( b , c ) Reduced viability of Bax / Bak − / − MEFs after exposure to etoposide . WT ( circles ) and Bax / Bak − / − ( squares ) MEFs were treated with 20 μM etoposide in the presence ( closed symbols ) or absence ( open symbols ) of 100 μM zVAD - fmk . Then cell viability was measured by the PI staining ( b ) and CTB assay ( c ) , and expressed as a percentage of the initial value without etoposide . Data are shown as mean ± s . d . ( n = 4 ) .

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(b, c) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide. WT (circles) and Bax/Bak−/− (squares) MEFs were treated with 20 μM etoposide in the presence (closed symbols) or absence (open symbols) of 100 μM zVAD-fmk. Then cell viability was measured by the PI staining (b) and CTB assay (c), and expressed as a percentage of the initial value without etoposide. Data are shown as mean ± s.d. (n = 4).


( d ) Reduced viability of etoposide - treated Bax / Bak − / − MEFs as assessed by proliferation assay . Bax / Bak − / − MEFs were either not treated ( 0 h ) or were treated with 20 μM etoposide for 12 and 24 h , then all cells were recovered and 5 , 000 cells were reseeded . Viable cell numbers were measured on the indicated days by the CTB assay . Results were normalized by adjusting the day 0 value . n = 4 .

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(d) Reduced viability of etoposide-treated Bax/Bak−/− MEFs as assessed by proliferation assay. Bax/Bak−/− MEFs were either not treated (0 h) or were treated with 20 μM etoposide for 12 and 24 h, then all cells were recovered and 5,000 cells were reseeded. Viable cell numbers were measured on the indicated days by the CTB assay. Results were normalized by adjusting the day 0 value. n = 4.


e ) Reduced viability of Bax / Bak − / − MEFs after exposure to etoposide and staurosporine , as assessed by clonogenicity assay . MEFs were treated with etoposide ( 20 μM ) or staurosporine ( 1 μM ) at the indicated times , collected , and 2 , 000 cells were seeded in the normal medium . After 1 week , colonies were counted . n =

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e) Reduced viability of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine, as assessed by clonogenicity assay. MEFs were treated with etoposide (20 μM) or staurosporine (1 μM) at the indicated times, collected, and 2,000 cells were seeded in the normal medium. After 1 week, colonies were counted. n = 4


f - i ) Induction of autophagy in Bax / Bak − / − MEFs by etoposide . ( f ) Electron micrograph ( × 14 , 800 ) of Bax / Bak − / − MEF treated with etoposide ( 20 μM ) for 18 h . Representative features of Bax / Bak − / − MEFs that have received no treatment ( NT ; that is , are healthy ) are also shown ( NT : × 14 , 000 ) .

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f-i) Induction of autophagy in Bax/Bak−/− MEFs by etoposide. (f) Electron micrograph (×14,800) of Bax/Bak−/− MEF treated with etoposide (20 μM) for 18 h. Representative features of Bax/Bak−/− MEFs that have received no treatment (NT; that is, are healthy) are also shown (NT: ×14,000).


( g ) In magnified photographs , a large number of autolysosomes / autophagosomes ( left : × 26 , 000 ) were observed , but the mitochondria were unaffected ( right : × 17 , 000 ) .

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(g) In magnified photographs, a large number of autolysosomes/autophagosomes (left: ×26,000) were observed, but the mitochondria were unaffected (right: ×17,000).


( h , i ) Punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs treated with etoposide . GFP - LC3 - transfected cells were incubated with and without etoposide ( 20 μM ) for 24 h ( h ) or the indicated times ( i ) , and then examined by fluorescent microscopy . ( h ) Representative photographs of healthy ( NT ) and etoposide - treated Bax / Bak − / − MEFs are shown . ( i ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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(h, i) Punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs treated with etoposide. GFP-LC3-transfected cells were incubated with and without etoposide (20 μM) for 24 h (h) or the indicated times (i), and then examined by fluorescent microscopy. (h) Representative photographs of healthy (NT) and etoposide-treated Bax/Bak−/− MEFs are shown. (i) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-positive cells. Data are shown as mean ± s.d. (n = 4).


( a , b ) Bax / Bak − / − MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3 - MA for 24 h , and then were examined by phase - contrast microscopy ( a ) and electron microscopy ( × 8 , 400 ) ( b ) . Autolysosomes / autophagosomes were hardly observed . (

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(a, b) Bax/Bak−/− MEFs were treated with 20 μM etoposide in the absence or presence of 10 mM 3-MA for 24 h, and then were examined by phase-contrast microscopy (a) and electron microscopy (×8,400) (b). Autolysosomes/autophagosomes were hardly observed. (


c , d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by 3 - MA . Bax / Bak − / − MEFs that were transfected with GFP - LC3 were incubated without ( NT ) or with 20 μM etoposide in the presence or absence of 10 mM 3 - MA for 24 h , and then were examined by confocal fluorescent microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in the presence of 3 - MA . ( d ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).


( c , d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by 3 - MA . Bax / Bak − / − MEFs that were transfected with GFP - LC3 were incubated without ( NT ) or with 20 μM etoposide in the presence or absence of 10 mM 3 - MA for 24 h , and then were examined by confocal fluorescent microscopy . ( c ) Representative photograph of etoposide - treated Bax / Bak − / − MEFs in the presence of 3 - MA . ( d ) The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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(c, d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by 3-MA. Bax/Bak−/− MEFs that were transfected with GFP-LC3 were incubated without (NT) or with 20 μM etoposide in the presence or absence of 10 mM 3-MA for 24 h, and then were examined by confocal fluorescent microscopy. (c) Representative photograph of etoposide-treated Bax/Bak−/− MEFs in the presence of 3-MA. (d) The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).


( e - g ) Inhibition of etoposide - and staurosporine - induced cell death by 3 - MA . Bax / Bak − / − MEFs were treated with 20 μM etoposide ( e - g ) or 1 μM staurosporine ( g ) in the absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA for the indicated time . Cell viability was measured by PI staining ( e ) , CTB assay ( f ) and clonogenicity assay ( g ) ( as in Fig . 1e ) . Upper panels in g : viable cells were visualized by staining with calcein - AM . n = 4 .

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(e-g) Inhibition of etoposide- and staurosporine-induced cell death by 3-MA. Bax/Bak−/− MEFs were treated with 20 μM etoposide (e-g) or 1 μM staurosporine (g) in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA for the indicated time. Cell viability was measured by PI staining (e), CTB assay (f) and clonogenicity assay (g) (as in Fig. 1e). Upper panels in g: viable cells were visualized by staining with calcein-AM. n = 4.


( a , b ) Bax / Bak − / − MEFs were treated with the indicated siRNAs ( 10 μg ) for 24 h and then incubated with 20 μM etoposide for the indicated times . Expression of APG5 - APG12 complex ( a ) , Beclin 1 ( b ) and VDAC ( loading control ) was analysed by Western blot analysis .

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(a, b) Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex (a), Beclin 1 (b) and VDAC (loading control) was analysed by Western blot analysis.


( c ) Inhibition of etoposide - induced autophagy by silencing of Beclin 1 . Bax / Bak − / − MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy ( × 8 , 500 ) .

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(c) Inhibition of etoposide-induced autophagy by silencing of Beclin 1. Bax/Bak−/− MEFs with silencing of Beclin 1 were treated with 20 μM etoposide at 18 h and were analysed by electron microscopy (×8,500).


d ) Reduction of punctate GFP - LC3 fluorescence in Bax / Bak − / − MEFs by silencing of Beclin 1 and APG5 . Bax / Bak − / − MEFs that were transfected with GFP - LC3 together with the indicated siRNAs , were incubated without ( NT ) or with 20 μM etoposide for 24 h , and then examined by confocal fluorescent microscopy . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . Data are shown as mean ± s . d . ( n = 4 ) .

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d) Reduction of punctate GFP-LC3 fluorescence in Bax/Bak−/− MEFs by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were transfected with GFP-LC3 together with the indicated siRNAs, were incubated without (NT) or with 20 μM etoposide for 24 h, and then examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. Data are shown as mean ± s.d. (n = 4).


( e , f ) Inhibition of etoposide - and staurosporine - induced cell death by silencing of Beclin 1 and APG5 . Bax / Bak − / − MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide ( e , f ) or 1 μM staurosporine ( f ) for the indicated time . Cell viability was measured by CTB assay ( e ) and clonogenicity assay ( f ) . n = 4 .

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(e, f) Inhibition of etoposide- and staurosporine-induced cell death by silencing of Beclin 1 and APG5. Bax/Bak−/− MEFs that were treated with the indicated siRNAs were incubated with 20 μM etoposide (e, f) or 1 μM staurosporine (f) for the indicated time. Cell viability was measured by CTB assay (e) and clonogenicity assay (f). n = 4.


