Figure 5D - E ) From one time - course experiment , ions , cytosolic proteins and total protein were extracted in parallel samples . The presented experiment is representative of 3 separate time - course experiments that were carried out ( N = 3 ) . Representative experiment shown ( n = 4 technical replicates for [ K + ] , n = 3 technical replicates for the other traces ) . Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration . Mean ± SEM , p - values from RAIN , red lines are fits by a damped cosine compared with a straight line ( null hypothesis ) . Parallel PER2 : : LUC recordings were also performed and plotted as a phase marker . F ) Examples of key ion transporters are shown , as detected in the proteomics experiment . P values show the results of an F test comparing fits of damped cosine against straight line . All proteins except WT NKCC1 had RAIN p - values < 0 . 05 .

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 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

Figure 5D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05.


Figure 6A ) WT and CKO cells were treated with 500 nM tunicamycin ( TUN ) and lysed in RIPA buffer at time points between 0 - 24 hours afterwards . Western blots were carried out , probing for phosphorylated eIF2α , total eIF2α and actin . Representative blots shown , n = 4 . B ) Quantification of all replicates represented in ( A ) , where phosphorylated eIF2α is normalised to total eIF2α . Mean ± SD . 2 - way ANOVA ( TWA ) with Holm - Sidak ' s multiple comparisons . C ) Mice were housed in 12h : 12h light - dark cycles , with unrestricted feeding . Mouse lungs were collected 7 hours after transition to the light phase , 5 hours after intraperitoneal injection of mice with bortezomib ( BTZ , 2 . 5 mg / kg ) or vehicle ( Veh , 1 % DMSO in sterile PBS ) . Tissues were lysed in RIPA buffer and western blots were performed , probing for phosphorylated STAT3 , total STAT3 and actin . Male ( left ) and female ( right ) replicates shown , N = 2 mice for each condition . D ) Quantification of the blots shown in ( C ) , where phosphorylated STAT3 is normalised to total STAT3 . Mean ± SD . Multiple t - tests , corrected for multiple comparisons with the Holm - Sidak method , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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 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

Figure 6A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at time points between 0-24 hours afterwards. Western blots were carried out, probing for phosphorylated eIF2α, total eIF2α and actin. Representative blots shown, n=4. B) Quantification of all replicates represented in (A), where phosphorylated eIF2α is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.C) Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots were performed, probing for phosphorylated STAT3, total STAT3 and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition. D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.


Figure 7A ) WT PER2 : : LUC cells were treated with epoxomicin or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . B ) Quantification of damping rate of the PER2 : : LUC recordings shown in A ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . C ) WT PER2 : : LUC cells were treated with tunicamycin or vehicle control . Mean ( solid lines ) ± SD ( dotted lines ) . D ) Quantification of damping rate of the PER2 : : LUC recordings shown in C ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 . E ) WT PER2 : : LUC cells were treated with radicicol or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . F ) Quantification of damping rate of the PER2 : : LUC recordings shown in E ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 . I - J ) Representative double - plotted actograms of WT mice receiving drinking water with DMSO vehicle control ( N = 7 mice ) or 35 µg / mL ixazomib ( N = 8 mice ) . Two representative actograms are shown per condition . For the first 7 days the mice were in 12 : 12h light - dark cycles ( LD ) , and had drinking water supplemented with blackcurrant squash . Ixazomib / DMSO was added to this , and the lighting was changed to constant darkness ( DD ) . After 7 days the drug / vehicle was removed , and the mice remained in DD until the end of the experiment .

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

Figure 7A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.


Figure 1CRH - Cre : : Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c - Fos expression in the PVH in 24hr fasting or refed after 24hr fasting . A ) Representative images showing CRH neurons ( red in the first colomn ) , c - Fos expression ( green in the second colomn ) , merged of red and green ( the third colomn ) and the amplied pictures for the boxed areas in the merged images ( the forth colomn ) . The experimental conditions for each group ( fasting verus refed , chow versus HFD ) are labelled on the left . Scale bar = 200µm . B - C ) Comparison of average neuron numbers from cell counting on total number of c - Fos positive neurons in the PVH ( B , * * p = 0 . 0032 : chow fasting versus chow refed and * p = 0 . 023 , HFD fasting versus HFD refed ) and total number of c - Fos positive CRH neurons ( C , * * p < 0 . 0001 : chow fasting versus chow refed and p = 0 . 7158 : HFD fasting versus HFD refed ) , n = 3 - 4 each , data presented as mean + / - SEM , two way ANOVA tests . Effects of HFD feeding on diurnal rhythmicity in feeding ( D and E ) The measurement was collected with male mice fed chow or after 4 weeks HFD feeding , n = 6 each , data presented as mean + / - SEM , * * p = 0 . 0066 in E and * * p = 0 . 0 . 0007 in G , unpaired Student ' s t tests . Effects of HFD feeding on diurnal rhythmicity in energy expenditure ( F and G ) . The measurement was collected with male mice fed chow or after 4 weeks HFD feeding , n = 6 each , data presented as mean + / - SEM , * * p = 0 . 0066 in E and * * p = 0 . 0 . 0007 in G , unpaired Student ' s t tests . CRH - Cre mice received delivery of AAV - Flex - GCaMP6m viral vectors to bilateral PVH and optic fiber implantation targeting PVH neurons , and were used for in vivo live recording . ( H - I ) Representative traces showing recordings of activity changes in CRH neuron in response to stressor ( water spray ) in chow - fed controls ( H ) , and 4 weeks after HFD ( I ) , ( J ) Comparison of average responses in the two conditions , n = 4 each , * * p = 0 . 0006 , paired Student ' s t test . K - L ) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone ( Dex ) injections in chow - fed controls ( K ) and after 4 weeks on HFD ( L ) . M ) Comparison of average responses in the two conditions , n = 4 each , data presented as mean + / - SEM , * * P = 0 . 0012 , paired Student ' s t tests .

