level in AKG treated male mice 3 hrs after i . p . injection ( n = 8 per group ) .

(E). Serum E level in AKG treated male mice 3 hrs after i.p. injection (n = 8 per group).


Oxygen consumption ( F - G ) in male C57BL / 6 mice ( 10 weeks ) i . p . injected with saline , 10 mg / kg AKG , 1 mg / kg SR59230A ( ADRB3 inhibitor ) or AKG + SR59230A ( n = 8 per group ) . All injections were performed at 7 : 00 am of second day . Data was summarized in bar graph ( G and I ) by light or dark cycle of second day .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Oxygen consumption (F-G) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.


RER ( H - I ) in male C57BL / 6 mice ( 10 weeks ) i . p . injected with saline , 10 mg / kg AKG , 1 mg / kg SR59230A ( ADRB3 inhibitor ) or AKG + SR59230A ( n = 8 per group ) . All injections were performed at 7 : 00 am of second day . Data was summarized in bar graph ( G and I ) by light or dark cycle of second day .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RER (H-I) in male C57BL/6 mice (10 weeks) i.p. injected with saline, 10 mg/kg AKG, 1 mg/kg SR59230A (ADRB3 inhibitor) or AKG + SR59230A (n = 8 per group). All injections were performed at 7:00 am of second day. Data was summarized in bar graph (G and I) by light or dark cycle of second day.


( J - N ) . Body weight gain ( J ) , cumulative food intake ( K ) , body composition ( L ) , fat weight ( M ) of sham or adrenalectomized male C57BL / 6 mice . Male mice were adrenalectomized at 8 weeks of age . Two weeks after surgeries , male mice were switched to HFD and given free access to tap water or 2 % AKG for 9 weeks ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J-N). Body weight gain (J), cumulative food intake (K), body composition (L), fat weight (M) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).


serum NEFA ( N ) of sham or adrenalectomized male C57BL / 6 mice . Male mice were adrenalectomized at 8 weeks of age . Two weeks after surgeries , male mice were switched to HFD and given free access to tap water or 2 % AKG for 9 weeks ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

serum NEFA (N) of sham or adrenalectomized male C57BL/6 mice. Male mice were adrenalectomized at 8 weeks of age. Two weeks after surgeries, male mice were switched to HFD and given free access to tap water or 2% AKG for 9 weeks (n = 8 per group).


( O - P ) . Representative images ( O ) and quantification ( P ) of BAT thermogenesis after 6 hrs cold exposure at 4 ℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(O-P). Representative images (O) and quantification (P) of BAT thermogenesis after 6 hrs cold exposure at 4℃ in sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 8 per group).


( Q ) . The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 6 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(Q). The mRNA expression of themogenic genes in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 6 per group).


( R - S ) . Immunoblots ( R ) and quantification ( S ) of p - HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group ) .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(R-S). Immunoblots (R) and quantification (S) of p-HSL and ATGL protein in the gWAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).


( T - U ) . Immunoblots ( T ) and quantification ( U ) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks ( n = 4 per group ) .

O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O

(T-U). Immunoblots (T) and quantification (U) of UCP1 protein in the BAT of sham or adrenalectomized male mice treated with AKG for 9 weeks (n = 4 per group).


( A ) . The mRNA expression of AKG - sensing genes in the adrenal gland tissue of 12 - weeks male C57BL / 6 mice ( n = 4 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A). The mRNA expression of AKG-sensing genes in the adrenal gland tissue of 12-weeks male C57BL/6 mice (n = 4 per group).


( B ) . The mRNA expression of OXGR1 in different tissues of 12 - weeks male C57BL / 6 mice ( n = 3 per group ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B). The mRNA expression of OXGR1 in different tissues of 12-weeks male C57BL/6 mice (n = 3 per group).


( C ) . OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 ( green ) .

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(C). OXGR1 localization in adrenal gland medulla indicated by fluorescent staining of OXGR1 (green).


( D ) . The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i . p . injection of saline or 10 mg / kg AKG , or immediately after 40 - mins resistance exercise ( n = 8 per group ) .

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(D). The mRNA expression of OXGR1 in the adrenal gland of male mice 3 hrs after i.p. injection of saline or 10 mg/kg AKG, or immediately after 40-mins resistance exercise (n=8 per group).


( E ) . Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control ( NC ) siRNA or siOXGR1 ( n = 3 per group ) .

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(E). Immunoblots and quantification of OXGR1 protein expression in adrenal chromaffin cells treated with negative control (NC) siRNA or siOXGR1 (n = 3 per group).


( F ) . E level in the medium from adrenal chromaffin cell cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 30 mins ( n = 8 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F). E level in the medium from adrenal chromaffin cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 30 mins (n = 8 per group).


( G ) . Intracellular calcium ion [ Ca2 + ] changes in adrenal medulla cell cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 ( n = 30 per group ) .

(G). Intracellular calcium ion [Ca2+] changes in adrenal medulla cell cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 (n = 30 per group).


( H - I ) . Body weight gain ( H ) and cumulative food intake ( I ) of male WT control ( littermates ) or OXGR1 global knock out ( OXGR1KO ) mice . At 12 weeks of age , both control and KO mice were switched to HFD and further divided into two groups , receiving tap water or water supplemented with 2 % AKG for 13 weeks ( n = 8 per group ) .

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(H-I). Body weight gain (H) and cumulative food intake (I) of male WT control (littermates) or OXGR1 global knock out (OXGR1KO) mice. At 12 weeks of age, both control and KO mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 13 weeks (n = 8 per group).


( J - K ) . Representative images of body composition ( J ) and fat and lean mass index ( K ) of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

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(J-K). Representative images of body composition (J) and fat and lean mass index (K) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


( L - M ) . Weight index of iWAT ( L ) and gWAT ( M ) in male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

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(L-M). Weight index of iWAT (L) and gWAT (M) in male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


( N - O ) . Immunoblots ( N ) and quantification ( O ) of p - HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 4 per group ) .

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(N-O). Immunoblots (N) and quantification (O) of p-HSL and ATGL protein in gWAT of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 4 per group).


Oxygen consumption ( P - Q ) of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

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Oxygen consumption (P-Q) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


RER ( R - S ) of male WT or OXGR1KO mice treated with AKG for 13 weeks ( n = 8 per group ) .

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RER (R-S) of male WT or OXGR1KO mice treated with AKG for 13 weeks (n = 8 per group).