( a , b ) APG5 - dependent punctate GFP - LC3 fluorescence in Bcl - 2 - transfected MEFs after etoposide treatment . APG5 + / + and APG5 − / − MEFs that were transfected with both GFP - LC3 and the indicated plasmids ( Bcl - 2 or vector ) were incubated without or with 20 μM etoposide for 24 h , then examined by confocal fluorescent microscopy ( a ) . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells ( b ) .

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(a, b) APG5-dependent punctate GFP-LC3 fluorescence in Bcl-2-transfected MEFs after etoposide treatment. APG5+/+ and APG5−/− MEFs that were transfected with both GFP-LC3 and the indicated plasmids (Bcl-2 or vector) were incubated without or with 20 μM etoposide for 24 h, then examined by confocal fluorescent microscopy (a). The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells (b).


( c ) Accumulation of Beclin 1 and APG5 - APG12 complex in Bcl - 2 - transfected MEFs after etoposide treatment . WT MEFs were transfected with the indicated plasmids , and were incubated without ( NT ) or with 20 μM etoposide for 24 h .

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(c) Accumulation of Beclin 1 and APG5-APG12 complex in Bcl-2-transfected MEFs after etoposide treatment. WT MEFs were transfected with the indicated plasmids, and were incubated without (NT) or with 20 μM etoposide for 24 h.


( d , e ) Occurrence of etoposide - induced 3 - MA - inhibitable non - apoptotic death in Bcl - xL - overexpressing APG5 + / + MEFs , but not APG5 − / − MEFs . APG5 + / + ( open columns ) and APG5 − / − ( closed columns ) MEFs were transfected with the indicated plasmids ( 2 μg ) . After 24 h , transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3 - MA for 24 h , and cell viability was measured by the CTB assay ( d ) and apoptotic nuclear morphology ( e ) . Data are shown as mean ± s . d . ( n = 4 ) .

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(d, e) Occurrence of etoposide-induced 3-MA-inhibitable non-apoptotic death in Bcl-xL-overexpressing APG5+/+ MEFs, but not APG5−/− MEFs. APG5+/+ (open columns) and APG5−/− (closed columns) MEFs were transfected with the indicated plasmids (2 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 100 μM of zVAD or 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay (d) and apoptotic nuclear morphology (e). Data are shown as mean ± s.d. (n = 4).


( f ) Overexpression of Bcl - xL does not sensitize Beclin 1 - silenced MEFs to etoposide - induced 3 - MA - inhibitable non - apoptotic death . WT MEFs with silencing of control GFP ( open columns ) or Beclin 1 ( closed columns ) were transfected with the indicated plasmids ( 1 μg ) . After 24 h , transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3 - MA for 24 h , and cell viability was measured by the CTB assay . n = 3 .

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(f) Overexpression of Bcl-xL does not sensitize Beclin 1-silenced MEFs to etoposide-induced 3-MA-inhibitable non-apoptotic death. WT MEFs with silencing of control GFP (open columns) or Beclin 1 (closed columns) were transfected with the indicated plasmids (1 μg). After 24 h, transfected cells were incubated with 20 μM of etoposide in the presence or absence of 10 mM of 3-MA for 24 h, and cell viability was measured by the CTB assay. n = 3.


( a ) Enhancement of etoposide - induced non - apoptotic death by overexpression of human Bcl - 2 and Bcl - xL in Bax / Bak − / − MEFs in a Beclin 1 - dependent manner . Bax / Bak − / − MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids ( 1 μg ) . After 24 h , the transfected cells were incubated with 20 μM etoposide for 24 h , and cell viability was assessed by the CTB assay . Data are shown as mean ± s . d . ( n = 3 ) .

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(a) Enhancement of etoposide-induced non-apoptotic death by overexpression of human Bcl-2 and Bcl-xL in Bax/Bak−/− MEFs in a Beclin 1-dependent manner. Bax/Bak−/− MEFs with silencing of control Bax or Beclin 1 were transfected with the indicated plasmids (1 μg). After 24 h, the transfected cells were incubated with 20 μM etoposide for 24 h, and cell viability was assessed by the CTB assay. Data are shown as mean ± s.d. (n = 3).


( b ) Bax / Bak − / − MEFs were transiently transfected with 10 μg of siRNA for Bcl - 2 , Bcl - x , or Bax ( negative control ) for 24 h . Expression levels of Bcl - 2 , Bcl - xL and Lamin B ( loading control ) were analysed by Western blot analysis .

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(b) Bax/Bak−/− MEFs were transiently transfected with 10 μg of siRNA for Bcl-2, Bcl-x, or Bax (negative control) for 24 h. Expression levels of Bcl-2, Bcl-xL and Lamin B (loading control) were analysed by Western blot analysis.


( c - e ) Inhibition of etoposide - induced autophagy in Bax / Bak − / − cells treated with siRNA for Bcl - x . ( c ) Bax / Bak − / − MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h , then examined by phase - contrast microscopy .

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(c-e) Inhibition of etoposide-induced autophagy in Bax/Bak−/− cells treated with siRNA for Bcl-x. (c) Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide for 24 h, then examined by phase-contrast microscopy.


( d ) Bax / Bak − / − MEFs that were transfected with siRNA for Bcl - x and then treated with etoposide for 18 h were analysed by electron microscopy ( × 8 , 500 )

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(d) Bax/Bak−/− MEFs that were transfected with siRNA for Bcl-x and then treated with etoposide for 18 h were analysed by electron microscopy (×8,500)


( e ) Reduction of punctate GFP - LC3 fluorescence in etoposide - treated Bax / Bak − / − MEFs by silencing of Bcl - x . Bax / Bak − / − MEFs that were transfected with both GFP - LC3 and the indicated siRNAs were incubated without ( NT ) or with etoposide for 24 h , and then were examined by confocal fluorescent microscopy . The percentage of cells with punctate GFP - LC3 fluorescence was calculated relative to all GFP - LC3 - positive cells . n = 4 .

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(e) Reduction of punctate GFP-LC3 fluorescence in etoposide-treated Bax/Bak−/− MEFs by silencing of Bcl-x. Bax/Bak−/− MEFs that were transfected with both GFP-LC3 and the indicated siRNAs were incubated without (NT) or with etoposide for 24 h, and then were examined by confocal fluorescent microscopy. The percentage of cells with punctate GFP-LC3 fluorescence was calculated relative to all GFP-LC3-positive cells. n = 4.


f ) Inhibition of etoposide - induced cell death by silencing of Bcl - x . Bax / Bak − / − MEFs were transfected with siRNA ( circles , siRNA for Bax ; squares , siRNA for Bcl - x ) were treated with 20 μM etoposide in the absence ( open symbols ) or presence ( closed symbols ) of 10 mM 3 - MA , and cell viability was assessed by the CTB assay at the indicated times . n = 4 .

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f) Inhibition of etoposide-induced cell death by silencing of Bcl-x. Bax/Bak−/− MEFs were transfected with siRNA (circles, siRNA for Bax; squares, siRNA for Bcl-x) were treated with 20 μM etoposide in the absence (open symbols) or presence (closed symbols) of 10 mM 3-MA, and cell viability was assessed by the CTB assay at the indicated times. n = 4.


( g ) Clonogenicity assay of Bax / Bak − / − MEFs after exposure to etoposide and staurosporine . Bax / Bak − / − MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h . After collection , 2 , 000 cells were seeded in the normal medium . After one week , colonies were counted .

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(g) Clonogenicity assay of Bax/Bak−/− MEFs after exposure to etoposide and staurosporine. Bax/Bak−/− MEFs that were transfected with the indicated siRNAs were treated with 20 μM etoposide or 1 μM staurosporine at 24 h. After collection, 2,000 cells were seeded in the normal medium. After one week, colonies were counted.


h , i ) Inhibition of APG5 - APG12 induction by silencing of Bcl - x or Beclin 1 . Bax / Bak − / − MEFs were treated with the indicated siRNAs ( 10 μg ) for 24 h and then incubated with 20 μM etoposide for the indicated times . Expression of APG5 - APG12 complex , Beclin 1 and GAPDH / VDAC ( loading control ) was analysed by Western blot analysis .

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h, i) Inhibition of APG5-APG12 induction by silencing of Bcl-x or Beclin 1. Bax/Bak−/− MEFs were treated with the indicated siRNAs (10 μg) for 24 h and then incubated with 20 μM etoposide for the indicated times. Expression of APG5-APG12 complex, Beclin 1 and GAPDH/VDAC (loading control) was analysed by Western blot analysis.