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

Figure 1CRH-Cre::Ai9 reporter male mice fed on chow or 4 weeks on HFD were used to examine c-Fos expression in the PVH in 24hr fasting or refed after 24hr fasting. A) Representative images showing CRH neurons (red in the first colomn), c-Fos expression (green in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed, chow versus HFD) are labelled on the left. Scale bar=200µm.B-C) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (B, **p=0.0032: chow fasting versus chow refed and *p=0.023, HFD fasting versus HFD refed) and total number of c-Fos positive CRH neurons (C, **p<0.0001: chow fasting versus chow refed and p=0.7158: HFD fasting versus HFD refed), n=3-4 each, data presented as mean +/-SEM, two way ANOVA tests.Effects of HFD feeding on diurnal rhythmicity in feeding (D and E) The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.Effects of HFD feeding on diurnal rhythmicity in energy expenditure (F and G). The measurement was collected with male mice fed chow or after 4 weeks HFD feeding, n=6 each, data presented as mean +/- SEM, **p=0.0066 in E and **p=0.0.0007 in G, unpaired Student's t tests.CRH-Cre mice received delivery of AAV-Flex-GCaMP6m viral vectors to bilateral PVH and optic fiber implantation targeting PVH neurons, and were used for in vivo live recording. (H-I) Representative traces showing recordings of activity changes in CRH neuron in response to stressor (water spray) in chow-fed controls (H), and 4 weeks after HFD (I), (J) Comparison of average responses in the two conditions, n=4 each, **p=0.0006, paired Student's t test.K-L) Representative traces showing recordings of activity changes in CRH neurons in response to Dexamethasone (Dex) injections in chow-fed controls (K) and after 4 weeks on HFD (L). M) Comparison of average responses in the two conditions, n=4 each, data presented as mean +/- SEM, **P=0.0012, paired Student's t tests.


Figure 2CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac vectors at 7 - 8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac ( one side ) or control neurons ( contralateral side ) of the same animal ( A ) ; typical action potentials ( APs ) in controls ( B , top panel ) and typical NachBac - mediated APs showing lower threshold and long duration ( B , bottom , the inset highlighting the long duration ) in NachBac micethe distribution of the membrane potential and firing cells in the two groups ( C , ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior ) . CRH - Cre male mice were injected with either AAV - Flex - GFP or AAV - EF1a - Flex - EGFP - P2A - mNachBac to examine c - Fos expression in the PVH in 24hr fasting or refed after fasting . ( D ) Representative images showing CRH neurons expressing GFP or NachBac ( GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn ) , c - Fos expression ( red in the second colomn ) , merged of red and green ( the third colomn ) and the amplied pictures for the boxed areas in the merged images ( the forth colomn ) . The experimental conditions for each group ( fasting verus refed ) are labelled on the left . ( E - F ) Comparison of average neuron numbers from cell counting on total number of c - Fos positive neurons in the PVH ( E , * * p < 0 . 0001 : fasting versus refed ; * * p = 0 . 0026 : fasting versus fasting / NachBac ; * * p < 0 . 0001 : fasting / NachBac versus refed / NachBac ) and total number of c - Fos positive CRH neurons ( F , * * p < 0 . 0001 : fasting versus refed ; * * p < 0 . 0001 : fasting versus fasting / NachBac ; p = 0 . 3423 : fasting / NachBac versus refed / NachBac ) , n = 3 - 4 each , data presented as mean + / - SEM , two way ANOVA tests . CRH - Cre male mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m and AAV - EF1a - Flex - EGFP - P2A - mNachBac vectors at 7 - 8 weeks of age with optic fiber implantation targeting PVH neurons . ( G - H ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in NachBac mice ( G ) , and comparison of responsiveness to water spray between NachBac and control mice ( H ) . ( I - J ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in NachBac mice ( I ) , and comparison of responsiveness to water spray between NachBac and control mice ( J ) . Gray traces showed Ca2 + independent signal for system stability and red ones showed Ca2 + dependent signals for neuron activity , N = 5 - 7 each , males , data presented as mean + / - SEM , * * p < 0 . 0001 in both ( H and J ) , unpaired Student ' s t tests .

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

Figure 2CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age and recordings were made at least 4 weeks after viral delivery on neurons with NachBac (one side) or control neurons (contralateral side) of the same animal (A);typical action potentials (APs) in controls (B, top panel) and typical NachBac-mediated APs showing lower threshold and long duration (B, bottom, the inset highlighting the long duration) in NachBac micethe distribution of the membrane potential and firing cells in the two groups (C, ratios in the bracket denoting neuron number out of total number recorded with the indicated behavior).CRH-Cre male mice were injected with either AAV-Flex-GFP or AAV-EF1a-Flex-EGFP-P2A-mNachBac to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing GFP or NachBac (GFP in the top 2 rows and NachBac in the bottom 2 rows of the first colomn), c-Fos expression (red in the second colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left.(E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p<0.0001: fasting versus refed; **p=0.0026: fasting versus fasting/NachBac; **p<0.0001: fasting/NachBac versus refed/NachBac) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; **p<0.0001: fasting versus fasting/NachBac; p=0.3423: fasting/NachBac versus refed/NachBac), n=3-4 each, data presented as mean +/- SEM, two way ANOVA tests.CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-Flex-EGFP-P2A-mNachBac vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (G), and comparison of responsiveness to water spray between NachBac and control mice (H).(I-J) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in NachBac mice (I), and comparison of responsiveness to water spray between NachBac and control mice (J).Gray traces showed Ca2+ independent signal for system stability and red ones showed Ca2+ dependent signals for neuron activity, N=5-7 each, males, data presented as mean +/-SEM, **p<0.0001 in both (H and J), unpaired Student's t tests.


Figure 3CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac or control GFP vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . Weekly body weight gain after viral delivery ( A ) at 11 weeks after viral delivery . CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - Flex - EGFP - P2A - mNachBac or control GFP vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . measurements of fat mass ( B ) and lean mass ( C ) at 11 weeks after viral delivery . Measurements of energy expenditure during the 7th week after viral delivery . Comparison of real time O2 consumption traces during the 4 - day measurement period ( D ) , of average O2 consumption between the 2 groups ( E ) , and of the difference between day and night periods between the 2 groups ( F ) . ( G - I ) Measurements of feeding during the 7th week after viral delivery . Comparison of real time feeding c traces during the 4 - day measurement period ( G ) , of average food intake between the 2 groups ( H ) , and of the difference in food intake between day and night periods between the 2 groups ( I ) .

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 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 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 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 3CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A) at 11 weeks after viral delivery.CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-Flex-EGFP-P2A-mNachBac or control GFP vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B) and lean mass (C) at 11 weeks after viral delivery.Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E), and of the difference between day and night periods between the 2 groups (F).(G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H), and of the difference in food intake between day and night periods between the 2 groups (I).


