Figure 1 ( 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.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.


Figure 2Relative 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 . 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 . 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 . 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 testRelative 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Relative 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.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.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.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 testRelative 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.


Figure 3Relative 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Relative 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.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.


Figure 4Left : 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 . 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 . 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 ) . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Left: 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.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.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).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.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.


Figure 5WT 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 . 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5WT 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.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 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.


Figure 6Wild - 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 . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Wild-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.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.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.


Figure 1A ) 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 ) . 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 ) 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A) 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).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)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.(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).


Figure 2 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(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.


Figure 3 - 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 . 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 . 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 ) 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 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3-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.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.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)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 (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).


Figure 4 ( 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 ) . 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 ) , 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 ) . ( 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 ) ( 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 ) . ( P ) Fecal energy density ( % recovered in feces ) after 3 - weeks of CTRL or BAM15 treatment ( N = 8 per group ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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).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),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).(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)(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).(P) Fecal energy density (% recovered in feces) after 3-weeks of CTRL or BAM15 treatment (N=8 per group).


Figure 5 ( 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 ) . ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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).(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).(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).


Figure 6 ( 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 . 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 B-PANEL_START O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(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.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).


Figure 7A - 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 ) . ( 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 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-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).(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 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).(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).


Figure 1 ( 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 .

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Figure 1(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.


Figure 2 ( 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 .

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Figure 2(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.


Figure 3 ( 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 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 . ( I ) WB showing SOX9 protein was rescued after constitutively activated β - catenin overexpression in organoids cultured without WNT activators .

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Figure 3(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 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.(I) WB showing SOX9 protein was rescued after constitutively activated β-catenin overexpression in organoids cultured without WNT activators.


Figure 4 ( 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 . ( 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 .

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Figure 4(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.(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.


Figure 1Ten - 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 ) .

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Figure 1Ten-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).


Figure 2A - 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 ) .

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Figure 2A-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).


Figure 3Serial dilutions of the strains indicated were spotted on SD C + / - I + / - and incubated at 30 oC for 4 d .

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Figure 3Serial dilutions of the strains indicated were spotted on SD C+/- I+/- and incubated at 30 oC for 4 d.


Figure 4Membrane 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 .

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Figure 4Membrane 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.


Figure 5Generation 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 .

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Figure 5Generation 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.


Figure 6ACC1 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 .

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Figure 6ACC1 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.


Figure 7POX1 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 .

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Figure 7POX1 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.


Figure 8Growth of co S ( 2n - 1 ) in the absence of choline is lost when GPT2 or DGA1 is overexpressed , and restored by SorA . Plasmids pHEYg - 1 - DGA1 , pHEYg - 1 - GPT2 and the empty vector control ( pEV ) were transformed into the strains indicated . After preculture in SD C + , ten - fold serial dilutions were spotted on SD C + / - with or without 0 . 1 μg / mL SorA and incubated at 30 oC for 3 d .

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Figure 8Growth of co S(2n-1) in the absence of choline is lost when GPT2 or DGA1 is overexpressed, and restored by SorA. Plasmids pHEYg-1-DGA1, pHEYg-1-GPT2 and the empty vector control (pEV) were transformed into the strains indicated. After preculture in SD C+, ten-fold serial dilutions were spotted on SD C+/- with or without 0.1 μg/mL SorA and incubated at 30 oC for 3 d.


Figure 9Intensity encoded GP - images of the indicated C - Laurdan - stained yeast strains cultured with or without choline , accompanied by the corresponding intensity weighted GP - histogram fitted to a single Gaussian function ( light blue line ) , and transmission image . White arrows point to plasma membranes exhibiting higher lipid packing than internal , organellar membranes ; blue horizontal arrows indicate highly packed lipid material in co S ( 2n - 1 ) . To deplete the cho2opi3 parent of PC , cells were transferred to SD C - at OD600 0 . 1 , and cultured into mid - log phase . White scale bars correspond to 10 µm ; red scale bars in transmission images correspond to 5 µm .

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Figure 9Intensity encoded GP-images of the indicated C-Laurdan-stained yeast strains cultured with or without choline, accompanied by the corresponding intensity weighted GP-histogram fitted to a single Gaussian function (light blue line), and transmission image. White arrows point to plasma membranes exhibiting higher lipid packing than internal, organellar membranes; blue horizontal arrows indicate highly packed lipid material in co S(2n-1). To deplete the cho2opi3 parent of PC, cells were transferred to SD C- at OD600 0.1, and cultured into mid-log phase. White scale bars correspond to 10 µm; red scale bars in transmission images correspond to 5 µm.