( A ) Profile of transcript levels of autophagy - related genes in iBAT which are significantly modified in cold exposed mice ( 4ºC , 24h ) , compared with mice maintained at thermoneutral temperature ( 29ºC ) . ( Fisher ' s exact test corrected by the Benjamini - Hochberg method , p < 0 . 05 ) . Heatmap represents log - fold change of RPKM values of cold versus thermoneutral ( TN ) sample data obtained from RNAseq analysis . Each square represents replicates ( n = 4 ) . The most repressed genes are shown on the bottom of the heatmap .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)Profile of transcript levels of autophagy-related genes in iBAT which are significantly modified in cold exposed mice (4ºC, 24h), compared with mice maintained at thermoneutral temperature (29ºC). (Fisher's exact test corrected by the Benjamini-Hochberg method, p< 0.05). Heatmap represents log-fold change of RPKM values of cold versus thermoneutral (TN) sample data obtained from RNAseq analysis. Each square represents replicates (n=4). The most repressed genes are shown on the bottom of the heatmap.


Parkin expression in iBAT from mice exposed to cold ( 4ºC ) for 1 day ( 1d ) or 21 days ( 21d ) compared with control mice ( ctl ) maintained at 22ºC . Left : Relative transcript levels . Right : representative immunoblot and Parkin protein quantification . Ponceau staining ( PS ) was used as loading control . ( n = 6 ; transcript levels ) ( n = 3 ; protein levels ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , cold vs . control and # # p < 0 . 01 , 1d vs . 21d . One - way ANOVA with Tukey ' s post hoc test .

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

Parkin expression in iBAT from mice exposed to cold (4ºC) for 1 day (1d) or 21 days (21d) compared with control mice (ctl) maintained at 22ºC. Left: Relative transcript levels. Right: representative immunoblot and Parkin protein quantification. Ponceau staining (PS) was used as loading control. (n=6; transcript levels) (n=3; protein levels). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ##p<0.01, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.


Relative transcript levels of Park2 in sWAT and eWAT . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , cold vs . control and # # # p < 0 . 001 , 1d vs . 21d . One - way ANOVA with Tukey ' s post hoc test .

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

Relative transcript levels of Park2 in sWAT and eWAT. (n= 6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, cold vs. control and ###p<0.001, 1d vs. 21d. One-way ANOVA with Tukey's post hoc test.


Relative transcript levels of Parkin in iBAT , sWAT and eWAT of mice injected with the β3 - AR agonist CL316 , 243 , every day for 1 week . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * * p < 0 . 001 , CL316 , 243 treatment vs . saline . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Parkin in iBAT, sWAT and eWAT of mice injected with the β3-AR agonist CL316,243, every day for 1 week. (n=6). Data are presented as means ±s.e.m. *p<0.05, ***p<0.001, CL316,243 treatment vs. saline. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in mature brown adipocytes compared with the stromal vascular fraction ( SVF ) in iBAT . ( n = 3 ) . Data are presented as means ± s . e . m . * * p < 0 . 01 , * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in mature brown adipocytes compared with the stromal vascular fraction (SVF) in iBAT. (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


mRNA expression profiles of Park2 and Ucp1 during differentiation . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to day ( d ) 4 . Two - tailed unpaired Student ' s t - test .

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

mRNA expression profiles of Park2 and Ucp1 during differentiation. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to day (d) 4. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture with fetal bovine serum ( FBS , to trigger in vitro differentiation ) or charcoal - stripped serum ( CSS , non - permissive for differentiation ) . ( n = 3 ) . Data are presented as means ± s . e . m . * * p < 0 . 01 , * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture with fetal bovine serum (FBS, to trigger in vitro differentiation) or charcoal-stripped serum (CSS, non-permissive for differentiation). (n=3). Data are presented as means ±s.e.m. **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine ( NE ) or cAMP for indicated times . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to non - treated cells . One - way ANOVA with Dunnett ' s post hoc test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes in culture treated with norepinephrine (NE) or cAMP for indicated times. (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to non-treated cells. One-way ANOVA with Dunnett's post hoc test.


Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3 - methyladenine ( 3 - MA ) . Top : Representative immunoblots . p62 and LC3BII proteins are shown as positive controls of 3 - MA treatment . β - actin was use as loading control . Bottom : Parkin protein quantification ( performed using 3 immunoblots processed in parallel due to sample size ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 compared to NE / cAMP non - treated cells . One - way ANOVA with Dunnett ' s post hoc test .

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

Parkin protein levels in brown adipocytes treated with NE or cAMP for the indicated times in the presence or absence of 3-methyladenine (3-MA). Top: Representative immunoblots. p62 and LC3BII proteins are shown as positive controls of 3-MA treatment. β-actin was use as loading control. Bottom: Parkin protein quantification (performed using 3 immunoblots processed in parallel due to sample size). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to NE/cAMP non-treated cells. One-way ANOVA with Dunnett's post hoc test.


Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide ( CHX ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 in brown adipocytes treated with NE in the presence or absence of cycloheximide (CHX). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001. Two-tailed unpaired Student's t-test.


Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D ( n = 3 ) . Data are presented as means ± s . e . m . One - way ANOVA with Tukey ' s post hoc test comparing treatments .

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

Relative mRNA expression levels of Parkin in brown adipocytes treated with NE in the presence or absence of actinomycin D (n=3). Data are presented as means ±s.e.m. One-way ANOVA with Tukey's post hoc test comparing treatments.


Park2 gene promoter activity in HIB - 1B cells transfected with a pGL4 - Park2 - Luc reporter construct treated with or without NE or cAMP . Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 compared to non - treated cells . ANOVA with Dunnett ' s post hoc

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

Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct treated with or without NE or cAMP. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that in untreated cells. (n=6). Data are presented as means ±s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test


Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP ( 12 h ) plus H89 ( a PKA inhibitor ) or SB202190 ( a p38 MAPK inhibitor ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 with vs . without inhibitor and # p < 0 . 05 , and # # p < 0 . 01 , # # # p < 0 . 001 with vs . without cAMP . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated with cAMP (12 h) plus H89 (a PKA inhibitor) or SB202190 (a p38 MAPK inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 , and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP ( 24h ) plus Atglistatin ( an adipose triglyceride lipase inhibitor ) or CAY10499 ( a hormone sensitive lipase inhibitor ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 with vs . without inhibitor and # p < 0 . 05 , and # # p < 0 . 01 , # # # p < 0 . 001 with vs . without cAMP . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2, and concentration of glycerol in the culture medium of brown adipocytes treated with cAMP (24h) plus Atglistatin (an adipose triglyceride lipase inhibitor) or CAY10499 (a hormone sensitive lipase inhibitor). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001 with vs. without inhibitor and #p<0.05, and ##p<0.01, ###p<0.001 with vs. without cAMP. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator ( GW7647 ) , a PPARα inhibitor ( GW6471 ) with or without norepinephrine ( NE ) , or all - trans - retinoic acid ( RA ) . ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 and Ucp1 in brown adipocytes treated for 24h with a PPARα activator (GW7647), a PPARα inhibitor (GW6471) with or without norepinephrine (NE), or all-trans-retinoic acid (RA). (n=3). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 ***p<0.001. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 in wild - type and PPARα - KO brown adipocytes treated for 24h with NE ( n = 3 ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

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

Relative transcript levels of Park2 in wild-type and PPARα-KO brown adipocytes treated for 24h with NE (n=3). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Park2 gene promoter activity in HIB - 1B cells transfected with a pGL4 - Park2 - Luc reporter construct and , when indicated , with a pSG5 - Ppara expression vector . Cells were treated with the PPARα activator GW7647 or NE . Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4 - Park2 - Luc construct ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 compared to non - treated cells . ANOVA with Dunnett ' s post hoc test .

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

Park2 gene promoter activity in HIB-1B cells transfected with a pGL4-Park2-Luc reporter construct and, when indicated, with a pSG5-Ppara expression vector. Cells were treated with the PPARα activator GW7647 or NE. Data was normalized using Renilla luciferase activity and are expressed as the relative firefly luciferase activity compared to that only transfected with the pGL4-Park2-Luc construct (n=6). Data are presented as means ± s.e.m. *p<0.05 compared to non-treated cells. ANOVA with Dunnett's post hoc test.


Left : Representative optical microscopic images of H & E - stained iBAT ( scale bars , 110µm ) and electron microscopic images of iBAT ( scale bars , 20µm ) from wild - type ( WT ) and Parkin - KO ( KO ) mice . Right : Quantification of lipid droplet area from electron microscopic images ( 10 images per condition were used , n = 3 WT , n = 3 Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . Two - tailed unpaired Student ' s t - test .

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

Left: Representative optical microscopic images of H&E-stained iBAT (scale bars, 110µm) and electron microscopic images of iBAT (scale bars, 20µm) from wild-type (WT) and Parkin-KO (KO) mice. Right: Quantification of lipid droplet area from electron microscopic images (10 images per condition were used, n =3 WT, n=3 Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. Two-tailed unpaired Student's t-test.


Representative thermal images ( left ) and quantification of surface temperature ( right ) for the interscapular region . ( n = 6 ) . Data are presented as means ± s . e . m . * * * p < 0 . 001 . Two - tailed unpaired Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 thermal images (left) and quantification of surface temperature (right) for the interscapular region. (n=6). Data are presented as means ±s.e.m. ***p<0.001. Two-tailed unpaired Student's t-test.