Figure 4CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors and AAV - Flex - GFP at 7 - 8 weeks of age and recording was made at least 4 weeks after viral delivery on neurons with Kir2 . 1 ( one side ) or control GFP neurons ( contralateral side ) of the same animal ( A ) typical spontaneous neuron activity recorded in GFP controls ( B , top panel ) and Kir2 . 1 neurons ( B , bottom ) ; and comparison in resting membrane potentials between the two groups ( C , * * p < 0 . 0001 , unpaired Student ' s t test ) . CRH - Cre male mice were injected with either AAV - Flex - mCherry or AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato to examine c - Fos expression in the PVH in 24hr fasting or refed after fasting . ( D ) Representative images showing CRH neurons expressing mCherry or Kir2 . 1 ( mCherry in the top 2 rows and Kir2 . 1 in the bottom 2 rows of the second colomn ) , c - Fos expression ( green in the first colomn ) , merged of red and green ( the third colomn ) and the amplied pictures for the boxed areas in the merged images ( the forth colomn ) . The experimental conditions for each group ( fasting verus refed ) are labelled on the left . CRH - Cre male mice were injected with either AAV - Flex - mCherry or AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato to examine c - Fos expression in the PVH in 24hr fasting or refed after fasting . ( E - F ) Comparison of average neuron numbers from cell counting on total number of c - Fos positive neurons in the PVH ( E , * * p = 0 . 0001 : fasting versus refed ; * p = 0 . 0497 : refed versus refed / Kir2 . 1 ; * * p = 0 . 0025 : fasting / Kir2 . 1 versus refed Kir2 . 1 ) and total number of c - Fos positive CRH neurons ( F , * * p < 0 . 0001 : fasting versus refed ; p = 0 . 9586 : fasting / Kir2 . 1 versus refed / Kir2 . 1 ) , n = 3 - 4 each , two way ANOVA tests . All data presented as mean + / - SEM . CRH - Cre male mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m and AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors at 7 - 8 weeks of age with optic fiber implantation targeting PVH neurons . ( G - H ) Representative traces showing typical responses of PVH CRH neurons to water spay ( arrows ) in Kir2 . 1 mice ( G ) , and comparison of responsiveness to water spray between Kir2 . 1 and control mice ( H ) . CRH - Cre male mice received stereotaxic delivery of a mixture of AAV - Flex - GCaMP6m and AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato vectors at 7 - 8 weeks of age with optic fiber implantation targeting PVH neurons .

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 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 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 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 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 4CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors and AAV-Flex-GFP at 7-8 weeks of age and recording was made at least 4 weeks after viral delivery on neurons with Kir2.1 (one side) or control GFP neurons (contralateral side) of the same animal (A)typical spontaneous neuron activity recorded in GFP controls (B, top panel) and Kir2.1 neurons (B, bottom); and comparison in resting membrane potentials between the two groups (C, **p<0.0001, unpaired Student's t test).CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (D) Representative images showing CRH neurons expressing mCherry or Kir2.1 (mCherry in the top 2 rows and Kir2.1 in the bottom 2 rows of the second colomn), c-Fos expression (green in the first colomn), merged of red and green (the third colomn) and the amplied pictures for the boxed areas in the merged images (the forth colomn). The experimental conditions for each group (fasting verus refed) are labelled on the left.CRH-Cre male mice were injected with either AAV-Flex-mCherry or AAV-EF1a-DIO-Kir2.1-P2A-dTomato to examine c-Fos expression in the PVH in 24hr fasting or refed after fasting. (E-F) Comparison of average neuron numbers from cell counting on total number of c-Fos positive neurons in the PVH (E, **p=0.0001: fasting versus refed; *p=0.0497: refed versus refed/Kir2.1; **p=0.0025: fasting/Kir2.1 versus refed Kir2.1) and total number of c-Fos positive CRH neurons (F, **p<0.0001: fasting versus refed; p=0.9586: fasting/Kir2.1 versus refed/Kir2.1), n=3-4 each, two way ANOVA tests. All data presented as mean +/- SEM.CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons. (G-H) Representative traces showing typical responses of PVH CRH neurons to water spay (arrows) in Kir2.1 mice (G), and comparison of responsiveness to water spray between Kir2.1 and control mice (H).CRH-Cre male mice received stereotaxic delivery of a mixture of AAV-Flex-GCaMP6m and AAV-EF1a-DIO-Kir2.1-P2A-dTomato vectors at 7-8 weeks of age with optic fiber implantation targeting PVH neurons.


Figure 5CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato or control AAV - Flex - mCherry vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . Weekly body weight gain after viral delivery ( A , * p < 0 . 0001 , unpaired Student ' s t test ) , at 15 weeks after viral delivery . CRH - Cre male mice received stereotaxic delivery of AAV - EF1a - DIO - Kir2 . 1 - P2A - dTomato or control AAV - Flex - mCherry vectors at 7 - 8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD . measurements of fat mass ( B , * * p = 0 . 0006 , unpaired Student ' s t test ) and lean mass ( C , p = 0 . 3728 , unpaired Student ' s t test ) at 15 weeks after viral delivery . ( D - F ) Measurements of energy expenditure during the 7th week after viral delivery . Comparison of real time O2 consumption traces during the 4 - day measurement period ( D ) , of average O2 consumption between the 2 groups ( E , * p = 0 . 0007 , unpaired Student ' s t test ) , and of the difference between day and night periods between the 2 groups ( F , p = 0 . 0791 , unpaired Student ' s t test ) . ( G - I ) Measurements of feeding during the 7th week after viral delivery . Comparison of real time feeding c traces during the 4 - day measurement period ( G ) , of average food intake between the 2 groups ( H , * * p = 0 . 0019 , unpaired Student ' s t test ) , and of the difference in food intake between day and night periods between the 2 groups ( I , * p = 0 . 0363 , unpaired Student ' s t test ) . n = 6 each . All data presented as mean + / - SEM .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. Weekly body weight gain after viral delivery (A, *p<0.0001, unpaired Student's t test), at 15 weeks after viral delivery.CRH-Cre male mice received stereotaxic delivery of AAV-EF1a-DIO-Kir2.1-P2A-dTomato or control AAV-Flex-mCherry vectors at 7-8 weeks of age and were maintained on chow diet for 7 weeks before switching to HFD. measurements of fat mass (B, **p=0.0006, unpaired Student's t test) and lean mass (C, p=0.3728, unpaired Student's t test) at 15 weeks after viral delivery.(D-F) Measurements of energy expenditure during the 7th week after viral delivery. Comparison of real time O2 consumption traces during the 4-day measurement period (D), of average O2 consumption between the 2 groups (E, *p=0.0007, unpaired Student's t test), and of the difference between day and night periods between the 2 groups (F, p=0.0791, unpaired Student's t test).(G-I) Measurements of feeding during the 7th week after viral delivery. Comparison of real time feeding c traces during the 4-day measurement period (G), of average food intake between the 2 groups (H, **p=0.0019, unpaired Student's t test), and of the difference in food intake between day and night periods between the 2 groups (I, *p=0.0363, unpaired Student's t test). n=6 each. All data presented as mean +/- SEM.