Body weight gain and cumulative food intake of male OXGR1 adrenal - specific reexpression mice ( OXGR1REAG ) . Male OXGR1KO mice ( 8 weeks ) were adrenal - specifically injected with control HBAAV2 / 9 - GFP ( OXGR1KO control ) or HBAAV2 / 9 - OXGR1 ( OXGR1REAG ) . Two weeks after injections , mice were switched to HFD and further divided into two groups , receiving tap water or water supplemented with 2 % AKG for 12 weeks ( n = 8 per group ) .

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Body weight gain and cumulative food intake of male OXGR1 adrenal-specific reexpression mice (OXGR1REAG). Male OXGR1KO mice (8 weeks) were adrenal-specifically injected with control HBAAV2/9-GFP (OXGR1KO control) or HBAAV2/9-OXGR1 (OXGR1REAG). Two weeks after injections, mice were switched to HFD and further divided into two groups, receiving tap water or water supplemented with 2% AKG for 12 weeks (n = 8 per group).


Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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Representative image of body composition and fat and lean mass index of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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Weight index of gWAT and iWAT in male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Immunoblots and quantification of p - HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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Immunoblots and quantification of p-HSL and ATGL protein in gWAT of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

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Oxygen consumption of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


RER of male OXGR1REAG mice treated with AKG for 12 weeks ( n = 8 per group ) .

O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

RER of male OXGR1REAG mice treated with AKG for 12 weeks (n = 8 per group).


Body weight gain in male WT littermates and OXGR1KO mice . At 8 weeks of age , male C57BL / 6 WT control or OXGR1KO mice were switched to HFD . After 12 weeks of HFD feeding , mice were further divided into two groups , receiving non - exercise or resistance exercise for 14 days . ( n = 8 per group ) .

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Body weight gain in male WT littermates and OXGR1KO mice. At 8 weeks of age, male C57BL/6 WT control or OXGR1KO mice were switched to HFD. After 12 weeks of HFD feeding, mice were further divided into two groups, receiving non-exercise or resistance exercise for 14 days. (n = 8 per group).


Exercise - induced body weight loss in male WT littermates and OXGR1KO mice . Body weights from exercise mice were subtracted by the average body weight of non - exercise control group for each genotype ( n = 8 per group ) .

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Exercise-induced body weight loss in male WT littermates and OXGR1KO mice. Body weights from exercise mice were subtracted by the average body weight of non-exercise control group for each genotype (n = 8 per group).


Exercise - induced fat mass loss in male WT littermates and OXGR1KO mice . Fat mass from exercise mice were subtracted by the average fat mass of non - exercise control group for each genotype ( n = 8 per group ) .

Exercise-induced fat mass loss in male WT littermates and OXGR1KO mice. Fat mass from exercise mice were subtracted by the average fat mass of non-exercise control group for each genotype (n = 8 per group).


Cumulative food intake of male WT littermates and OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

Cumulative food intake of male WT littermates and OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


Weight index of gWAT ( E ) and iWAT ( F ) of male OXGR1KO mice after 14 - days resistance exercise ( n = 8 per group ) .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O

Weight index of gWAT (E) and iWAT (F) of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).


Body composition of male OXGR1KO mice after 14 - days resistance exercise ( n = 8 per group ) .

Body composition of male OXGR1KO mice after 14-days resistance exercise (n = 8 per group).


Serum AKG levels of male WT and OXGR1KO mice after resistance exercise . Male WT and OXGR1KO mice ( 10 weeks ) fed with normal chow were receiving resistance exercise for 40 min ( n = 8 per group ) . The serum AKG levels were tested before and immediately after exercise .

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Serum AKG levels of male WT and OXGR1KO mice after resistance exercise. Male WT and OXGR1KO mice (10 weeks) fed with normal chow were receiving resistance exercise for 40 min (n = 8 per group). The serum AKG levels were tested before and immediately after exercise.


Serum E level in male OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

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Serum E level in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


The mRNA expression in the BAT of male OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

The mRNA expression in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


protein expression of UCP1 in the BAT of male OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

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protein expression of UCP1 in the BAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


the mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14 - day resistance exercise ( n = 4 per group ) .

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the mRNA expression of HSL and ATGL n the gWAT of male OXGR1KO mice after 14-day resistance exercise (n = 4 per group).


Oxygen consumption in male OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

Oxygen consumption in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


RER in male OXGR1KO mice after 14 - day resistance exercise ( n = 8 per group ) .

RER in male OXGR1KO mice after 14-day resistance exercise (n = 8 per group).


Ingenuity Pathway Analysis ( IPA ) of AKG - induced transcriptome signature in adrenal chromaffin cell treated with AKG . The mRAN was extracted from adrenal chromaffin cells after 3 - hrs incubation of vehicle or AKG ( 100 μM ) . ( n = 3 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Ingenuity Pathway Analysis (IPA) of AKG-induced transcriptome signature in adrenal chromaffin cell treated with AKG. The mRAN was extracted from adrenal chromaffin cells after 3-hrs incubation of vehicle or AKG (100 μM). (n = 3 per group).


Immunoblots and quantification of p - IKK / IKK , p65 , p - IκB / IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 3 hrs ( n = 3 per group ) .

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Immunoblots and quantification of p-IKK/IKK, p65, p-IκB/IκB and OXGR1 protein in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group).


p65 translocation in adrenal chromaffin cells cultured with vehicle + NC , vehicle + siOXGR1 , AKG ( 100 μM ) + NC or AKG + siOXGR1 for 3 hrs ( n = 3 per group ) . Scale bars , 100 μm .

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p65 translocation in adrenal chromaffin cells cultured with vehicle + NC, vehicle + siOXGR1, AKG (100 μM) + NC or AKG + siOXGR1 for 3 hrs (n = 3 per group). Scale bars, 100 μm.


Immunoblots and quantification of p - IKK / IKK , p65 and p - IκB / IκB protein in adrenal chromaffin cells cultured with vehicle , AKG ( 100 μM ) , IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs .

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Immunoblots and quantification of p-IKK/IKK, p65 and p-IκB/IκB protein in adrenal chromaffin cells cultured with vehicle, AKG (100 μM), IKK inhibitor IKK16 or AKG + IKK16 for 3 hrs.


E level in the medium from adrenal chromaffin cell cultured with vehicle , AKG ( 100 μM ) , IKK16 or AKG + IKK16 for 3 hrs ( n = 10 per group ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E level in the medium from adrenal chromaffin cell cultured with vehicle, AKG (100 μM), IKK16 or AKG + IKK16 for 3 hrs (n = 10 per group).


Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L + W133V DHFR strains after being grown at 30 ° C , 37 ° C , or 42 ° C ( I91L + W133V was grown at 40 ° C ) in M9 medium supplemented with amino acids for 4 hours ( see Methods ) .