Representative immunoblot of UCP1 protein and the loading control ( Ponceau staining , PS ) in iBAT samples .

Representative immunoblot of UCP1 protein and the loading control (Ponceau staining, PS) in iBAT samples.


Relative transcript levels of thermogenesis - related genes of iBAT from WT and Parkin - KO mice exposed to acute cold ( 4ºC , 24h ) compared to those maintained at the control temperature ( 22ºC ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , CTL vs . cold ; # p < 0 . 05 , WT vs . Parkin - KO . ANOVA with Tukey ' s post hoc test .

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

Relative transcript levels of thermogenesis-related genes of iBAT from WT and Parkin-KO mice exposed to acute cold (4ºC, 24h) compared to those maintained at the control temperature (22ºC). (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001, CTL vs. cold; #p<0.05, WT vs. Parkin-KO. ANOVA with Tukey's post hoc test.


Weight gain from WT and Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

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

Weight gain from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


, blood glucose levels and plasma insulin levels from WT and Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

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

, blood glucose levels and plasma insulin levels from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


insulin tolerance test ( ITT ) from WT and Parkin - KO mice fed with chow ( CD ) or high fat diet ( HFD ) . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) .

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

insulin tolerance test (ITT) from WT and Parkin-KO mice fed with chow (CD) or high fat diet (HFD). (n=8; WT) (n=6; Parkin-KO).


Relative transcript levels of thermogenesis - related genes in iBAT from WT and Parkin - KO mice given HFD . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . # p < 0 . 05 . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of thermogenesis-related genes in iBAT from WT and Parkin-KO mice given HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. #p<0.05. Two-tailed unpaired Student's t-test.


Relative transcript levels of Park2 in iBAT from WT mice fed CD or HFD . ( n = 8 ; WT ) ( n = 6 ; Parkin - KO ) . Data are presented as means ± s . e . m . Two - tailed unpaired Student ' s t - test .

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

Relative transcript levels of Park2 in iBAT from WT mice fed CD or HFD. (n=8; WT) (n=6; Parkin-KO). Data are presented as means ±s.e.m. Two-tailed unpaired Student's t-test.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Top : Total protein content in iBAT during cold deaclimation . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 compared to 21d . ANOVA with Dunnett ' s post hoc test . Bottom : Representative optical microscopic images of H & E - stained iBAT ( scale bars , 100µm ) and electron microscopic images of iBAT ( scale bars , 10µm ) .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Top: Total protein content in iBAT during cold deaclimation. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01 compared to 21d. ANOVA with Dunnett's post hoc test. Bottom: Representative optical microscopic images of H&E-stained iBAT (scale bars, 100µm) and electron microscopic images of iBAT (scale bars, 10µm).


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Relative transcript levels of thermogenesis - related genes and Park2 in iBAT . ( n = 6 ) . Data are presented as means ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 compared to 21d . ANOVA with Dunnett ' s post hoc test .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Relative transcript levels of thermogenesis-related genes and Park2 in iBAT. (n=6). Data are presented as means ±s.e.m. *p<0.05, **p<0.01, ***p<0.001 compared to 21d. ANOVA with Dunnett's post hoc test.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative immunoblot of the indicated thermogenic and autophagic proteins in iBAT during cold acclimation and deacclimation . PS : Ponceau staining .

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of the indicated thermogenic and autophagic proteins in iBAT during cold acclimation and deacclimation. PS: Ponceau staining.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation . Arrowheads indicate Parkin 52kDa band and asterisk indicates a non - specific band .

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative immunoblot of Parkin phosphoSer65 and total Parkin in whole lysates of iBAT and after Parkin immunoprecipitation. Arrowheads indicate Parkin 52kDa band and asterisk indicates a non-specific band.


WT mice were acclimated to cold for 21 days ( 21d , 4ºC ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( 1d ) or 7 days ( 7d ) . Representative electron microscopic images of autophagic and mitophagic events in iBAT after 1 day of cold deacclimation . Yellow arrowheads indicate mitochondrial cristae , blue arrowheads indicate double - membrane autophagosomes , and red arrowheads indicate structures compatible with mitochondria - derived vesicles . LD : lipid droplet . Scale bars , 1 µm .

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

WT mice were acclimated to cold for 21 days (21d, 4ºC) and then deacclimated at thermoneutrality (29ºC) for 1 day (1d) or 7 days (7d). Representative electron microscopic images of autophagic and mitophagic events in iBAT after 1 day of cold deacclimation. Yellow arrowheads indicate mitochondrial cristae, blue arrowheads indicate double-membrane autophagosomes, and red arrowheads indicate structures compatible with mitochondria-derived vesicles. LD: lipid droplet. Scale bars, 1 µm.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Representative immunoblot of LC3B in iBAT , and the loading control ( Ponceau staining , PS ) .

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Representative immunoblot of LC3B in iBAT, and the loading control (Ponceau staining, PS).


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Immunoblot for p62 and OPTN ( left ) and its quantification ( right ) in iBAT . PS was used as the loading control . Arrowhead indicates p62 band and asterisk indicates a non - specific band . ( n = 4 ; cold ) ( n = 3 ; deacclimation ) . Data are presented as means ± s . e . m . * * p < 0 . 01 . ANOVA with Tukey ' s post hoc test .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Immunoblot for p62 and OPTN (left) and its quantification (right) in iBAT. PS was used as the loading control. Arrowhead indicates p62 band and asterisk indicates a non-specific band. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. **p<0.01. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Quantification of the total protein amounts of the indicated OXPHOS protein subunits and UCP1 in iBAT . ( n = 4 ; cold ) ( n = 3 ; deacclimation ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Quantification of the total protein amounts of the indicated OXPHOS protein subunits and UCP1 in iBAT. (n=4; cold) (n=3; deacclimation). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Citrate synthase activity in iBAT protein extracts . ( n = 6 ; WT ) ( n = 8 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Citrate synthase activity in iBAT protein extracts. (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Mitochondrial DNA ( mtDNA ) content in iBAT relative to nuclear DNA ( nDNA ) . ( n = 6 ; WT ) ( n = 8 ; Parkin - KO ) . Data are presented as means ± s . e . m . * p < 0 . 05 . ANOVA with Tukey ' s post hoc test .

O O O O O O O O O O O O O O O O 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-MEASURED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Mitochondrial DNA (mtDNA) content in iBAT relative to nuclear DNA (nDNA). (n=6; WT) (n=8; Parkin-KO). Data are presented as means ±s.e.m. *p<0.05. ANOVA with Tukey's post hoc test.


Wild - type ( WT ) and Parkin - KO ( KO ) mice were acclimated to cold for 21 days ( c ) and then deacclimated at thermoneutrality ( 29ºC ) for 1 day ( d ) . Representative electron microscopic images of mitochondrial integrity and autophagic / mitophagic events in iBAT after 1 day of cold - deacclimation . Yellow arrowheads indicate mitochondria cristae and blue arrowheads indicate double - membrane autophagosomes . Left panels scale bars , 2 µm . Right panels scale bars , 1 µm .

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

Wild-type (WT) and Parkin-KO (KO) mice were acclimated to cold for 21 days (c) and then deacclimated at thermoneutrality (29ºC) for 1 day (d). Representative electron microscopic images of mitochondrial integrity and autophagic/mitophagic events in iBAT after 1 day of cold-deacclimation. Yellow arrowheads indicate mitochondria cristae and blue arrowheads indicate double-membrane autophagosomes. Left panels scale bars, 2 µm. Right panels scale bars, 1 µm.


A ) Caspase 3 / 7 activation ± 16 - hour treatment with varying concentrations of BAM15 , DNP , or FCCP ( N = 3 per condition ) . 5 μM : BAM15 vs . DNP ( P = 0 . 030 ) . 10 μM : BAM15 vs . DNP ( P = 0 . 013 ) . 20 μM : BAM15 vs . DNP ( P = 0 . 0005 ) , BAM15 vs . FCCP ( P = 0 . 036 ) . 40 μM : BAM15 vs . DNP ( P = 0 . 018 ) , BAM15 vs . FCCP ( P = 0 . 021 ) . 80 μM : BAM15 vs . DNP ( P = 0 . 018 ) , DNP vs . FCCP ( P = 0 . 004 ) .

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

A) Caspase 3/7 activation ± 16-hour treatment with varying concentrations of BAM15, DNP, or FCCP (N=3 per condition). 5 μM: BAM15 vs. DNP (P=0.030). 10 μM: BAM15 vs. DNP (P=0.013). 20 μM: BAM15 vs. DNP (P=0.0005), BAM15 vs. FCCP (P=0.036). 40 μM: BAM15 vs. DNP (P=0.018), BAM15 vs. FCCP (P=0.021). 80 μM: BAM15 vs. DNP (P=0.018), DNP vs. FCCP (P=0.004).