Figure 1 ( B ) BAX / BAK - deficient HeLa cells reconstituted with BAX and where indicated HA - Parkin were treated with antimycin A and oligomycin ( AO ) for 2 h and then 1 µM of each of the BH3 mimetics ABT - 737 and S63485 for 6 h . Cell death was assessed at the indicated times by LDH assay . Data is mean + / - SD of four independent experiments . ( C ) BAX / BAK - deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin ( AO , 3 h ) , MG132 ( 20 µM , 3 . 5 h ) and BH3 mimetics ( 1 µM of each of ABT - 737 and S63485 , 1 h ) . Cytosol and membrane fractions were immunoblotted as indicated . Graph shows densitometric analysis of non - ubiquitinated membrane proteins ( top , relative to untreated control ) or cytochrome c release ( bottom ; cytosol / cytosol + membrane , relative to untreated controls ) from three independent experiments . Error bars represent mean + / - SD . ( D ) BAX / BAK - deficient HeLa cells ectopically expressing BAX were treated and analysed ( E ) UBA pull - down of ubiquitinated proteins following antimycin A and oligomycin ( AO ) treatment of BAX / BAK - deficient HeLa cells ectopically expressing HA - Parkin and BAX for 3 h . Representative immunoblot from two independent experiments .

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

Figure 1(B) BAX/BAK-deficient HeLa cells reconstituted with BAX and where indicated HA-Parkin were treated with antimycin A and oligomycin (AO) for 2 h and then 1 µM of each of the BH3 mimetics ABT-737 and S63485 for 6 h. Cell death was assessed at the indicated times by LDH assay. Data is mean +/- SD of four independent experiments.(C) BAX/BAK-deficient HeLa cells ectopically expressing BAX and Parkin were treated with antimycin A and oligomycin (AO, 3 h), MG132 (20 µM, 3.5 h) and BH3 mimetics (1 µM of each of ABT-737 and S63485, 1 h). Cytosol and membrane fractions were immunoblotted as indicated. Graph shows densitometric analysis of non-ubiquitinated membrane proteins (top, relative to untreated control) or cytochrome c release (bottom; cytosol/cytosol+membrane, relative to untreated controls) from three independent experiments. Error bars represent mean +/- SD.(D) BAX/BAK-deficient HeLa cells ectopically expressing BAX were treated and analysed(E) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment of BAX/BAK-deficient HeLa cells ectopically expressing HA-Parkin and BAX for 3 h. Representative immunoblot from two independent experiments.


Figure 2 ( A ) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA - Parkin following 2 h of antimycin A and oligomycin ( AO ) treatment . ( B ) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin ( AO ) for 3 h with MG132 ( 20 µM ) . Graph shows densitometric analysis of non - ubiquitinated proteins relative to untreated control from three independent experiments . Error bars represent mean + / - SD . ( C ) UBA pull - down of ubiquitinated proteins following antimycin A and oligomycin ( AO ) treatment for 3 h and treatment with the non - specific DUB USP2 for 30 min at 37 ° C , representative immunoblot from two independent experiments . ( E ) Whole cell lysates of HA - Parkin and BAK variant - expressing HeLa cells following 3 h antimycin A and oligomycin ( AO ) treatment with 30 min pre - treatment with MG132 ( 20 µM ) , representative immunoblots from three independent experiments .

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 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 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

Figure 2(A) Subcellular fractionation of cytosol and membrane fractions of HeLa cells expressing HA-Parkin following 2 h of antimycin A and oligomycin (AO) treatment.(B) Immunoblotting of whole cell lysates immunoblotted following treatment with antimycin A and oligomycin (AO) for 3 h with MG132 (20 µM). Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/- SD.(C) UBA pull-down of ubiquitinated proteins following antimycin A and oligomycin (AO) treatment for 3 h and treatment with the non-specific DUB USP2 for 30 min at 37°C, representative immunoblot from two independent experiments.(E) Whole cell lysates of HA-Parkin and BAK variant-expressing HeLa cells following 3 h antimycin A and oligomycin (AO) treatment with 30 min pre-treatment with MG132 (20 µM), representative immunoblots from three independent experiments.


Figure 3 ( A ) HeLaParkin cells were treated with CCCP ( 10 μM ) , antimycin A and oligomycin ( AO ) for 2h or GTPP ( 10 μM ) for 6h . Immunoblots are representative of three independent experiments . Graph shows densitometric analysis of non - ubiquitinated proteins relative to untreated control from three independent experiments . Error bars represent mean + / - ( B ) SH - SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN ( sgPRKN ) or a non - targeting control ( sgCon ) were assessed for Parkin expression by immunoblotting of cytosolic fractions . SH - SY5Y cells generated in ( B ) were treated with antimycin A and oligomycin ( AO ) for 3 h prior to cell lysis and immunoblotting . Mono and di - ubiquitinated BAK in Parkin - expressing cells following AO are indicated ( asterisk ) . Data is representative of three independent experiments . ( D ) Domain architecture of human Parkin with positions of selected PD - associated mutations . Ubl , ubiquitin - like ; IBR , in - between - RING HeLa cells expressing wild - type Parkin or the indicated PD - associated Parkin mutants were treated with antimycin A and oligomycin ( AO ) for 2h . Immunoblots are representative of three independent experiments . Graph shows densitometric analysis of non - ubiquitinated proteins in AO - treated samples relative to untreated control from three independent experiments . Error bars represent mean + / - SD . All Parkin constructs were N - terminally FLAG - tagged except R104W .