Figure 1C Time - lapse photographs of typical patterns obtained for bacteria alone and bacteria with phage , with corresponding model simulations . Scale bar represents 1 cm . F Visualization of infected region . E . coli FM15 was inoculated at the center of a semi - solid agar plate and the reporter phage M13 - sfGFP was inoculated 1 - cm away from the center . Fluorescence images were captured after overnight incubation using FITC channel and an exposure time of 200 ms . Scale bar represents 1 cm . G Relative fluorescence intensity at the center and edge positions of the infection zone as shown in ( F ) . The relative intensity was obtained by dividing the detected values with the maximum value of green fluorescence intensity on the plate . Data represent mean values ± s . d . for three values of the center and six values of the edge from three biological replicates . Two - tailed t - test was used to compare two groups . * * * , p = 0 . 0005 .

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Figure 1C Time-lapse photographs of typical patterns obtained for bacteria alone and bacteria with phage, with corresponding model simulations. Scale bar represents 1 cm.F Visualization of infected region. E. coli FM15 was inoculated at the center of a semi-solid agar plate and the reporter phage M13-sfGFP was inoculated 1-cm away from the center. Fluorescence images were captured after overnight incubation using FITC channel and an exposure time of 200 ms. Scale bar represents 1 cm. G Relative fluorescence intensity at the center and edge positions of the infection zone as shown in (F). The relative intensity was obtained by dividing the detected values with the maximum value of green fluorescence intensity on the plate. Data represent mean values ± s.d. for three values of the center and six values of the edge from three biological replicates. Two-tailed t-test was used to compare two groups. ***, p = 0.0005.


Figure 2B Microscopic images of E . coli FM15 cells carrying plasmids with gfp gene downstream of the psp promoter with or without wild - type M13 phage infection . Scale bar : 1 μm . D Photographs of a quarter of the semi - solid agar plate of a SPACE experiment using high - copy accessory plasmid to improve within - cell activity of T7 RNAP on a synthetic promoter 1D8 . An E . coli FM15 strain carrying no MP was used as control . Scale bar : 1 cm . E Activity in cells of T7 RNAP and its mutants from the SPACE experiment in ( D ) . Two clones of the ancestor and five clones of the mutants obtained from the evolution towards 1D8 recognition were measured by in vivo transcriptional assay Two - tailed t - test was used to compare the two groups . * * * , p = 0 . 0001 . Data represent mean values ± s . d . .

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Figure 2B Microscopic images of E. coli FM15 cells carrying plasmids with gfp gene downstream of the psp promoter with or without wild-type M13 phage infection. Scale bar: 1 μm.D Photographs of a quarter of the semi-solid agar plate of a SPACE experiment using high-copy accessory plasmid to improve within-cell activity of T7 RNAP on a synthetic promoter 1D8. An E. coli FM15 strain carrying no MP was used as control. Scale bar: 1 cm.E Activity in cells of T7 RNAP and its mutants from the SPACE experiment in (D). Two clones of the ancestor and five clones of the mutants obtained from the evolution towards 1D8 recognition were measured by in vivo transcriptional assay Two-tailed t-test was used to compare the two groups. ***, p=0.0001. Data represent mean values ± s.d..


Figure 3B Microscopic images of E . coli FM15 cells with or without phage infection . Scale bar represents 1 μm . C The experimental result shows raw photographs of a representative two - phage competition 20 h after initial inoculation with 105 : 1 mixture of M13w : M13s at 1 cm away from the center . The simulation result is the outcome of competition between two phages PW and PS with relative production rates of 40 and 100 , respectively . Scale bar represents 1 cm . D Phage titer ratio of M13s to M13w along the central line and the edge of the fan - shaped infection zone in the experimental result in ( C ) . Samples were collected from different positions as shown in the schematic and phage titer was quantified by qPCR Data represent mean values ± s . d . for three independent assays . Two - tailed t - test was used to compare two groups . * , p = 0 . 028 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B Microscopic images of E. coli FM15 cells with or without phage infection. Scale bar represents 1 μm. C The experimental result shows raw photographs of a representative two-phage competition 20 h after initial inoculation with 105:1 mixture of M13w: M13s at 1 cm away from the center. The simulation result is the outcome of competition between two phages PW and PS with relative production rates of 40 and 100, respectively. Scale bar represents 1 cm.D Phage titer ratio of M13s to M13w along the central line and the edge of the fan-shaped infection zone in the experimental result in (C). Samples were collected from different positions as shown in the schematic and phage titer was quantified by qPCR Data represent mean values ± s.d. for three independent assays. Two-tailed t-test was used to compare two groups. *, p = 0.028.