B ) The rate of oxygen consumption ( OCR ) , ( C ) ratio of oxygen consumption to extracellular acidification ( OCR / ECAR ) , ollowing acute injection of 1 μM BAM15 , DNP , or FCCP ( BAM15 vs . DNP , P < 0 . 0001 ; BAM15 vs . FCCP ( P < 0 . 0001 ) ( N = 6 per condition ) .

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

B) The rate of oxygen consumption (OCR), (C) ratio of oxygen consumption to extracellular acidification (OCR/ECAR), ollowing acute injection of 1 μM BAM15, DNP, or FCCP (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001) (N=6 per condition).


D ) maximal rate of OCR ( BAM15 vs . DNP , P < 0 . 0001 ; DNP vs . FCCP ( P < 0 . 0001 ) ( E ) time to maximal uncoupling rate ( BAM15 vs . DNP , P < 0 . 0001 ; BAM15 vs . FCCP ( P < 0 . 0001 ) , and ( F ) mitochondrial respiratory half - life following acute injection of 1 μM BAM15 , DNP , or FCCP ( BAM15 vs . DNP , P < 0 . 0001 ; BAM15 vs . FCCP ( P < 0 . 0001 ) ( N = 6 per condition ) .

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

D) maximal rate of OCR (BAM15 vs. DNP, P<0.0001; DNP vs. FCCP (P<0.0001) (E) time to maximal uncoupling rate (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001), and (F) mitochondrial respiratory half-life following acute injection of 1 μM BAM15, DNP, or FCCP (BAM15 vs. DNP, P<0.0001; BAM15 vs. FCCP (P<0.0001) (N=6 per condition).


G ) Representative respirometry plot of intact cells ± 16 - hour treatment with 20 μM BAM15 sequentially injected with oligomycin , FCCP , and a cocktail of rotenone and antimycin a ( N = 5 per condition )

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

G) Representative respirometry plot of intact cells ± 16-hour treatment with 20 μM BAM15 sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)


H ) basal ( P = 0 . 002 ) and ATP - linked respiration , proton leak ( P = 0 . 0003 ) , maximal uncoupling , spare respiratory capacity ( P < 0 . 0001 ) , and non - mitochondrial respiration ( N = 5 per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) basal (P=0.002) and ATP-linked respiration, proton leak (P=0.0003), maximal uncoupling, spare respiratory capacity (P<0.0001), and non-mitochondrial respiration (N=5 per condition).


I ) Assessment of routine ( R ) oxygen flux ( P = 0 . 030 ) , leak ( L ) ( P = 0 . 010 ) , OXPHOS ( P ) and ETC ( E ) capacity in permeabilized C2C12 cells ± 16 - hour treatment with BAM15 ( N = 4 per condition ) . PM : pyruvate and malate , ADP : adenosine diphosphate , GS : glutamate and succinate , Tm : tetramethyl - p - phenylenediamine .

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

I) Assessment of routine (R) oxygen flux (P=0.030), leak (L) (P=0.010), OXPHOS (P) and ETC (E) capacity in permeabilized C2C12 cells ± 16-hour treatment with BAM15 (N=4 per condition). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine.


( J ) Citrate synthase activity ± 16 - hour treatment with 20 μM BAM15 ( N = 10 per condition ) . ( K ) mtDNA ( COX2 : 18S ) ± 16 - hour treatment with 20 μM BAM15 ( N = 6 per condition ) .

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 B-CONTROLLED_VAR O O O O O O O O

(J) Citrate synthase activity ± 16-hour treatment with 20 μM BAM15 (N=10 per condition). (K) mtDNA (COX2:18S) ± 16-hour treatment with 20 μM BAM15 (N=6 per condition).


( A ) Heat map visualization of top 30 differentially regulated mRNA transcripts . Color represents significant down - ( blue ) or up - regulated ( red ) mRNA expressed as a log2 fold change relative to vehicle corrected for the false discovery rate .

O O O O O O O O O O O O O O O O O O O O O O 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) Heat map visualization of top 30 differentially regulated mRNA transcripts. Color represents significant down- (blue) or up-regulated (red) mRNA expressed as a log2 fold change relative to vehicle corrected for the false discovery rate.


- D ( A ) Representative immunoblots and ( B ) quantitative analysis of AKT ( 1 μM insulin : P = 0 . 038 ) , ( C ) AS160 ( - insulin : P = 0 . 036 , 1 μM insulin : P = 0 . 0038 ) , and ( D ) AMPK ( - insulin : P = 0 . 018 ) phosphorylation ± 16 hour BAM15 treatment and 15 minute insulin stimulation ( N = 3 per condition ) in C2C12 myotubes .

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

-D (A) Representative immunoblots and (B) quantitative analysis of AKT (1 μM insulin: P=0.038), (C) AS160 (- insulin: P=0.036, 1 μM insulin: P=0.0038), and (D) AMPK (- insulin: P=0.018) phosphorylation ± 16 hour BAM15 treatment and 15 minute insulin stimulation (N=3 per condition) in C2C12 myotubes.


, F ( E ) Representative immunoblots and quantitative analysis of ( F ) plasma membrane GLUT4 translocation ( P = 0 . 0139 ) ± 16 - hour BAM15 treatment ( N = 3 per condition ) in C2C12 myotubes .

O O O O O 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

, F (E) Representative immunoblots and quantitative analysis of (F) plasma membrane GLUT4 translocation (P=0.0139) ± 16-hour BAM15 treatment (N=3 per condition) in C2C12 myotubes.


G ) [ 3 - 3H ] glucose uptake ± 16 hour BAM15 treatment and 30 minute insulin stimulation ( N = 4 per condition ; vehicle vs . vehicle + insulin : P = 0 . 0016 , vehicle vs . BAM15 : P < 0 . 0001 , vehicle vs . BAM15 + insulin : P < 0 . 0001 , vehicle + insulin vs . BAM15 + insulin : P = 0 . 0018 , BAM15 vs . BAM15 + insulin : P = 0 . 0024 )

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

G) [3-3H]glucose uptake ± 16 hour BAM15 treatment and 30 minute insulin stimulation (N=4 per condition; vehicle vs. vehicle + insulin: P=0.0016, vehicle vs. BAM15: P<0.0001, vehicle vs. BAM15 + insulin: P<0.0001, vehicle + insulin vs. BAM15 + insulin: P=0.0018, BAM15 vs. BAM15 + insulin: P=0.0024)


H ) [ 1 - 14C ] palmitate oxidation ± 16 hour BAM15 treatment and 5 mM glucose and 1 mM pyruvate ( N = 6 per condition ; no glucose / pyruvate : P < 0 . 0001 ) in C2C12 myotubes .

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

H) [1-14C]palmitate oxidation ± 16 hour BAM15 treatment and 5 mM glucose and 1 mM pyruvate (N=6 per condition; no glucose/pyruvate: P<0.0001) in C2C12 myotubes.


I ) Representative immunoblot and quantitative analysis ( N = 5 per condition ; P < 0 . 0001 ) of AMPK demonstrating loss of function following transfection with an shAMPK construct in C2C12 myotubes .

O O O O O O O O O O O O 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) Representative immunoblot and quantitative analysis (N=5 per condition; P<0.0001) of AMPK demonstrating loss of function following transfection with an shAMPK construct in C2C12 myotubes.


J ) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin , FCCP , and a cocktail of rotenone and antimycin a ( N = 5 per condition )

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

J) Representative respirometry plot of intact cells ± shAMPK and 16 hour BAM15 treatment in C2C12 myotubes cells sequentially injected with oligomycin, FCCP, and a cocktail of rotenone and antimycin a (N=5 per condition)


K ) basal and ATP - linked respiration , proton leak , maximal uncoupling , and spare respiratory capacity ( N = 5 per condition ) . Basal : shEV vs . shEV + BAM15 ( P = 0 . 0317 ) , shEV + BAM15 vs . shAMPK ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) . ATP : shEV vs . shAMPK + BAM15 ( P = 0 . 0026 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P = 0 . 0003 ) , shAMPK vs . shAMPK + BAM15 ( P = 0 . 040 ) . Proton leak : shEV vs . shEV + BAM15 ( P = 0 . 0052 ) , shAMPK vs . shAMPK + BAM15 ( P = 0 . 0394 ) . Maximal : shEV vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shAMPK vs . shAMPK + BAM15 ( P < 0 . 0001 ) . Spare Capacity : shEV vs . shAMPK ( P = 0 . 0028 ) , shEV vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK ( P < 0 . 0001 ) , shEV + BAM15 vs . shAMPK + BAM15 ( P < 0 . 0001 ) , shAMPK vs . shAMPK + BAM15 ( P < 0 . 0001 ) .