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 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 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

Figure 3(A) HeLaParkin cells were treated with CCCP (10 μM), antimycin A and oligomycin (AO) for 2h or GTPP (10 μM) for 6h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins relative to untreated control from three independent experiments. Error bars represent mean +/-(B) SH-SY5Y neuroblastoma cells expressing Cas9 together with a sgRNA targeting PRKN (sgPRKN) or a non-targeting control (sgCon) were assessed for Parkin expression by immunoblotting of cytosolic fractions.SH-SY5Y cells generated in (B) were treated with antimycin A and oligomycin (AO) for 3 h prior to cell lysis and immunoblotting. Mono and di-ubiquitinated BAK in Parkin-expressing cells following AO are indicated (asterisk). Data is representative of three independent experiments.(D) Domain architecture of human Parkin with positions of selected PD-associated mutations. Ubl, ubiquitin-like; IBR, in-between-RING HeLa cells expressing wild-type Parkin or the indicated PD-associated Parkin mutants were treated with antimycin A and oligomycin (AO) for 2h. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of non-ubiquitinated proteins in AO-treated samples relative to untreated control from three independent experiments. Error bars represent mean +/- SD. All Parkin constructs were N-terminally FLAG-tagged except R104W.


Figure 4 ( A ) BN - PAGE and SDS - PAGE of HeLa + HA - Parkin cells treated with 1 µM of each of ABT - 737 and S63485 for 3 h ( BH3 ) , antimycin A and oligomycin ( AO ) for 2 h or a combination of the two . Experiment was performed in the presence of 10 µM QVD . oph and immunoblots are representative of three independent experiments . ( B ) UBA enrichment of ubiquitinated proteins from HeLa + HA - Parkin cells , BAX - / - BAK - / - ( DKO ) HeLa cells or wild - type ( WT ) HeLa cells expressing HA - Parkin in response to 1 µM of each of ABT - 737 and S63485 for 3 h ( BH3 ) in the presence of QVD . oph ( 10 μM ) . Representative of three independent experiments . ( C ) HeLa cells expressing HA - Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non - targeting sgRNA control , treated with AO ( 2 h ) to stabilise PINK1 expression and immunoblotted for PINK1 . ( D ) Parkin activity induced by apoptotic mitochondrial damage is PINK1 - dependent . UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT - 737 and S63485 for 3 h ( BH3 ) in the presence of Q - VD . oph ( 10 μM ) . * , ubiquitinated protein . Representative of two independent experiments .

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 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

Figure 4(A) BN-PAGE and SDS-PAGE of HeLa+HA-Parkin cells treated with 1 µM of each of ABT-737 and S63485 for 3 h (BH3), antimycin A and oligomycin (AO) for 2 h or a combination of the two. Experiment was performed in the presence of 10 µM QVD.oph and immunoblots are representative of three independent experiments.(B) UBA enrichment of ubiquitinated proteins from HeLa+HA-Parkin cells, BAX-/- BAK-/- (DKO) HeLa cells or wild-type (WT) HeLa cells expressing HA-Parkin in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of QVD.oph (10 μM). Representative of three independent experiments.(C) HeLa cells expressing HA-Parkin and Cas9 were transduced with sgRNA targeting PINK1 or a non-targeting sgRNA control, treated with AO (2 h) to stabilise PINK1 expression and immunoblotted for PINK1.(D) Parkin activity induced by apoptotic mitochondrial damage is PINK1-dependent. UBA enrichment of ubiquitinated proteins from HeLa cells generated in response to 1 µM of each of ABT-737 and S63485 for 3 h (BH3) in the presence of Q-VD.oph (10 μM). *, ubiquitinated protein. Representative of two independent experiments.


Figure 5 ( B ) Non - reducing SDS - PAGE and immunoblot of recombinant BAK and BAK - Ub following treatment with 25 mM TCEP ( C ) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID - BH3 peptide to 50 nM BAK or BAK - Ub in the presence or absence of 25 mM TCEP . Data is mean + / - SD of three independent experiments . ( D ) BN - PAGE of BAK and BAK - Ub on liposomes following incubation with 4 µM BID - BH3 peptide in the presence or absence of 25 mM TCEP , immunoblotted for BAK . Data representative of three independent experiments . Graphs shows densitometric analysis of the BAK tetramer ( 4x ) from three or two independent experiments . Error bars represent mean + / - SD for n = 3 or mean and range for n = 2 .

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 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 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 5(B) Non-reducing SDS-PAGE and immunoblot of recombinant BAK and BAK-Ub following treatment with 25 mM TCEP(C) Timecourse monitoring fluorescence increase on liposomes following addition of 4 µM BID-BH3 peptide to 50 nM BAK or BAK-Ub in the presence or absence of 25 mM TCEP. Data is mean +/- SD of three independent experiments.(D) BN-PAGE of BAK and BAK-Ub on liposomes following incubation with 4 µM BID-BH3 peptide in the presence or absence of 25 mM TCEP, immunoblotted for BAK. Data representative of three independent experiments. Graphs shows densitometric analysis of the BAK tetramer (4x) from three or two independent experiments. Error bars represent mean +/- SD for n=3 or mean and range for n=2.


Figure 6 ( A ) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK . ( B ) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function . Mitochondrially - enriched membranes from MEFs expressing BAK or BAK K113C were cross - linked ( CuPhe ) with UbG76C and treated with cBID for 30 min at 30 ° C . Supernatant ( S ) and pellet ( P ) fractions were then immunoblotted for BAK under non - reducing conditions , or cytochrome c or ubiquitin under reducing conditions . Representative of three independent experiments . Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS - PAGE . ( C ) Parkin - mediated ubiquitination limits BAK activation . HeLa + HA - Parkin cells were treated with antimycin A and oligomycin ( AO ) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT - 737 and S63485 ( BH3 mim ) for 1 h . Intracellular flow cytometry was performed using the conformation - specific anti - BAK antibody G317 - 2 . Representative histograms are shown and the fold increase in mean fluorescence intensity ( MFI ) is plotted from three independent experiments with error bars showing SD . Data are normalised to untreated control . ( D ) Parkin - mediated ubiquitination limits BAK apoptotic activity . BAK - / - BAX - / - HeLa cells expressing Parkin and BAK were treated with AO ( 2 h ) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release . Immunoblots are representative of three independent experiments . Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction . Error bars represent mean + / - SD . ( E ) Parkin activity limits cytochrome c release . HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments . Ubiquitinated BAK indicated ( arrow ) . Graph shows densitometric analysis of three independent experiments showing cytochrome c release ( supernatant / supernatant + membrane ) relative to 100 nM cBID as 100 % release . Error bars represent mean + / - SD .