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Live cells DIC images and DAPI nucleoids staining of WT DHFR and I91L+W133V DHFR strains after being grown at 30°C, 37°C, or 42°C (I91L+W133V was grown at 40°C) in M9 medium supplemented with amino acids for 4 hours (see Methods).


Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42 ° C ( for WT , W133V and V75H + I155A strains ) or at 40 ° C ( for I91L + W133V and V75H + I91L + I155A ) in M9 medium supplemented with amino acids . For the boxplots in panels , F the central band represents the median of the distribution , the box ends represent the 25th and 75th percentile , the whiskers represent the 10th and 90th percentile , while the dots represent the 5th and 95th percentile . Data was usually obtained from 2 - 3 biological replicates . The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 ( please refer to Figure 1 source data for exact number of cells for each dataset ) . See related Figure EV1B .

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Functional complementation of WT DHFR from an arabinose inducible pBAD plasmid rescues filamentation of mutant strains grown at 42°C (for WT, W133V and V75H+I155A strains) or at 40°C (for I91L+W133V and V75H+I91L+I155A) in M9 medium supplemented with amino acids. For the boxplots in panels , F the central band represents the median of the distribution, the box ends represent the 25th and 75th percentile, the whiskers represent the 10th and 90th percentile, while the dots represent the 5th and 95th percentile. Data was usually obtained from 2-3 biological replicates. The number of cells used to derive the boxplot distributions in the different panels range usually between 200 to 450 (please refer to Figure 1 source data for exact number of cells for each dataset). See related Figure EV1B.


( A ) and ( B ) shows abundance of selected nucleotides and amino acids for mutants I91L + W133V and W133V as well as WT strain treated with 0 . 5μg / ml of Tmp ( Trimethoprim ) after 4 hours of growth at 40 / 42 ° C in M9 minimal medium ( condition of no filamentation ) , Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions . In minimal medium ( B ) Methionine levels are extremely low Levels of purines ( IMP , AMP ) are also largely rescued with amino acid supplementation , however dTMP , dTDP and dTTP levels remain extremely low . Error bars represent SEM of at least three biological replicates ( see Methods ) . The dashed line in each plot represents value of 1 for WT .

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(A) and (B) shows abundance of selected nucleotides and amino acids for mutants I91L+W133V and W133V as well as WT strain treated with 0.5μg/ml of Tmp (Trimethoprim) after 4 hours of growth at 40/42°C in M9 minimal medium (condition of no filamentation), Concentration of all metabolites were normalized to WT levels at 4 hours when grown under similar conditions. In minimal medium (B) Methionine levels are extremely low Levels of purines (IMP, AMP) are also largely rescued with amino acid supplementation, however dTMP, dTDP and dTTP levels remain extremely low. Error bars represent SEM of at least three biological replicates (see Methods). The dashed line in each plot represents value of 1 for WT.


( A ) recA ( B ) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP . WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37 ° C , while WT treated with different concentrations of Tmp were grown for 4 hours at 42 ° C . Brown bars ( M9 + AA ) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes . On the other hand , conditions with loss of filamentation ( with dTMP or no supplementation ) show much less expression . Error bars represent SD of 2 - 3 biological replicates ( see Methods ) .

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(A) recA (B) recN genes measured by quantitative PCR when WT and mutant strains are grown in M9 medium with or without supplementation of amino acids or dTMP. WT and mutant strains were grown for 4 hours of growth in the indicated medium at 37°C, while WT treated with different concentrations of Tmp were grown for 4 hours at 42°C. Brown bars (M9+AA) in the gray shaded area correspond to filamentation conditions and these are associated with pronounced upregulation of all three SOS genes. On the other hand, conditions with loss of filamentation (with dTMP or no supplementation) show much less expression. Error bars represent SD of 2-3 biological replicates (see Methods).


WT cells were treated with different concentrations of Tmp at 42 ° C for varying amounts of time in amino acid supplemented M9 medium ( panel B ) or in M9 media supplemented with both amino acids and GMP ( panel C ) , following which they were spotted on M9 + AA plates and allowed to grow at 30 ° C . Colonies were counted next day . In the presence of only amino acids , there was no loss in viability for any concentration of Tmp ( panel B ) , despite extensive filamentation ( Figure EV1E ) . In contrast , in the presence of amino acids and GMP , the cells showed sharp loss in viability when grown at high Tmp concentrations . In both panels , error bars represent SD of two biological replicates . The dashed line represents cfu at one - hour timepoint .

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WT cells were treated with different concentrations of Tmp at 42°C for varying amounts of time in amino acid supplemented M9 medium (panel B) or in M9 media supplemented with both amino acids and GMP (panel C), following which they were spotted on M9+AA plates and allowed to grow at 30°C. Colonies were counted next day. In the presence of only amino acids, there was no loss in viability for any concentration of Tmp (panel B), despite extensive filamentation (Figure EV1E). In contrast, in the presence of amino acids and GMP, the cells showed sharp loss in viability when grown at high Tmp concentrations. In both panels, error bars represent SD of two biological replicates. The dashed line represents cfu at one-hour timepoint.


Expression of SOS response genes recA , recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids ( black bars ) or in the presence of both amino acids and GMP ( gray bars ) at 42 ° C ( I91L + W133V was grown at 40 ° C ) . Error bars represent SD of 2 - 3 biological replicates .

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Expression of SOS response genes recA, recN and sulA when mutant strains and Tmp treated WT cells are grown in M9 medium supplemented with amino acids (black bars) or in the presence of both amino acids and GMP (gray bars) at 42°C (I91L+W133V was grown at 40°C). Error bars represent SD of 2-3 biological replicates.


Comparison of growth rates of WT and mutant DHFR strains at 42 ° C ( 40 ° C for I91L + W133V and V75H + I91L + I155A ) in minimal medium that is supplemented with amino acids and / or 1mM dTMP . Except for W133V and to a lesser extent for V75H + I155A , the effect of dTMP on growth rates is only modest . Error bars represent SEM of 3 biological replicates .

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Comparison of growth rates of WT and mutant DHFR strains at 42°C (40°C for I91L+W133V and V75H+I91L+I155A) in minimal medium that is supplemented with amino acids and/or 1mM dTMP. Except for W133V and to a lesser extent for V75H+I155A, the effect of dTMP on growth rates is only modest. Error bars represent SEM of 3 biological replicates.


in vitro activity of purified Thymidylate Kinase ( Tmk ) as a function of dTMP concentration shows Michaelis - Menten ( MM ) like kinetics . ATP concentration is saturating at 1mM . The KM of dTMP is 13μM . The error bars represent SD of three technical replicates .