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

K) basal and ATP-linked respiration, proton leak, maximal uncoupling, and spare respiratory capacity (N=5 per condition). Basal: shEV vs. shEV + BAM15 (P=0.0317), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001). ATP: shEV vs. shAMPK + BAM15 (P=0.0026), shEV + BAM15 vs. shAMPK + BAM15 (P=0.0003), shAMPK vs. shAMPK + BAM15 (P=0.040). Proton leak: shEV vs. shEV + BAM15 (P=0.0052), shAMPK vs. shAMPK + BAM15 (P=0.0394). Maximal: shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001). Spare Capacity: shEV vs. shAMPK (P=0.0028), shEV vs. shAMPK + BAM15 (P<0.0001), shEV + BAM15 vs. shAMPK (P<0.0001), shEV + BAM15 vs. shAMPK + BAM15 (P<0.0001), shAMPK vs. shAMPK + BAM15 (P<0.0001).


L ) [ 3 - 3H ] glucose uptake ± shAMPK , 16 hour BAM15 treatment , and 30 minute insulin stimulation in C2C12 myotubes ( N = 3 per condition ) . No insulin : shEV vs . shEV + BAM15 ( P = 0 . 0001 ) . Insulin : shEV vs . shEV + BAM15 ( P = 0 . 0142 ) .

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

L) [3-3H]glucose uptake ± shAMPK, 16 hour BAM15 treatment, and 30 minute insulin stimulation in C2C12 myotubes (N=3 per condition). No insulin: shEV vs. shEV + BAM15 (P=0.0001). Insulin: shEV vs. shEV + BAM15 (P=0.0142).


- O ( M ) Representative immunoblots and quantitative analysis of ( N ) AKT ( shEV + insulin : P = 0 . 0128 , shAMPK - insulin : P = 0 . 0018 , shAMPK + insulin , P = 0 . 0124 ) , and ( O ) AS160 ( shEV - insulin : P = 0 . 0283 , shEV + insulin : P = 0 . 0137 ) phosphorylation ± shAMPK , 16 hour BAM15 treatment , and 15 minute insulin stimulation in C2C12 myotubes ( N = 3 per condition ) .

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

-O (M) Representative immunoblots and quantitative analysis of (N) AKT (shEV + insulin: P=0.0128, shAMPK - insulin: P=0.0018, shAMPK + insulin, P=0.0124), and (O) AS160 (shEV - insulin: P=0.0283, shEV + insulin: P=0.0137) phosphorylation ± shAMPK, 16 hour BAM15 treatment, and 15 minute insulin stimulation in C2C12 myotubes (N=3 per condition).


( A ) Serum concentrations of BAM15 over a 24 - hour period ( N = 3 animals per time point )

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

(A) Serum concentrations of BAM15 over a 24-hour period (N=3 animals per time point)


( B ) peak tissue concentrations of BAM15 ( N = 6 per tissue ) .

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

(B) peak tissue concentrations of BAM15 (N=6 per tissue).


C , D ( C ) Daily body weight ( day 8 : P = 0 . 0454 , day 9 : P = 0 . 0353 , day 10 : P = 0 . 0251 , day 11 : P = 0 . 0131 , day 12 : 0 . 0038 , day 13 : P = 0 . 0008 , day 14 : P = 0 . 0005 , day 15 : P = 0 . 0003 , day 16 - 21 : P < 0 . 0001 ) and ( D ) cumulative food intake over the 3 - week treatment in CTRL and BAM15 treated animals ( N = 16 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 B-CONTROLLED_VAR O O O O O O O O O O

C, D (C) Daily body weight (day 8: P=0.0454, day 9: P=0.0353, day 10: P=0.0251, day 11: P=0.0131, day 12: 0.0038, day 13: P=0.0008, day 14: P=0.0005, day 15: P=0.0003, day 16-21: P<0.0001) and (D) cumulative food intake over the 3-week treatment in CTRL and BAM15 treated animals (N=16 per group).


( E ) Whole body temperature before ( pre ) , one hour ( 1 hr Post ) , and 24 hours ( 24 hrs Post ) after intraperitoneal injection of corn oil , vehicle ( corn oil and saline ) , 0 . 1 , 0 . 5 , and , 1 mg BAM15 per kg of body weight ( N = 11 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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

(E) Whole body temperature before (pre), one hour (1 hr Post), and 24 hours (24 hrs Post) after intraperitoneal injection of corn oil, vehicle (corn oil and saline), 0.1, 0.5, and, 1 mg BAM15 per kg of body weight (N=11 per group),


Whole body temperature ( F ) daily during chronic BAM15 treatment ( N = 8 per group ) .

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

Whole body temperature (F) daily during chronic BAM15 treatment (N=8 per group).


G , H ( G ) Change in fat mass ( % ) ( P < 0 . 0001 ) and ( H ) lean mass ( % ) ( P < 0 . 0001 ) from baseline to 3 - weeks in CTRL and BAM15 treated animals ( 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 B-CONTROLLED_VAR O O O O O O O O O O

G, H (G) Change in fat mass (%) (P<0.0001) and (H) lean mass (%) (P<0.0001) from baseline to 3-weeks in CTRL and BAM15 treated animals (N=8 per group).


( I ) Fasting plasma glucose ( N = 8 per group ; P = 0 . 007 ) and ( J ) insulin ( N = 7 per group ; P = 0 . 0024 ) after 3 weeks of treatment in CTRL and BAM15 treated animals .

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 B-MEASURED_VAR 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

(I) Fasting plasma glucose (N=8 per group; P=0.007) and (J) insulin (N=7 per group; P=0.0024) after 3 weeks of treatment in CTRL and BAM15 treated animals.


( K ) Plasma glucose concentrations at 0 , 15 , 30 , 60 , 90 , and 120 minutes following intraperitoneal injection of glucose ( 2 g glucose / kg of body weight ; N = 8 per group )

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

(K) Plasma glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)


( L ) total area under the curve glucose after 3 - weeks in CTRL and BAM15 treated animals ( N = 8 per group ; P = 0 . 0106 ) .

O O O O O O O O B-MEASURED_VAR 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

(L) total area under the curve glucose after 3-weeks in CTRL and BAM15 treated animals (N=8 per group; P=0.0106).


M - O ( M ) Oxygen consumption rates ( N ) energy expenditure ( daily : P = 0 . 0357 , dark phase : P = 0 . 030 ) , and ( O ) respiratory exchange ratio ( daily : P = 0 . 0424 , dark phase : P = 0 . 0016 ) measured by continuous high - resolution indirect calorimetry after 3 - weeks of CTRL or BAM15 treatment ( 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 B-CONTROLLED_VAR O O O O O O O O O

M-O (M) Oxygen consumption rates (N) energy expenditure (daily: P=0.0357, dark phase: P=0.030), and (O) respiratory exchange ratio (daily: P=0.0424, dark phase: P=0.0016) measured by continuous high-resolution indirect calorimetry after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


( P ) Fecal energy density ( % recovered in feces ) after 3 - weeks of CTRL or BAM15 treatment ( N = 8 per group ) .

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

(P) Fecal energy density (% recovered in feces) after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


( A ) Change in body weight ( CTRL vs . BAM15 : P < 0 . 0001 , CTRL vs . CR : P < 0 . 0001 ) , ( B ) fat mass ( CTRL vs . BAM15 : P = 0 . 0005 , CTRL vs . CR : P = 0 . 0041 , BAM15 vs . CR : P = 0 . 043 ) , and ( C ) lean mass ( CTRL vs . BAM15 : P = 0 . 0014 , CTRL vs . CR : P < 0 . 0001 , BAM15 vs . CR : P = 0 . 0047 ) from baseline to 2 - weeks in CTRL , BAM15 , and CR treated

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

(A) Change in body weight (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P<0.0001), (B) fat mass (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0041, BAM15 vs. CR: P=0.043), and (C) lean mass (CTRL vs. BAM15: P=0.0014, CTRL vs. CR: P<0.0001, BAM15 vs. CR: P=0.0047) from baseline to 2-weeks in CTRL, BAM15, and CR treated animals


( D ) daily food intake ( CTRL vs . CR : P = 0 . 0092 , BAM15 vs . CR : P = 0 . 0175 ) in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 ) .

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

(D) daily food intake (CTRL vs. CR: P=0.0092, BAM15 vs. CR: P=0.0175) in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( E ) Blood glucose concentrations at 0 , 15 , 30 , 60 , 90 , and 120 minutes following intraperitoneal injection of glucose ( 2 g glucose / kg of body weight ; N = 8 per group )

(E) Blood glucose concentrations at 0, 15, 30, 60, 90, and 120 minutes following intraperitoneal injection of glucose (2 g glucose/kg of body weight; N=8 per group)


( F ) total area under the curve glucose ( CTRL vs . BAM15 : P < 0 . 0001 , CTRL vs . CR : P = 0 . 003 , BAM15 vs . CR : P = 0 . 0261 ) after 2 - weeks of treatment in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 ) .