O O B-PANEL_START 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 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 O O O 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

Figure 6(A) Immunoblot of whole cell lysates of MEFs stably expressing ΔCys variants of BAK.(B) Conjugation of ubiquitin to BAK K113C impairs BAK apoptotic function. Mitochondrially-enriched membranes from MEFs expressing BAK or BAK K113C were cross-linked (CuPhe) with UbG76C and treated with cBID for 30 min at 30°C. Supernatant (S) and pellet (P) fractions were then immunoblotted for BAK under non-reducing conditions, or cytochrome c or ubiquitin under reducing conditions. Representative of three independent experiments. Note that the incorporation of recombinant UbG76C on higher molecular weight proteins is likely due to incomplete reduction on reducing SDS-PAGE.(C) Parkin-mediated ubiquitination limits BAK activation. HeLa+HA-Parkin cells were treated with antimycin A and oligomycin (AO) for 2 h prior to induction of apoptosis with 1 µM of each of the BH3 mimetics ABT-737 and S63485 (BH3 mim) for 1 h. Intracellular flow cytometry was performed using the conformation-specific anti-BAK antibody G317-2. Representative histograms are shown and the fold increase in mean fluorescence intensity (MFI) is plotted from three independent experiments with error bars showing SD. Data are normalised to untreated control.(D) Parkin-mediated ubiquitination limits BAK apoptotic activity. BAK-/-BAX-/- HeLa cells expressing Parkin and BAK were treated with AO (2 h) prior to isolation of mitochondria and treatment with recombinant cBID and analysis of cytochrome c release. Immunoblots are representative of three independent experiments. Graph shows densitometric analysis of three independent experiments or cytochrome c in the supernatant fraction. Error bars represent mean +/- SD.(E) Parkin activity limits cytochrome c release. HeLa cells or HeLa cells expressing Parkin were treated and analysed Immunoblots are representative of three independent experiments. Ubiquitinated BAK indicated (arrow). Graph shows densitometric analysis of three independent experiments showing cytochrome c release (supernatant/supernatant+membrane) relative to 100 nM cBID as 100% release. Error bars represent mean +/- SD.


Figure 3 ( D ) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein ( Kd = 9 . 5 + / - 2 . 9 µM ) , in strength similar to the binding of the known PTS1 motif from Idp3 ( Kd = 5 . 5 + / - 0 . 5 µM ) . The plots represent the mean of three independent experiments , and the error bars represent the standard deviation . ( F ) Subcellular fractionations followed by enzymatic activity assays show that untagged , native , Glr1 is active in peroxisomes , in addition to its known activity in mitochondria . Fum1 and Pox2 were used as markers for mitochondrial , and peroxisomal fractions , respectively . ( H ) Yeast - 2 - hybrid assay shows Glr1 interacts in vivo with both Pex5 full - length and Pex5 N ' domain .

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Figure 3(D) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein (Kd = 9.5 +/-2.9 µM), in strength similar to the binding of the known PTS1 motif from Idp3 (Kd = 5.5 +/- 0.5 µM). The plots represent the mean of three independent experiments, and the error bars represent the standard deviation.(F) Subcellular fractionations followed by enzymatic activity assays show that untagged, native, Glr1 is active in peroxisomes, in addition to its known activity in mitochondria. Fum1 and Pox2 were used as markers for mitochondrial, and peroxisomal fractions, respectively.(H) Yeast-2-hybrid assay shows Glr1 interacts in vivo with both Pex5 full-length and Pex5 N' domain.


Figure 4 ( B ) Metabolomic analysis focused on strains with over - expression or deletion of the AYT1 gene shows a significant reduction or accumulation , respectively , in acetyl - glutamate compared to the matched control strain of each mutant . Only metabolites changing significantly in at least one of the two conditions are presented . D - EIP is D - erythro - 1 - ( Imidazole - 4 - yl ) glycerol 3 - phosphate . ( D ) A growth assay in a condition that does not induce high levels of acetyl - CoA in peroxisomes ( glucose ) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital . In stationary phase , the over - expression ( OE ) of AYT1 grows to a lower density than the control strain , plausibly due to burdening of peroxisomal functions . ( E ) A growth assay in a condition expected to elevate acetyl - glutamate levels in peroxisomes ( oleate as a sole carbon source and glutamate as a nitrogen source ) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control . Data information : The growth assays in D and E were done in three biologically independent replicates and error bars were plotted . Note that some error bars are shorter than the symbol , hence are not visible in the graph . ( G ) A β - oxidation activity assay of Δlpx1 , Δfsh3 , and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon - or 18 carbon - free fatty acids in media supplemented with 0 . 5 % glucose shows a significant reduction in β - oxidation activity compared to the wild type and the two single mutants , suggesting an overlapping role for Fsh3 with Lpx1 . This assay was done in three biologically independent replicates . Bars represent the mean and error bars represent the standard deviation .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(B) Metabolomic analysis focused on strains with over-expression or deletion of the AYT1 gene shows a significant reduction or accumulation, respectively, in acetyl-glutamate compared to the matched control strain of each mutant. Only metabolites changing significantly in at least one of the two conditions are presented. D-EIP is D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate.(D) A growth assay in a condition that does not induce high levels of acetyl-CoA in peroxisomes (glucose) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital. In stationary phase, the over-expression (OE) of AYT1 grows to a lower density than the control strain, plausibly due to burdening of peroxisomal functions. (E) A growth assay in a condition expected to elevate acetyl-glutamate levels in peroxisomes (oleate as a sole carbon source and glutamate as a nitrogen source) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control. Data information: The growth assays in D and E were done in three biologically independent replicates and error bars were plotted. Note that some error bars are shorter than the symbol, hence are not visible in the graph.(G) A β-oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon- or 18 carbon-free fatty acids in media supplemented with 0.5% glucose shows a significant reduction in β-oxidation activity compared to the wild type and the two single mutants, suggesting an overlapping role for Fsh3 with Lpx1.This assay was done in three biologically independent replicates. Bars represent the mean and error bars represent the standard deviation.


Figure 5 ( E ) Western blot analysis of Fbp1 - HA levels in mutants Δpex3 ( no peroxisomes ) , Δpex5 ( abolished targeting of GID subunits to peroxisomes ) , and Δpex7 ( no β - oxidation due to loss of Pot1 targeting ) demonstrates that two hours after the transition from glucose to ethanol ( gluconeogenic conditions ) , control and Δpex7 cells upregulated Fbp1 - HA levels , while for Δpex3 and Δpex5 cells Fbp1 - HA levels remain low . Antibodies were used against the HA tag ( for Fbp1 ) and Actin as a loading control .