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in vitro activity of purified Thymidylate Kinase (Tmk) as a function of dTMP concentration shows Michaelis-Menten (MM) like kinetics. ATP concentration is saturating at 1mM. The KM of dTMP is 13μM. The error bars represent SD of three technical replicates.


The green points were acquired during growth of WT , W133V and I91L + W133V mutants in the presence of different concentrations of thymidine , which follow the red curve . The inset plot shows the same graph with selected datapoints for I91L + W133V mutant . The cyan circle shows I91L + W133V mutant in the absence of any metabolite supplementation , while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The green points were acquired during growth of WT, W133V and I91L+W133V mutants in the presence of different concentrations of thymidine, which follow the red curve. The inset plot shows the same graph with selected datapoints for I91L+W133V mutant. The cyan circle shows I91L+W133V mutant in the absence of any metabolite supplementation, while red and black points indicate metabolite levels following thymidine and dTMP supplementation respectively.


Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for ( A ) Adenylate Kinase ( AK ) ( B ) Uridylate Kinase ( PyrH ) ( C ) Adenylosuccinate Lyase ( PurB ) and ( D ) GAR Transformylase ( PurN ) . AK , PyrH and PurB appear to follow hyperbolic MM kinetics in vivo , while PurN shows Hill - like dependence with cooperativity . In panel ( A ) , the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK ( Adkar et al . , 2019 ) as well as DHFR mutants used in the present study , while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP ( Adkar et al . , 2019 ) . Data points for all other panels ( B - D ) are derived from mutant DHFR strains in the present study . Since SAICAR , AICAR , GAR and FGAR levels were undetectable in WT , all data points in ( C - D ) are relative to those observed in I91L + W133V mutant following 4 hours of growth at 40 ° C . Data points in all panels represent metabolite levels for individual biological replicates without averaging .

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Steady state levels of different metabolites obtained from metabolomics were used to derive activity curves for (A) Adenylate Kinase (AK) (B) Uridylate Kinase (PyrH) (C) Adenylosuccinate Lyase (PurB) and (D) GAR Transformylase (PurN). AK, PyrH and PurB appear to follow hyperbolic MM kinetics in vivo, while PurN shows Hill-like dependence with cooperativity. In panel (A), the red data points were acquired during growth in amino acid supplemented M9 medium in the absence of external AMP and are derived from WT and various stabilizing and destabilizing point mutations of AK (Adkar et al., 2019) as well as DHFR mutants used in the present study, while black points were derived exclusively from cultures of AK mutants supplemented with 1mM external AMP (Adkar et al., 2019). Data points for all other panels (B-D) are derived from mutant DHFR strains in the present study. Since SAICAR, AICAR, GAR and FGAR levels were undetectable in WT, all data points in (C-D) are relative to those observed in I91L+W133V mutant following 4 hours of growth at 40°C. Data points in all panels represent metabolite levels for individual biological replicates without averaging.


( b ) in N2 wild type ( black diamonds ) and ung - 1 mutants ( red squares ) , ( c ) following the depletion of EXO - 3 ( red triangles ) , APN - 1 ( blue circles ) or control RNAi ( L4440 , black diamonds ) in the N2 wild - type background , and ( d ) in msh - 2 ( ok2410 ) ( orange triangles ) , msh - 6 ( pk2504 ) ( green circles ) and mlh - 1 ( ok1917 ) ( grey squares ) mutants .

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(b) in N2 wild type (black diamonds) and ung-1 mutants (red squares), (c) following the depletion of EXO-3 (red triangles), APN-1 (blue circles) or control RNAi (L4440, black diamonds) in the N2 wild-type background, and (d) in msh-2(ok2410) (orange triangles), msh-6(pk2504) (green circles) and mlh-1(ok1917) (grey squares) mutants.


( e ) Western blottings showing expression of MSH - 6 protein in the wild type strain grown on E . coli expressing control ( N2 ) or the indicated RNAi , as well as in msh - 6 ( pk2504 ) and msh - 2 ( ok2410 ) mutants ( top panel ) , and expression of MSH - 2 : : GFP fusion protein in transgenic worms grown on E . coli expressing RNAi for MSH - 2 , MSH - 6 or empty vector control as indicated ( lower panel ) . Actin was used as the loading control .

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(e) Western blottings showing expression of MSH-6 protein in the wild type strain grown on E. coli expressing control (N2) or the indicated RNAi, as well as in msh-6(pk2504) and msh-2(ok2410) mutants (top panel), and expression of MSH-2::GFP fusion protein in transgenic worms grown on E. coli expressing RNAi for MSH-2, MSH-6 or empty vector control as indicated (lower panel). Actin was used as the loading control.


( f ) F1 survival after depletion of MSH - 2 and MSH - 6 in the mlh - 1 ( ok1917 ) mutant . ( g , h ) F1 survival after depletion of the AP endonucleases EXO - 3 and APN - 1 in ( g ) the msh - 6 ( pk2504 ) or ( h ) msh - 2 ( ok2410 ) mutant background . Kruskal - Wallis one - way analysis on ranks ( P = 0 . 963 and P = 0 . 985 in h and g , respectively ) . Mann - Whitney rank - sum test failed to identify a difference in median survival after depleting EXO - 3 in the msh - 6 mutant ( g , P = 0 . 929 ) . ( i ) F1 survival following depletion of the indicated genes in exo - 3 ( tm4374 ) mutants . ( j ) F1 survival in msh - 6 ( pk2504 ) , exo - 3 ( tm4374 ) and exo - 3 ; msh - 6 double mutants . ( k ) F1 survival in exo - 3 ( tm4374 ) , mlh - 1 ( ok1917 ) and exo - 3 ; mlh - 1 double mutants . All survival curves ( b - d , f - k ) show the mean ± s . d . for each data point from three independent experiments .

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(f) F1 survival after depletion of MSH-2 and MSH-6 in the mlh-1(ok1917) mutant. (g,h) F1 survival after depletion of the AP endonucleases EXO-3 and APN-1 in (g) the msh-6(pk2504) or (h) msh-2(ok2410) mutant background. Kruskal-Wallis one-way analysis on ranks (P=0.963 and P=0.985 in h and g, respectively). Mann-Whitney rank-sum test failed to identify a difference in median survival after depleting EXO-3 in the msh-6 mutant (g, P=0.929). (i) F1 survival following depletion of the indicated genes in exo-3(tm4374) mutants. (j) F1 survival in msh-6(pk2504), exo-3(tm4374) and exo-3;msh-6 double mutants. (k) F1 survival in exo-3(tm4374), mlh-1(ok1917) and exo-3;mlh-1 double mutants. All survival curves (b-d,f-k) show the mean±s.d. for each data point from three independent experiments.