Figure 4C Fold change in the relative expression activity of the evolved RNAP mutants as compared with the wild - type T7 RNAP , for the 10 selected promoter sequences corresponding to the images in ( B ) . Results were obtained by in vitro transcriptional assay Data represent mean values ± s . d . for three biologically independent assays . D Expression activity of 10 RNAP mutants on T7 promoter and 10 promoter variants measured by in vivo transcriptional assay . Mutants are arranged in the same order as their original target promoters listed on the left side of the heatmap . Data are normalized so that the activity of wild - type RNAP on the T7 promoter is 100 ; the mean for three biologically independent replicates is

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C Fold change in the relative expression activity of the evolved RNAP mutants as compared with the wild-type T7 RNAP, for the 10 selected promoter sequences corresponding to the images in (B). Results were obtained by in vitro transcriptional assay Data represent mean values ± s.d. for three biologically independent assays.D Expression activity of 10 RNAP mutants on T7 promoter and 10 promoter variants measured by in vivo transcriptional assay. Mutants are arranged in the same order as their original target promoters listed on the left side of the heatmap. Data are normalized so that the activity of wild-type RNAP on the T7 promoter is 100; the mean for three biologically independent replicates is shown


Figure 1A . Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest , upon stimulation with 40 and 900 APs at 20Hz ( in the presence of bafilomycin A1 ) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation . B . Average traces of the normalised fluorescence change ( ΔF / F0 ) of sypHy in WT ( black ) and BsnGT ( red ) neurons as described in A . Intensities were normalized to the peak of NH4Cl response . C . Quantifications of mean RRP fraction in WT and BsnGT . D . Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta ( from 6 independent experiments ) to stimulation with 40 AP . The distribution is shifted to lower values in BsnGT . E . Quantifications of mean TRP fraction in WT and BsnGT . F . Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP . Note the shift in distribution between genotypes . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . In the frequency distribution histograms ( D and F ) black lines depict superimposed Gaussian fits for each group . The sample size n ( in parentheses ) corresponds to the number of analysed imaging experiments ( B , C , E ) In D and F , the data of 1050 synapses per genotype were processed . Data is obtained from 6 ( A - F ) independent culture preparations . Significance was assessed with Student ' s t - test ; * * * p < 0 . 001 . Scale bar is 2 µm in AG , Representative images of Syt1Ab uptake ( magenta ) driven by endogenous network activity in hippocampal neurons ( 18 DIV ) from WT and BsnGT mice . VGLUT1 ( green ) marks excitatory presynapses ( upper panels ) and VGAT the inhibitory ones ( lower panels ) . H . Quantification of normalized IF of Syt1Ab uptake in excitatory ( red boxes ) and inhibitory ( pink boxes ) synapses on experiments in G . I . Quantification of fraction of active ( Syt1Ab - labelled ) excitatory and inhibitory synapses on experiments illustrated in G . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The dashed lines in H depict IF in WT that were used for normalization . The sample size n ( in parentheses ) corresponds to the number of analysed images ( G , Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - test ; * * * p < 0 . 001 . Scale bar is 5 µm in GJ . Representative images of Syt1Ab uptake ( magenta ) upon depolarization with 50 mM KCl . Identical cultures and staining were applied as in G . K . Quantification of normalized IF of Syt1Ab uptake on experiments from J . L . Quantification of fraction of active excitatory and inhibitory synapses in J . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . The dashed lines in K depict IF in WT that were used for normalization . The sample size n ( in parentheses ) corresponds to the number of analysed images J ) . Data is obtained from independent culture preparations . Significance was assessed with Student ' s t - test ; * * * p < 0 . 001 . Scale bar is 5 µm in J .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Representative pseudo colour images of sypHy fluorescence in WT and BsnGT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20Hz (in the presence of bafilomycin A1) and upon application of NH4Cl to visualise SVs that were refractory to electrical stimulation. B. Average traces of the normalised fluorescence change (ΔF/F0) of sypHy in WT (black) and BsnGT (red) neurons as described in A. Intensities were normalized to the peak of NH4Cl response. C. Quantifications of mean RRP fraction in WT and BsnGT. D. Frequency distribution histograms of the response amplitudes of 1050 individual synaptic puncta (from 6 independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in BsnGT. E. Quantifications of mean TRP fraction in WT and BsnGT. F. Frequency distribution histograms of synaptic response amplitudes in BsnGT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) In D and F, the data of 1050 synapses per genotype were processed. Data is obtained from 6 (A-F) independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 2 µm in AG, Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and BsnGT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels). H. Quantification of normalized IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in G. I. Quantification of fraction of active (Syt1Ab-labelled) excitatory and inhibitory synapses on experiments illustrated in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in H depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images (G, Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in GJ. Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in G. K. Quantification of normalized IF of Syt1Ab uptake on experiments from J. L. Quantification of fraction of active excitatory and inhibitory synapses in J. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. The dashed lines in K depict IF in WT that were used for normalization. The sample size n (in parentheses) corresponds to the number of analysed images J). Data is obtained from independent culture preparations. Significance was assessed with Student's t-test; *** p < 0.001. Scale bar is 5 µm in J.