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Figure 5(E) Western blot analysis of Fbp1-HA levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1 targeting) demonstrates that two hours after the transition from glucose to ethanol (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA levels, while for Δpex3 and Δpex5 cells Fbp1-HA levels remain low. Antibodies were used against the HA tag (for Fbp1) and Actin as a loading control.


Figure 3A . Volcano plots of genes that are differentially expressed in infected cells relative to mock - infected cells at 12 hpi and 24 hpi in ileum organoids . The statistical significance ( - log10 of the adjusted p - value ) is shown as a function of the log2 fold change . MAST tests were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations . F . Multiplex in situ RNA hybridization of ACE2 and SARS - CoV - 2 of mock - infected and infected 2D ileum organoids at 12 hpi and 24 hpi . White arrows point at SARS - CoV - 2 - infected cells . A representative image is shown .

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 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

Figure 3A. Volcano plots of genes that are differentially expressed in infected cells relative to mock-infected cells at 12 hpi and 24 hpi in ileum organoids. The statistical significance (-log10 of the adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in all panels are gene names of selected differentially expressed genes between the compared two populations.F. Multiplex in situ RNA hybridization of ACE2 and SARS-CoV-2 of mock-infected and infected 2D ileum organoids at 12 hpi and 24 hpi. White arrows point at SARS-CoV-2-infected cells. A representative image is shown.


Figure 4Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS - CoV - 2 infection of colon organoids . infected vs . mock infected cells The statistical significance ( - log10 adjusted p - value ) is shown as a function of the log2 fold change . MAST tests were used to generate P ‐ values , bonferroni multiple hypotheses correction was used to compute FDR values . Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations . Labeled dots in red in all panels are gene corresponding to interferon if detected .

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Figure 4Volcano plots displaying the genes that are differentially expressed in immature enterocytes 2 upon SARS-CoV-2 infection of colon organoids. infected vs. mock infected cells The statistical significance (-log10 adjusted p-value) is shown as a function of the log2 fold change. MAST tests were used to generate P‐values, bonferroni multiple hypotheses correction was used to compute FDR values. Labeled dots in blue in all panels are gene names of selected differentially expressed genes between the compared two populations. Labeled dots in red in all panels are gene corresponding to interferon if detected.


Figure 6A . Human ileum - derived organoids were seeded in 2D on iBIDi chamber slides . 12 and 24 hpi cells were fixed and the amount of SARS - CoV - 2 infected cells ( red ) and the induction of ISG15 ( white ) was analyzed by single molecule RNA FISH . Nuclei were visualized with DAPI ( blue ) . White arrows indicate SARS - CoV - 2 positive cells . N = 3 biological samples . Representative image is shown . Scale bar = 100 um . D . T84 IRF3 knock - out cells were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , cells were incubated in the presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment RNA was harvested and the induction of ISG15 was analyzed by q - RT - PCR and normalized to the housekeeping gene TBP . N = 3 biological replicates . Error bar indicates standard deviation . Statistics were determined by unpaired t - test . E . T84 IRF3 knock - out cells were infected with SARS - CoV - 2 or human astrovirus 1 at an MOI = 3 . 24 hpi , cells were fixed and stained for either the SARS - CoV - 2 N protein or for human astrovirus 1 ( HAstV1 ) capsid protein . N = 3 biological replicates . Representative image is shown . Scale bar = 100 um . G . T84 MX - 1 mcherry were infected with SARS - CoV - 2 at an MOI = 3 or MOI = 1 . 24 hpi cells were incubated in the presence or absence of 2000 IU / mL of IFNβ1 . 12 h post - treatment cells were fixed and stained for SARS - CoV - 2 N protein . The number of MX - 1 positive , SARS - CoV - 2 positive and double positive cells was quantified . N = 3 biological replicates were performed . Error bar indicates standard deviation .

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 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 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

Figure 6A. Human ileum-derived organoids were seeded in 2D on iBIDi chamber slides. 12 and 24 hpi cells were fixed and the amount of SARS-CoV-2 infected cells (red) and the induction of ISG15 (white) was analyzed by single molecule RNA FISH. Nuclei were visualized with DAPI (blue). White arrows indicate SARS-CoV-2 positive cells. N=3 biological samples. Representative image is shown. Scale bar=100 um.D. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment RNA was harvested and the induction of ISG15 was analyzed by q-RT-PCR and normalized to the housekeeping gene TBP. N=3 biological replicates. Error bar indicates standard deviation. Statistics were determined by unpaired t-test.E. T84 IRF3 knock-out cells were infected with SARS-CoV-2 or human astrovirus 1 at an MOI=3. 24 hpi, cells were fixed and stained for either the SARS-CoV-2 N protein or for human astrovirus 1 (HAstV1) capsid protein. N=3 biological replicates. Representative image is shown. Scale bar=100 um.G. T84 MX-1 mcherry were infected with SARS-CoV-2 at an MOI=3 or MOI=1. 24 hpi cells were incubated in the presence or absence of 2000 IU/mL of IFNβ1. 12 h post-treatment cells were fixed and stained for SARS-CoV-2 N protein. The number of MX-1 positive, SARS-CoV-2 positive and double positive cells was quantified. N=3 biological replicates were performed. Error bar indicates standard deviation.


Figure 1 ( A ) Confocal microscopy of GFP - TRIM33 - overexpressing mESCs and differentiated cells . Scale bar = 5 µm . ( B ) Line scans of GFP - TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( C ) FRAP assays showing recovery time of normalized fluorescence signals for GFP - TRIM33 puncta in mESCs . Photobleaching occurs at t = 5s . Data are the mean ± S . D . of three biological experiments . ( D ) Sphericity analysis and 3D imaging by NIS - Elements 3D tools of TRIM33 puncta in live mESCs . Panels show the xz , xy , and yz planes , from left to right . Scale bar = 1 µm . Data information : All data are from three biological replicates . ( F ) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co - transfected with plasmids encoding Flag - PML and GFP - TRIM33 . GFP - TRIM33 was immunoprecipitated with anti - GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP . Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates . ( n = 3 ) . Data information : All data are from three biological replicates . ( G ) Lysates from mESCs and differentiated cells were immunoprecipitated with anti - PML antibody , and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML . ( n = 3 ) . Data information : All data are from three biological replicates . ( H ) Lysates from HEK293T cells co - transfected with plasmids encoding either full length Flag - TRIM33 or the Flag - TRIM33 - RBCC domain alone and full - length HA - PML or the HA - PML - RBCC domain alone immunoprecipitated with anti - Flag affinity beads . Protein complexes were detected using antibodies against Flag and HA . The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates . ( n = 3 ) . Data information : All data are from three biological replicates .