( a ) Immunofluorescence images showing anti - RPA - 1 - positive foci in response to 5 - FU after depleting the indicated genes by RNAi or empty vector control RNAi ( L4440 ; scale bars , 5 μm ) .

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(a) Immunofluorescence images showing anti-RPA-1-positive foci in response to 5-FU after depleting the indicated genes by RNAi or empty vector control RNAi (L4440; scale bars, 5 μm).


b ) Quantification of the fraction ( % ) of 5 - FU - treated embryos that contain RPA - 1 - positive foci .

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b) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci.


( c ) Quantification of the fraction ( % ) of 5 - FU - treated embryos that contain RPA - 1 - positive foci in N2 , mlh - 1 ( ok1917 ) and exo - 1 ( tm1842 ) mutants fed control ( L4440 ) or RNAi targeting APN - 1 ( apn - 1 ) .

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(c) Quantification of the fraction (%) of 5-FU-treated embryos that contain RPA-1-positive foci in N2, mlh-1(ok1917) and exo-1(tm1842) mutants fed control (L4440) or RNAi targeting APN-1 (apn-1).


( d ) Immunofluorescence showing CHK - 1 phosphorylation at Ser139 in response to 5 - FU in N2 but not in msh - 6 ( pk2504 ) mutants or in N2 after apn - 1 ( RNAi ) ( scale bars , 5 μm ) . ( e ) Quantification of the fraction ( % ) of 5 - FU - treated embryos with phospho - CHK - 1 foci .

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(d) Immunofluorescence showing CHK-1 phosphorylation at Ser139 in response to 5-FU in N2 but not in msh-6(pk2504) mutants or in N2 after apn-1(RNAi) (scale bars, 5 μm). (e) Quantification of the fraction (%) of 5-FU-treated embryos with phospho-CHK-1 foci.


( f ) Western blottings showing H1X . 101 levels in N2 and msh - 6 mutants , treated ( + ) or not ( − ) with 5 - FU . Actin was used as loading control . ( g ) Quantification of H1X . 101 levels relative to actin in control versus 5 - FU - treated worms . ( b , c , e , g ) Bar graphs show the mean ± s . d . from three independent experiments .

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(f) Western blottings showing H1X.101 levels in N2 and msh-6 mutants, treated (+) or not (−) with 5-FU. Actin was used as loading control. (g) Quantification of H1X.101 levels relative to actin in control versus 5-FU-treated worms. (b,c,e,g) Bar graphs show the mean±s.d. from three independent experiments.


a ) Autophagy induction in response to 5 - FU measured in a GFP : : LGG - 1 reporter strain fed control RNAi ( L4440 ) or RNAi targeting the indicated genes ( scale bars , 10 μm ) .

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a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm).


( b ) Immunofluorescence showing anti - VPS - 34 staining in dissected embryos in the absence or presence of 5 - FU ( scale bar , 5 μm ) .

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(b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm).


( c ) Western blot analysis of embryonic extracts with and without the addition of 5 - FU or the autophagy inhibitor 3 - MA , detecting the ~ 40 - kDa GFP : : LGG - 1 fusion protein and the cleaved ~ 25 - kDa product .

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(c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product.


( d ) F1 survival measured after depletion of BEC - 1 ( red triangles ) and ATG - 7 ( blue squares ) as compared with worms fed control RNAi ( black diamonds ) . The survival curve shows the mean ± s . d . for each data point from three independent experiments .

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(d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments.


( e ) Induction of autophagy ( % GFP - positive embryos ) following depletion of the indicated genes .

(e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes.


( f ) Immunofluorescence showing anti - VPS - 34 staining in N2 and atl - 1 ( tm853 ) embryos in the absence or presence of 5 - FU ( scale bar , 5 μm ) .

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(f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm).


( g ) The fraction ( as % of untreated control ) of embryos with VPS - 34 - positive foci following 5 - FU treatment .

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(g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment.


( h ) Induction of autophagy ( % GFP - positive embryos ) in control ( GFP : : LGG - 1 ) and atm - 1 ( gk186 ) mutants ( atm - 1 ; GFP : : LGG - 1 ) . ( e , g , h ) Bar graphs represent mean ± s . d . from three independent experiments .

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(h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.


a ) Induction of autophagy in response to 5 - FU in the GFP : : LGG - 1 reporter strain fed on RNAi targeting the indicated DNA repair genes ( scale bars , 10 μm ) .

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a) Induction of autophagy in response to 5-FU in the GFP::LGG-1 reporter strain fed on RNAi targeting the indicated DNA repair genes (scale bars, 10 μm).


( b ) The fraction ( as % of untreated control ) of hermaphrodites having embryos with excessive GFP expression following 5 - FU treatment . Autophagy induction in independent experiments ( circles ) and the mean ( line ) are presented .

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(b) The fraction (as % of untreated control) of hermaphrodites having embryos with excessive GFP expression following 5-FU treatment. Autophagy induction in independent experiments (circles) and the mean (line) are presented.


( c ) F1 survival after control ( black diamonds ) or bec - 1 RNAi ( red squares ) in N2 and msh - 6 ( pk2504 ) mutants . The survival curve shows the mean ± s . d . for each data point from three independent experiments .

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(c) F1 survival after control (black diamonds) or bec-1 RNAi (red squares) in N2 and msh-6(pk2504) mutants. The survival curve shows the mean±s.d. for each data point from three independent experiments.


( a ) Expression of ATF - 2 : : GFP in 5 - FU - treated hermaphrodites fed control or RNAi targeting the indicated genes ( scale bars , 20 μm ) . ( b ) The number of animals harbouring embryos with excessive ATF - 2 : : GFP expression was scored in at least 150 animals and given as the fraction ( % ) of the total .

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(a) Expression of ATF-2::GFP in 5-FU-treated hermaphrodites fed control or RNAi targeting the indicated genes (scale bars, 20 μm). (b) The number of animals harbouring embryos with excessive ATF-2::GFP expression was scored in at least 150 animals and given as the fraction (%) of the total.


( c ) N2 or msh - 6 ( pk2504 ) embryos were continuously monitored under differential interference contrast microscopy ( scale bars , 5 μm ) from the three - cell stage until completion of the four - cell stage . Magnified images of representative nuclei are shown . ( d ) The fraction of embryos with visible , enlarged nucleoli ( arrows ) after 5 - FU treatment was scored . Bar graphs represent mean ± s . d . from three independent experiments .