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

(F) total area under the curve glucose (CTRL vs. BAM15: P<0.0001, CTRL vs. CR: P=0.003, BAM15 vs. CR: P=0.0261) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( G ) Plasma insulin concentrations at baseline ( PRE ) , and 0 ( CTRL vs . BAM15 : P = 0 . 0154 , CTRL vs . CR : P = 0 . 0425 ) and 120 minutes ( CTRL vs . BAM15 : P = 0 . 0005 , CTRL vs . CR : P = 0 . 0211 , BAM15 vs . CR : P = 0 . 0264 ) after injection of glucose after 2 - weeks of treatment in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 )

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

(G) Plasma insulin concentrations at baseline (PRE), and 0 (CTRL vs. BAM15: P=0.0154, CTRL vs. CR: P=0.0425) and 120 minutes (CTRL vs. BAM15: P=0.0005, CTRL vs. CR: P=0.0211, BAM15 vs. CR: P=0.0264) after injection of glucose after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8)and


( H ) Homeostatic Model Assessment of Insulin Resistance ( HOMA - IR ) ( CTRL vs . BAM15 : P = 0 . 0259 , CTRL vs . CR : P = 0 . 040 ) after 2 - weeks of treatment in CTRL , BAM15 , and CR treated animals ( CTRL N = 7 , BAM15 and CR N = 8 ) .

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

(H) Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) (CTRL vs. BAM15: P=0.0259, CTRL vs. CR: P=0.040) after 2-weeks of treatment in CTRL, BAM15, and CR treated animals (CTRL N=7, BAM15 and CR N=8).


( A ) Representative H & E stained liver , adipose tissue , and skeletal muscle sections from CTRL and BAM15 treated animals . Scale bars ( black ) = 100 μm .

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

(A) Representative H&E stained liver, adipose tissue, and skeletal muscle sections from CTRL and BAM15 treated animals. Scale bars (black) = 100 μm.


( B ) Representative immunohistochemical analysis of pancreatic insulin ( red ) content counterstained with diaminobenzidine ( blue ) . Scale bars ( black ) = 100 μm .

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) Representative immunohistochemical analysis of pancreatic insulin (red) content counterstained with diaminobenzidine (blue). Scale bars (black) = 100 μm.


C - F ( C ) Quantitative analysis of liver steatosis ( μm2 ) ( N = 6 per group ; P < 0 . 0001 ) , ( D ) gastrocnemius muscle cross sectional area ( μm2 ) ( CTRL N = 7 , BAM15 N = 8 ) , ( E ) pancreatic insulin positive area ( % of total area ) ( N = 6 per group ; P = 0 . 0420 ) , ( F ) and β - cell mass ( N = 4 per group ; P = 0 . 0005 ) .

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

C-F (C) Quantitative analysis of liver steatosis (μm2) (N=6 per group; P<0.0001), (D) gastrocnemius muscle cross sectional area (μm2)(CTRL N=7, BAM15 N=8), (E) pancreatic insulin positive area (% of total area) (N=6 per group; P=0.0420), (F) and β-cell mass (N=4 per group; P=0.0005).


( G - I ) Size ( area ; μm2 ) and distributional ( number ) analysis of ( G ) iWAT , ( H ) gWAT , and ( I ) BAT from CTRL and BAM15 treated animals ( 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 B-CONTROLLED_VAR O O O O O O O O O O

(G-I) Size (area; μm2) and distributional (number) analysis of (G) iWAT, (H) gWAT, and (I) BAT from CTRL and BAM15 treated animals (N=8 per group).


A - D ( A ) Plasma concentrations of non - esterified fatty acids ( NEFA ) ( CTRL N = 7 , BAM15 N = 8 , P = 0 . 0073 ) , ( B ) leptin ( N = 8 per group , P < 0 . 0001 ) , ( C ) GDF15 ( N = 7 per group , P = 0 . 035 ) , and ( D ) FGF21 ( N = 7 per group , P = 0 . 0026 ) after 3 - weeks of CTRL or BAM15 treatment .

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

A-D (A) Plasma concentrations of non-esterified fatty acids (NEFA) (CTRL N=7, BAM15 N=8, P=0.0073), (B) leptin (N=8 per group, P<0.0001), (C) GDF15 (N=7 per group, P=0.035), and (D) FGF21 (N=7 per group, P=0.0026) after 3-weeks of CTRL or BAM15 treatment.


E , F ( E ) Total ( basal : P = 0 . 015 ) and ( F ) complete ( basal : P = 0 . 0137 ) oxidation of 200 μM [ 1 - 14C ] palmitate to CO2 from mixed gastrocnemius muscle homogenates ( N = 7 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

E, F (E) Total (basal: P=0.015) and (F) complete (basal: P=0.0137) oxidation of 200 μM [1-14C]palmitate to CO2 from mixed gastrocnemius muscle homogenates (N=7 per group).


( G ) Routine ( R ) oxygen flux , leak ( L ) , OXPHOS ( P ) and ETC ( E ) capacity in permeabilized mixed gastrocnemius fibers ( N = 6 per group ) . PM : pyruvate and malate , ADP : adenosine diphosphate , GS : glutamate and succinate , Tm : tetramethyl - p - phenylenediamine , PalM : palmitoylcarnitine and malate , Oct : octanoylcarnitine , and Dur : duroquinol .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Routine (R) oxygen flux, leak (L), OXPHOS (P) and ETC (E) capacity in permeabilized mixed gastrocnemius fibers (N=6 per group). PM: pyruvate and malate, ADP: adenosine diphosphate, GS: glutamate and succinate, Tm: tetramethyl-p-phenylenediamine, PalM: palmitoylcarnitine and malate, Oct: octanoylcarnitine, and Dur: duroquinol.


( H ) Representative immunoblots of phosphorylated and total AMPK and ACC from iWAT , and mixed gastrocnemius

(H) Representative immunoblots of phosphorylated and total AMPK and ACC from iWAT, and mixed gastrocnemius muscle


( I - K ) Densitometric quantification ( N = 4 per group ; iWAT p - AMPKT172 : P = 0 . 0186 , iWAT p - ACCS79 : P = 0 . 0083 , mixed gastrocnemius p - AMPKT172 : P = 0 . 0286 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) Densitometric quantification (N=4 per group; iWAT p-AMPKT172: P=0.0186, iWAT p-ACCS79: P=0.0083, mixed gastrocnemius p-AMPKT172: P=0.0286).


( L - N ) mRNA expression of transcriptional regulators of lipogenesis and lipolysis in iWAT ( N = 7 per group ) . Srebf1 ( P < 0 . 0001 ) , Mlxipl ( P = 0 . 0018 ) , Scd1 ( P < 0 . 0001 ) , FASN ( P = 0 . 0009 ) , LIPE ( P = 0 . 0015 ) , Pnpla2 ( P < 0 . 0001 ) , PPARg ( P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-N) mRNA expression of transcriptional regulators of lipogenesis and lipolysis in iWAT (N=7 per group). Srebf1 (P<0.0001), Mlxipl (P=0.0018), Scd1 (P<0.0001), FASN (P=0.0009), LIPE (P=0.0015), Pnpla2 (P<0.0001), PPARg (P<0.0001).


( C ) Representative flow cytometry results showing CD71 , a cell surface marker , can be efficiently knocked - down in the majority of organoid cells after 5 days of Dox and TMP treatment . N = 3 different organoid lines and 3 different gRNAs for CD71 were used . Mean ± SEM is labelled .

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

(C) Representative flow cytometry results showing CD71, a cell surface marker, can be efficiently knocked-down in the majority of organoid cells after 5 days of Dox and TMP treatment. N = 3 different organoid lines and 3 different gRNAs for CD71 were used. Mean ± SEM is labelled.


( A ) Human fetal lung tip organoids faithfully express in vivo tip progenitor markers , SOX2 and SOX9 . Upper panel : SOX2 / SOX9 dual expression in human fetal lungs . Lower panel : SOX2 / SOX9 in human fetal lung tip organoids . SOX9 green , SOX2 red , E - Cadherin white . Data information : Scale bars denote 50 μm (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Human fetal lung tip organoids faithfully express in vivo tip progenitor markers, SOX2 and SOX9. Upper panel: SOX2/SOX9 dual expression in human fetal lungs. Lower panel: SOX2/SOX9 in human fetal lung tip organoids. SOX9 green, SOX2 red, E-Cadherin white. Data information: Scale bars denote 50 μm (A


( B ) qRT - PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript levels using inducible CRISPRi after 5 days of Dox and TMP treatment . 3 different organoid lines and 2 different SOX9 gRNAs were used . Error bars : mean ± SEM . Two - sided Student ' s t - test with equal variance * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR showing that SOX9 gRNAs effectively knocked down SOX9 transcript levels using inducible CRISPRi after 5 days of Dox and TMP treatment. 3 different organoid lines and 2 different SOX9 gRNAs were used. Error bars: mean ± SEM. Two-sided Student's t-test with equal variance ***p < 0.001.