Figure 2A , B . Representative pseudo colour images ( A ) and average traces ( B ) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine ( rosc , 100µM , gray and pink trace ) or vehicle ( black and red trace ) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A . C , D . Plots show mean values of RRP ( C ) and TRP ( D ) fraction for both genotypes before and after treatment . E . Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT . Data information : n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis . In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers , and + indicates mean . Significance was assessed by two - way ANOVA with Tukey ´ s multiple comparison test ( C , D ) and by Student ' s t - test ( E ) * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 2µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A, B. Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and BsnGT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100µM, gray and pink trace) or vehicle (black and red trace) for 30 minutes before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D. Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E. Roscovitine treatment has a significantly higher effect on RRP and TRP in BsnGT neurons compared to WT. Data information: n corresponding to the number of imaging experiments done on 4 independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two-way ANOVA with Tukey´s multiple comparison test (C, D) and by Student's t-test (E) *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 2µm.


Figure 3A . Representative immunoblot with antibody against CDK5 - dependent pSer551 of Syn1 ( pSer551Syn1 ) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice . B , C . Quantification of pSer551Syn1 / Syn1 ratio ( in B ) and total Syn1 ( in C ) abundance on blots from A . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size n ( in brackets ) corresponds to the number of independently processed animals in B and C For statistics , student ' s t - test in B and C were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . E . Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1 . F . Quantification of IF intensity of pSer551Syn1 in synapses from WT , BsnGT , and WT cells treated with roscovitine . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size n ( in brackets ) corresponds to the number of independent images ( E , quantified from two ( F ) independent cultures per condition . For statistics , one - way ANOVA with Sidak ´ s multiple comparisons test in F were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm inG . Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity , upon network activity silencing ( TTX , 1 µM , 72 h ) or in conditions of increased activity ( BIC , 30 µM , 48 h ) H . Quantification of experiment in G . Data information : In the plots , the interquartile range and median are depicted as boxes , minimal and maximal values as whiskers and + indicates mean . The sample size n ( in brackets ) corresponds to the number of independent images , G ) quantified from four ( H ) independent cultures per condition . For statistics two - way ANOVA with Tukey ´ s multiple comparisons test in H were used , * p ≤ 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Scale bar is 5 µm in G .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Representative immunoblot with antibody against CDK5-dependent pSer551 of Syn1 (pSer551Syn1) and total Syn1 and total protein stain used as loading control for quantification of Syn1 on P2 fractions prepared from hippocampal tissue lysates of WT and BsnGT mice. B, C. Quantification of pSer551Syn1 / Syn1 ratio (in B) and total Syn1 (in C) abundance on blots from A. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independently processed animals in B and C For statistics, student's t-test in B and C were used, *p≤0.05, **p < 0.01, ***p< 0.001.E. Representative images of 19 DIV cultured hippocampal neurons from WT and BsnGT immunostained for pSer551Syn1 and total Syn1. F. Quantification of IF intensity of pSer551Syn1 in synapses from WT, BsnGT, and WT cells treated with roscovitine. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images (E, quantified from two (F) independent cultures per condition. For statistics, one-way ANOVA with Sidak´s multiple comparisons test in F were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm inG. Representative images of 21 DIV hippocampal WT and BsnGT neurons at baseline activity, upon network activity silencing (TTX, 1 µM, 72 h) or in conditions of increased activity (BIC, 30 µM, 48 h) H. Quantification of experiment in G. Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers and + indicates mean. The sample size n (in brackets) corresponds to the number of independent images , G) quantified from four (H) independent cultures per condition. For statistics two-way ANOVA with Tukey´s multiple comparisons test in H were used, *p≤0.05, **p < 0.01, ***p< 0.001. Scale bar is 5 µm in G.