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(c) N2 or msh-6(pk2504) embryos were continuously monitored under differential interference contrast microscopy (scale bars, 5 μm) from the three-cell stage until completion of the four-cell stage. Magnified images of representative nuclei are shown. (d) The fraction of embryos with visible, enlarged nucleoli (arrows) after 5-FU treatment was scored. Bar graphs represent mean±s.d. from three independent experiments.


( a ) Western blots showing autophagy induction by monitoring LC3 levels in whole - cell extracts from U2OS cells treated or not with 5 - FU alone or in combination with Mx for the indicated time . Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase - 3 ( 17 and 19 kDa ) and poly ( ADP - ribose ) polymerase 1 ( PARP1 ; ΔPARP , 85 kDa ) . α − Actin was used as loading control . ( b ) Quantification of LC3 - II levels in cells treated as in a . Intensities of the lower LC3 band ( LC3 - II ) was normalized to those of actin .

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(a) Western blots showing autophagy induction by monitoring LC3 levels in whole-cell extracts from U2OS cells treated or not with 5-FU alone or in combination with Mx for the indicated time. Staurosporin was used as a positive control for apoptosis induction shown by the appearance of cleaved fragments of caspase-3 (17 and 19 kDa) and poly (ADP-ribose) polymerase 1 (PARP1; ΔPARP, 85 kDa). α−Actin was used as loading control. (b) Quantification of LC3-II levels in cells treated as in a. Intensities of the lower LC3 band (LC3-II) was normalized to those of actin.


. ( c ) Autophagy induction demonstrated by accumulation of LC3 - positive puncta in U2OS cells treated or not with 5 - FU alone or in combination with Mx for 48 h . The cells were fixed and prepared for immunofluorescence of LC3 ( green ) . DAPI staining is shown in blue . Scale bar , 20 μm . ( d ) Quantification of LC3 - positive puncta in cells treated as in c . In each separate experiment , 200 - 1 , 600 cells were scored per condition .

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. (c) Autophagy induction demonstrated by accumulation of LC3-positive puncta in U2OS cells treated or not with 5-FU alone or in combination with Mx for 48 h. The cells were fixed and prepared for immunofluorescence of LC3 (green). DAPI staining is shown in blue. Scale bar, 20 μm. (d) Quantification of LC3-positive puncta in cells treated as in c. In each separate experiment, 200-1,600 cells were scored per condition.


e ) Western blots showing absence of LC3 - II accumulation in U2OS cells transfected with siRNA against MSH - 2 . The same blot probed with anti - MSH - 2 antibodies confirm efficient knockdown . ( f ) Quantification of LC3 - II levels in cells transfected with control or MSH - 2 siRNA with or without FU treatment for 24 h .

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e) Western blots showing absence of LC3-II accumulation in U2OS cells transfected with siRNA against MSH-2. The same blot probed with anti-MSH-2 antibodies confirm efficient knockdown. (f) Quantification of LC3-II levels in cells transfected with control or MSH-2 siRNA with or without FU treatment for 24 h.


( g ) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH - 2 . ( h ) Quantification of LC3 - positive puncta in cells treated as in g .

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(g) Immunofluorescence showing reduced accumulation of LC3 puncta in U2OS cells transfected with siRNA against MSH-2. (h) Quantification of LC3-positive puncta in cells treated as in g.


( i ) Quantification of normalized RPS3 protein level ( black bars ) or RPS3 mRNA level ( white bars ) in U2OS cells after 48 h 5 - FU treatment . RPS3 mRNA levels were determined by quantitative real - time reverse transcriptase - PCR .

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(i) Quantification of normalized RPS3 protein level (black bars) or RPS3 mRNA level (white bars) in U2OS cells after 48 h 5-FU treatment. RPS3 mRNA levels were determined by quantitative real-time reverse transcriptase-PCR.


( B ) When transposon insertion libraries subjected to ampicillin resistance selection , a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor ( pBR322 bla : : Im7 L53A I54A ) relative to a strain containing just pBR322 . Transposon insertion frequency increased 21 - , 24 - , and 8 - fold in the pstS , pstA , and pstC genes , respectively , compared to the insertion frequency found with no ampicillin selection .

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(B) When transposon insertion libraries subjected to ampicillin resistance selection, a substantial increase in transposon insertion frequency in the pstSCA genes was observed in strains containing the protein folding sensor (pBR322 bla::Im7 L53A I54A) relative to a strain containing just pBR322. Transposon insertion frequency increased 21-, 24-, and 8-fold in the pstS, pstA, and pstC genes, respectively, compared to the insertion frequency found with no ampicillin selection.


( A ) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla : : Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect , pBR322 and pBR322 bla : : Im7 L53A I54A were transformed into strains containing in - frame deletion mutants of each of the pst genes ( pstS , pstC , and pstA , respectively ) . The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla : : Im7 L53A I54A , but did not shower higher ampicillin resistance in the presence of pBR322 .

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(A) To verify that disruption of the pst genes is sufficient to increase ampicillin resistance of strains containing the pBR322 bla::Im7 L53A I54A folding biosensor and to test which of the genes in the pst operon are sufficient for this effect, pBR322 and pBR322 bla::Im7 L53A I54A were transformed into strains containing in-frame deletion mutants of each of the pst genes (pstS, pstC, and pstA, respectively). The deletion mutants of each of these pst genes showed higher ampicillin resistance than did the WT strain MG1655 in the presence of pBR322 bla::Im7 L53A I54A, but did not shower higher ampicillin resistance in the presence of pBR322.


( B ) Levels of β - lactamase ( Bla ) and β - lactamase - Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β - lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes ( CL248 ( pstS : : Tn5 ) , CL249 ( pstC : : Tn5 ) , CL250 ( pstA : : Tn5 ) ) . Maltose binding protein ( MBP ) was used as a control .

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(B) Levels of β-lactamase (Bla) and β-lactamase-Im7 L53A I54A were detected by Western blotting using a polyclonal antibody recognizing β-lactamase in the WT strain MG1655 and in isogenic strains containing insertions in the pstSCA genes (CL248 (pstS::Tn5), CL249 (pstC::Tn5), CL250 (pstA::Tn5)). Maltose binding protein (MBP) was used as a control.


( C ) β - lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay . Data are represented as mean ± SEM from three technical replicates ( unpaired t - test , * * P < 0 . 01 , * * * P < 0 . 001 comparison to MG1655 ; ns , not significant ) .

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(C) β-lactamase enzymatic activities present in periplasmic extracts of the Im7 folding biosensor were monitored in pst WT and pstSCA mutant strains using a spectrophotometric assay. Data are represented as mean ± SEM from three technical replicates (unpaired t-test,** P<0.01, *** P<0.001 comparison to MG1655; ns, not significant).