( D ) CD44 protein expression in SOX9 + tip progenitor cells . SOX9 green , SOX2 red , CD44 white . Data information : Scale bars = 50

O O O O O 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) CD44 protein expression in SOX9+ tip progenitor cells. SOX9 green, SOX2 red, CD44 white. Data information: Scale bars = 50 μm


( E ) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor cells . SOX9 red , LGR5 cyan . ( F ) Tip progenitor cells are of high WNT signalling activity . NOTUM yellow , SOX9 red . Data information : Scale bars = 50

O O O O O O O O O O O O O O O O O O O O O O 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) LGR5 mRNA expression enrichment in SOX9 expressing tip progenitor cells. SOX9 red, LGR5 cyan. (F) Tip progenitor cells are of high WNT signalling activity. NOTUM yellow, SOX9 red. Data information: Scale bars = 50 μm


( H ) qRT - PCR showing rescue of SOX9 transcription after constitutively activated β - catenin overexpression in organoids cultured without WNT activators . N = 3 different organoid lines . Error bars : mean ± SEM .

O O O O O O O O O O O O O O O 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) qRT-PCR showing rescue of SOX9 transcription after constitutively activated β-catenin overexpression in organoids cultured without WNT activators. N = 3 different organoid lines. Error bars: mean ± SEM.


( I ) WB showing SOX9 protein was rescued after constitutively activated β - catenin overexpression in organoids cultured without WNT activators .

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

(I) WB showing SOX9 protein was rescued after constitutively activated β-catenin overexpression in organoids cultured without WNT activators.


( A , B ) HCR images showing ETV5 ( A ) and ETV4 ( B ) expression in SOX9 expressing tip progenitors . ETV4 / 5 red , SOX9 cyan . Data information : Scale bars denote 50 μm ( A , B )

O O O O O O O O O O O O O O O O O O O O O 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) HCR images showing ETV5 (A) and ETV4 (B) expression in SOX9 expressing tip progenitors. ETV4/5 red, SOX9 cyan. Data information: Scale bars denote 50 μm (A, B)


( C ) qRT - PCR showing ETV4 and ETV5 dual knockdown in tip organoids . N = 3 different organoid lines and 2 different ETV4 and ETV5 gRNA combinations were used . Error bars : mean ± SEM . Two tailed paired t - test : * * p < 0 . 01 ; * * * p < 0 . 001 .

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

(C) qRT-PCR showing ETV4 and ETV5 dual knockdown in tip organoids. N =3 different organoid lines and 2 different ETV4 and ETV5 gRNA combinations were used. Error bars: mean ± SEM. Two tailed paired t-test: **p < 0.01; ***p < 0.001.


( F ) qRT - PCR of ETV4 , ETV5 and SOX9 expression level in organoids in self - renewing medium and self - renewing medium without RTK ligands . N = 3 different organoid lines used . Error bars : mean ± SEM . Two - tailed paired T - test : * * p < 0 . 01 ; n . s . non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR of ETV4, ETV5 and SOX9 expression level in organoids in self-renewing medium and self-renewing medium without RTK ligands. N = 3 different organoid lines used. Error bars: mean ± SEM. Two-tailed paired T-test: **p < 0.01; n.s. non-significant.


Ten - fold serial dilutions of 1 OD600 unit / mL of the indicated strains were spotted on SD plates containing 0 ( C - ) or 1 mM choline ( C + ) and 0 ( I - ) or 75 μM inositol ( I + ) and incubated at 30oC for 3 d . A representative experiment is shown ( from n = 5 ) .

O O O O O 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 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

Ten-fold serial dilutions of 1 OD600 unit/mL of the indicated strains were spotted on SD plates containing 0 (C-) or 1 mM choline (C+) and 0 (I-) or 75 μM inositol (I+) and incubated at 30oC for 3 d. A representative experiment is shown (from n=5).


A - L Wild type cultured in C - ( A ) , cho2opi3 cultured in C + ( B ) , coS # 4 ( C - D ) , coS # 3 ( E - G ) , and coS # 2 ( I - K ) cultured in C - were analyzed by electron microscopy . In addition , coS # 3 ( H ) and coS # 2 ( L ) are shown after culture in C + for 3 generations . The arrow heads ( F , I , J ) point to protrusions in the nuclear envelope . CW , cell wall ; ER , endoplasmic reticulum ; PM , plasma membrane ; M , mitochondria ; N , nucleus ; V , vacuole ; * , lipid droplet . Scale bars correspond to 200 nm ( A , B , D , F , G ) or 500 nm ( C , E , H , I , J , K , L ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-L Wild type cultured in C- (A), cho2opi3 cultured in C+ (B ), coS#4 (C-D), coS#3 (E-G), and coS#2 (I-K) cultured in C- were analyzed by electron microscopy. In addition, coS#3 (H) and coS#2 (L) are shown after culture in C+ for 3 generations. The arrow heads (F, I, J) point to protrusions in the nuclear envelope. CW, cell wall; ER, endoplasmic reticulum; PM, plasma membrane; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 200 nm (A, B, D, F, G) or 500 nm (C, E, H, I, J, K, L).


Serial dilutions of the strains indicated were spotted on SD C + / - I + / - and incubated at 30 oC for 4 d .

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

Serial dilutions of the strains indicated were spotted on SD C+/- I+/- and incubated at 30 oC for 4 d.


Membrane lipid and TAG content , and ergosterolester content ( EE , inset ) per OD600 unit of the yeast strains indicated after culture to mid - log phase in SD with or without 1 mM choline ( C ) ; * p < 0 . 05 , * * p < 0 . 01 , unpaired two - tailed t - test of the indicated bar compared to the C + condition . Data information : All data were obtained by mass spectrometry and are presented as mean ± SD ( n = 3 biological replicates ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , unpaired two - tailed t - test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise .

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

Membrane lipid and TAG content, and ergosterolester content (EE, inset) per OD600 unit of the yeast strains indicated after culture to mid-log phase in SD with or without 1 mM choline (C); * p < 0.05, ** p < 0.01, unpaired two-tailed t-test of the indicated bar compared to the C+ condition. Data information: All data were obtained by mass spectrometry and are presented as mean ±SD (n=3 biological replicates); * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, unpaired two-tailed t-test of the indicated bar compared to the cho2opi3 parent unless indicated otherwise.


Generation of suppressors of choline auxotrophy of cho2opi3 , cho2opi3lro1 , and cho2opi3lro1 pLRO1 on choline - free medium as indicated at 30 oC for 7 d .

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

Generation of suppressors of choline auxotrophy of cho2opi3, cho2opi3lro1, and cho2opi3lro1 pLRO1 on choline-free medium as indicated at 30 oC for 7 d.


Growth ( 30 oC for 6 d ) and phospholipid composition of co acc1N / H lro1 pLRO1 cultured in SD C - ura - , containing glucose / galactose mixtures ( 2 % , w / v ) as carbon source with the percentage of galactose indicated . Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated .

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

Growth (30 oC for 6 d) and phospholipid composition of co acc1N/H lro1 pLRO1 cultured in SD C- ura-, containing glucose/galactose mixtures (2%, w/v) as carbon source with the percentage of galactose indicated. Phospholipid composition analyzed by TLC is presented as mean percentage of total phospholipid of 3 biological replicates with the individual values indicated.


ACC1 transcript levels after switching the strains indicated from SD C + I + to the medium indicated at OD600 0 . 02 or 0 . 2 ( co diploid in C - ) , and culture for 24 h at 30 oC . Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C + I + , with the individual values indicated .

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

ACC1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.


( F - G ) EM analysis of cho2opi3 cells that after preculture in SD C + , were transferred to OD600 0 . 05 and subsequently cultured to mid - log phase in SD C + ( F ) or SD C - ( G ) both containing 0 . 05 μg / mL SorA . CW , cell wall ; ER , endoplasmic reticulum ; M , mitochondria ; N , nucleus ; V , vacuole ; * , lipid droplet . Scale bars correspond to 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) EM analysis of cho2opi3 cells that after preculture in SD C+, were transferred to OD600 0.05 and subsequently cultured to mid-log phase in SD C+ (F) or SD C- (G) both containing 0.05 μg/mL SorA. CW, cell wall; ER, endoplasmic reticulum; M, mitochondria; N, nucleus; V, vacuole; *, lipid droplet. Scale bars correspond to 500 nm.


POX1 transcript levels after switching the strains indicated from SD C + I + to the medium indicated at OD600 0 . 02 or 0 . 2 ( co diploid in C - ) , and culture for 24 h at 30 oC . Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C + I + , with the individual values indicated .

POX1 transcript levels after switching the strains indicated from SD C+I+ to the medium indicated at OD600 0.02 or 0.2 (co diploid in C-), and culture for 24 h at 30 oC. Data were normalized to ACT1 and expressed as means of 3 biological replicates relative to the corresponding strain cultured in C+I+, with the individual values indicated.