( D ) To test if levels of the Im7 L53A I54A protein itself , in the absence of the fusion , were also higher in the pstA mutant , expression of the Im7 L53A I54A protein from the IPTG - inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0 - 5 mM . Periplasmic fractions were isolated and examined by SDS - PAGE and subsequent Coomassie staining to determine overall protein expression patterns . Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7 . MBP was used as a loading control . Im7 L53A I54A was increased in the pstA strain relative to the WT control strain . Coomassie staining revealed that a number of periplasmic proteins , including UgpB , were also induced in the pstA strain .

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(D) To test if levels of the Im7 L53A I54A protein itself, in the absence of the fusion, were also higher in the pstA mutant, expression of the Im7 L53A I54A protein from the IPTG-inducible plasmid pCDFTrc was induced using IPTG concentrations ranging from 0-5 mM. Periplasmic fractions were isolated and examined by SDS-PAGE and subsequent Coomassie staining to determine overall protein expression patterns. Im7 L53A I54A levels were determined by Western blotting using an antibody for Im7. MBP was used as a loading control. Im7 L53A I54A was increased in the pstA strain relative to the WT control strain. Coomassie staining revealed that a number of periplasmic proteins, including UgpB, were also induced in the pstA strain.


( A ) MG1655 WT , single mutants of pstA and ugpB , and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322 - based folding biosensor constructs . The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations . Strains containing pBR322 bla : : Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation ( compare uppermost left and right panels ) . This resistance is completely lost upon transducing in a ugpB mutation ( compare next panels down ) , showing that ugpB is responsible for the resistance gained by the pstA mutation .

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(A) MG1655 WT, single mutants of pstA and ugpB, and a double mutant of pstA ugpB were transformed with pBR322 and various pBR322-based folding biosensor constructs. The transformants were examined by a spot titer assay on LB agar plates containing various ampicillin concentrations. Strains containing pBR322 bla::Im7 L53A I54A gain considerable ampicillin resistance in the presence of a pstA mutation (compare uppermost left and right panels). This resistance is completely lost upon transducing in a ugpB mutation (compare next panels down), showing that ugpB is responsible for the resistance gained by the pstA mutation.


( B ) Ampicillin sensitivity conferred by a ugpB deletion mutant ( CL543 ) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB ( pCL1 ) .

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(B) Ampicillin sensitivity conferred by a ugpB deletion mutant (CL543) is complemented by UgpB expression from the arabinose inducible expression vector pBAD33 ugpB (pCL1).


( C ) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein . Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain ( CL543 ) from pCDFTrc and pTrc99a vectors , respectively , both of which have an IPTG - inducible promoter . Periplasmic fractions were isolated and examined by Western blotting . " - " indicates strains containing empty vector and " + " indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB . MBP was used as a loading control . Data information : These experiments were repeated twice with similar results and one representative is shown .

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(C) UgpB expression is sufficient to lead to higher levels of Im7 L53A I54A protein. Im7 L53A I54A and UgpB were expressed in a ugpB mutant strain (CL543) from pCDFTrc and pTrc99a vectors, respectively, both of which have an IPTG-inducible promoter. Periplasmic fractions were isolated and examined by Western blotting. "-" indicates strains containing empty vector and "+" indicates strains containing a recombinant plasmid encoding either Im7 L53A I54A or UgpB. MBP was used as a loading control. Data information: These experiments were repeated twice with similar results and one representative is shown.


( A ) UgpB overexpression confers bile salt resistance , and a ugpB deletion mutant is slightly CHO sensitive .

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(A) UgpB overexpression confers bile salt resistance, and a ugpB deletion mutant is slightly CHO sensitive.


( B ) CHO - induced MDH protein aggregation analyzed by monitoring light scattering .

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(B) CHO-induced MDH protein aggregation analyzed by monitoring light scattering.


( C ) CHO - induced MDH protein aggregation analyzed by a solubility assay using centrifugation ( cfg ) at 16 , 000 g for 20 min . The supernatant ( Sup ) represents the soluble fraction and the pellet the insoluble fraction ; 12 µM MDH was used as a model aggregation - prone substrate ( see Methods ) . Addition of increasing quantities of UgpB increasingly solubilizes MDH . The molar ratios of MDH : UgpB that were used ( left to right ) are as follows : 1 : 0 , 1 : 0 . 1 , 1 : 0 . 2 , 1 : 0 . 5 , and 1 : 1 . 5 - fold more of the pellet fraction was loaded relative to the supernatant fraction .

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(C) CHO-induced MDH protein aggregation analyzed by a solubility assay using centrifugation (cfg) at 16,000 g for 20 min. The supernatant (Sup) represents the soluble fraction and the pellet the insoluble fraction; 12 µM MDH was used as a model aggregation-prone substrate (see Methods). Addition of increasing quantities of UgpB increasingly solubilizes MDH. The molar ratios of MDH:UgpB that were used (left to right) are as follows: 1:0, 1:0.1, 1:0.2, 1:0.5, and 1:1. 5-fold more of the pellet fraction was loaded relative to the supernatant fraction.


( D ) The supernatant / pellet assay was performed using a 1 : 1 MDH : chaperone ratio for Spy , SurA , and OsmY . The pellet fraction was concentrated 5 - fold .

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(D) The supernatant/pellet assay was performed using a 1:1 MDH:chaperone ratio for Spy, SurA, and OsmY. The pellet fraction was concentrated 5-fold.


( E ) 12 µM of MDH alone and a 1 : 1 ratio of either MDH : UgpB or MDH : MBP were incubated at 37 ° C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above ( C ) . The pellet fraction was concentrated 5 - fold . In some samples ( + G3P ) , 120 µM of G3P was added prior to the addition of any other protein . Addition of G3P inactivates UgpB ' s ability to inhibit protein aggregation .

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(E) 12 µM of MDH alone and a 1:1 ratio of either MDH:UgpB or MDH:MBP were incubated at 37°C for 60 min in the presence of CHO and then fractionated via centrifugation for use in a solubility assay as described above (C) . The pellet fraction was concentrated 5-fold. In some samples (+G3P), 120 µM of G3P was added prior to the addition of any other protein. Addition of G3P inactivates UgpB's ability to inhibit protein aggregation.


( F ) 800 μg of E . coli total lysate and 60 µM of UgpB / MBP were used for the supernatant / pellet assay . 600 µM of G3P was added before adding proteins . The pellet fraction was concentrated 10 - fold . As before , addition of UgpB inhibited aggregation , but only in the absence of G3P .

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(F) 800 μg of E. coli total lysate and 60 µM of UgpB/MBP were used for the supernatant/pellet assay. 600 µM of G3P was added before adding proteins. The pellet fraction was concentrated 10-fold. As before, addition of UgpB inhibited aggregation, but only in the absence of G3P.


( G ) The anti - aggregation activity of tryptophan single mutant ( W169S ) and double mutant ( W169S / W172S ) was examined by CHO - induced MDH aggregation assay . Light scattering values were monitored .

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(G) The anti-aggregation activity of tryptophan single mutant (W169S) and double mutant (W169S/W172S) was examined by CHO-induced MDH aggregation assay. Light scattering values were monitored.


( I ) The anti - aggregation activity of the UgpB variants ( E66A and R374V ) was examined by CHO - induced MDH aggregation assay . CHO - induced MDH protein aggregation analyzed by monitoring light scattering .

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(I) The anti-aggregation activity of the UgpB variants (E66A and R374V) was examined by CHO-induced MDH aggregation assay. CHO-induced MDH protein aggregation analyzed by monitoring light scattering.


( J ) Solubility of MDH is also monitored by supernatant / pellet assay . 12 µM of MDH alone and a 1 : 1 ratio of MDH : UgpB proteins , 120 µM of G3P were used . 15 mM CHO were used for all the aggregation and supernatant / pellet assays .

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(J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used. 15 mM CHO were used for all the aggregation and supernatant/pellet assays.


( J ) Solubility of MDH is also monitored by supernatant / pellet assay . 12 µM of MDH alone and a 1 : 1 ratio of MDH : UgpB proteins , 120 µM of G3P were used .

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(J) Solubility of MDH is also monitored by supernatant/pellet assay. 12 µM of MDH alone and a 1:1 ratio of MDH:UgpB proteins, 120 µM of G3P were used.


( A ) Acid denatures UgpB . The secondary structure of 2 µM UgpB was monitored by circular dichroism ( CD ) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7 .

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(A) Acid denatures UgpB. The secondary structure of 2 µM UgpB was monitored by circular dichroism (CD) spectroscopy in 10 mM sodium phosphate buffer at pH 2 and pH 7.


( B ) G3P binds to acid - stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum , either at pH7 or after transition from pH 2 to 7 ( see Methods ) . Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift , indicating G3P binding .

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(B) G3P binds to acid-stripped UgpB as observed by monitoring the tryptophan fluorescence spectrum, either at pH7 or after transition from pH 2 to 7 (see Methods). Transitions from pH 2 to 7 in the presence of G3P are accompanied by a blue shift, indicating G3P binding.


( C ) The anti - aggregation activity of UgpB is monitored by CHO - induced MDH aggregation assay . Light scattering values were monitored . The anti - aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P ( 120 µM ) can again inactivate its chaperone activity .

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(C) The anti-aggregation activity of UgpB is monitored by CHO-induced MDH aggregation assay. Light scattering values were monitored. The anti-aggregation activity of UgpB is activated after transition from pH 2 to 7 and the addition of G3P (120 µM) can again inactivate its chaperone activity.


( D ) Addition of G3P to UgpB inhibits its anti - aggregation chaperone activity . 12 µM of MDH and a 1 : 1 ratio of UgpB : MDH were used in a CHO - induced aggregation assay . At 30 min , the indicated concentrations of G3P were added to solutions containing MDH , and MDH aggregation was monitored by light scattering . Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan

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(D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity . 12 µM of MDH and a 1:1 ratio of UgpB:MDH were used in a CHO-induced aggregation assay. At 30 min, the indicated concentrations of G3P were added to solutions containing MDH, and MDH aggregation was monitored by light scattering. Note that G3P binding to UgpB still occurs in the presence of 15 mM CHO as monitored by tryptophan fluorescence


( D ) Addition of G3P to UgpB inhibits its anti - aggregation chaperone

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(D) Addition of G3P to UgpB inhibits its anti-aggregation chaperone activity


B Growth curves of the indicated strains shown as population doublings ( pds ) on the y axis and hours in liquid culture on the x axis . Before inoculation in liquid YES medium , single cells underwent approximately 25 pds to produce visible colonies on solid medium . For each strain , averages and SDs ( error bars ) of three biological replicates are shown . Arrows indicate times of harvesting and corresponding pds for experiments shown in ( C ) and ( D ) .

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B Growth curves of the indicated strains shown as population doublings (pds) on the y axis and hours in liquid culture on the x axis. Before inoculation in liquid YES medium, single cells underwent approximately 25 pds to produce visible colonies on solid medium. For each strain, averages and SDs (error bars) of three biological replicates are shown. Arrows indicate times of harvesting and corresponding pds for experiments shown in (C) and (D).


C Telo - PCR analysis of telomere length in wt ( ter1 + and est1 + ) and two independent clones ( a and b ) deleted for ter1 + or est1 + grown for ~ 25 + 10 ( ~ 35 ) , ~ 25 + 14 ( ~ 39 ) and ~ 25 + 18 ( ~ 43 ) pds in YES medium . PCRs were performed with the subtelomeric oligonucleotide oF5 , located 620 bp upstream of the first telomeric repeat , and the oligo ( dG ) - containing oligonucleotide odG18 . Marker ( m ) molecular weights are on the left in kilobases . tels : telomere - containing Telo - PCR products .

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C Telo-PCR analysis of telomere length in wt (ter1+ and est1+) and two independent clones (a and b) deleted for ter1+ or est1+ grown for ~ 25+10 (~ 35), ~ 25+14 (~ 39) and ~ 25+18 (~ 43) pds in YES medium. PCRs were performed with the subtelomeric oligonucleotide oF5, located 620 bp upstream of the first telomeric repeat, and the oligo(dG)-containing oligonucleotide odG18. Marker (m) molecular weights are on the left in kilobases. tels: telomere-containing Telo-PCR products.


D qRT - PCR analysis of G - rich TERRA , polyA + TERRA and ARRET / αARRET levels in cells as in ( B ) . Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains ( ter1 + and est1 + ) grown in parallel . Bars and error bars are averages and SD from 4 ( ter1Δ clones ) or 3 ( est1Δ clones ) independent experiments . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( relative to wt ; two - tailed Student ' s t test ) .

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D qRT-PCR analysis of G-rich TERRA, polyA+ TERRA and ARRET/αARRET levels in cells as in (B). Values are normalized to ACT1 mRNA and expressed as fold increase over control wt strains (ter1+ and est1+) grown in parallel. Bars and error bars are averages and SD from 4 (ter1Δ clones) or 3 (est1Δ clones) independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (relative to wt; two-tailed Student's t test).