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 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 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 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

Figure 1(A) Confocal microscopy of GFP-TRIM33-overexpressing mESCs and differentiated cells. Scale bar = 5 µm. (B) Line scans of GFP-TRIM33 and DAPI fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (C) FRAP assays showing recovery time of normalized fluorescence signals for GFP-TRIM33 puncta in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of three biological experiments.(D) Sphericity analysis and 3D imaging by NIS-Elements 3D tools of TRIM33 puncta in live mESCs. Panels show the xz, xy, and yz planes, from left to right. Scale bar = 1 µm. Data information: All data are from three biological replicates.(F) Western blot analysis of TRIM33 interactions with PML in lysates of HEK293T cells co-transfected with plasmids encoding Flag-PML and GFP-TRIM33. GFP-TRIM33 was immunoprecipitated with anti-GFP affinity beads and immune complexes were detected using antibodies targeting Flag and GFP. Protein inputs were detected using antibodies against Flag and GFP in the same amount of cell lysates. (n = 3). Data information: All data are from three biological replicates.(G) Lysates from mESCs and differentiated cells were immunoprecipitated with anti-PML antibody, and protein complexes and inputs were analyzed by immunoblotting with antibodies against TRIM33 and PML. (n = 3). Data information: All data are from three biological replicates.(H) Lysates from HEK293T cells co-transfected with plasmids encoding either full length Flag-TRIM33 or the Flag-TRIM33-RBCC domain alone and full-length HA-PML or the HA-PML-RBCC domain alone immunoprecipitated with anti-Flag affinity beads. Protein complexes were detected using antibodies against Flag and HA. The protein inputs were detected using antibodies against Flag and HA in the same amount of cell lysates. (n = 3). Data information: All data are from three biological replicates.


Figure 2 ( A ) Representative images of immunofluorescence staining of PML in WT and Pml null mESCs . Scale bar = 5 µm . ( B ) Representative confocal microscopy images of mESCs overexpressing mCherry - PML or GFP - PML ( top ) . Representative confocal microscopy images of mESCs overexpressing mCherry - EV or GFP - EV ( bottom ) . Scale bar = 5 µm . ( C ) FRAP assays quantifying normalized fluorescence recovery of GFP - PML signal in mESCs . Photobleaching occurs at t = 5s . Data are the mean ± S . D . of four biological experiments . ( D , G , I , Representative images of co - localization assays with mCherry - PML and GFP - TRIM33 in mESCs ( D ) , differentiated cells ( G ) , NaAsO2 - treated mESCs ( 200 µM for 2 hr ) ( I ) Scale bar = 5 µm . ( E , H , J Line scans of GFP - TRIM33 and mCherry - PML fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( F ) Column graph of the numbers of GFP - TRIM33 puncta and mCherry - PML NBs in mESCs . K ) Representative images of co - localization assays with GFP - TRIM33 in mESCs Pml null ( K ) . Scale bar = 5 µm . L ) Line scans of GFP - TRIM33 fluorescence imaging in each cell context at the position depicted by the white line ( from left to right ) . These line scans were processed by plot profile using image J software . ( M ) Column graph of the numbers of GFP - TRIM33 puncta and DAPI in mESCs . ( N ) Scatter plot of the numbers of GFP - TRIM33 puncta ( per cell ) in WT and Pml KO mESCs . ( n = 20 ) . ( O ) Scatter plot of the volume of GFP - TRIM33 puncta ( per cell ) in WT and Pml KO mESCs . ( n = 20 ) . Data information : In panels ( N , O , 20 individual puncta with three biological experiments were analyzed . Significant differences were determined by t - tests . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) . ( P ) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs . Scale bar = 10 µm . ( Q ) Scatter plot of the numbers of PML NBs ( per cell ) in WT and Trim33 KO mESCs . ( n = 20 ) . ( R ) Scatter plot of the proportion of PML NBs ( per cell ) in WT and Trim33 KO mESCs . ( n = 20 ) . Data information : In panels Q and R ) , 20 individual puncta with three biological experiments were analyzed . Significant differences were determined by t - tests . ( * p < 0 . 05 , * * * * p < 0 . 0001 ) .

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

Figure 2(A) Representative images of immunofluorescence staining of PML in WT and Pml null mESCs. Scale bar = 5 µm.(B) Representative confocal microscopy images of mESCs overexpressing mCherry-PML or GFP-PML (top). Representative confocal microscopy images of mESCs overexpressing mCherry-EV or GFP-EV (bottom). Scale bar = 5 µm. (C) FRAP assays quantifying normalized fluorescence recovery of GFP-PML signal in mESCs. Photobleaching occurs at t = 5s. Data are the mean ± S.D. of four biological experiments.(D, G, I, Representative images of co-localization assays with mCherry-PML and GFP-TRIM33 in mESCs (D), differentiated cells (G), NaAsO2-treated mESCs (200 µM for 2 hr) (I) Scale bar = 5 µm. (E, H, J Line scans of GFP-TRIM33 and mCherry-PML fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (F) Column graph of the numbers of GFP-TRIM33 puncta and mCherry-PML NBs in mESCs.K) Representative images of co-localization assays with GFP-TRIM33 in mESCs Pml null (K). Scale bar = 5 µm. L) Line scans of GFP-TRIM33 fluorescence imaging in each cell context at the position depicted by the white line (from left to right). These line scans were processed by plot profile using image J software. (M) Column graph of the numbers of GFP-TRIM33 puncta and DAPI in mESCs.(N) Scatter plot of the numbers of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20). (O) Scatter plot of the volume of GFP-TRIM33 puncta (per cell) in WT and Pml KO mESCs. (n = 20). Data information: In panels (N, O, 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001). (P) Representative images of immunofluorescence staining of PML in WT and Trim33 null mESCs. Scale bar = 10 µm. (Q) Scatter plot of the numbers of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20). (R) Scatter plot of the proportion of PML NBs (per cell) in WT and Trim33 KO mESCs. (n = 20). Data information: In panels Q and R), 20 individual puncta with three biological experiments were analyzed. Significant differences were determined by t-tests. (*p < 0.05, ****p < 0.0001).


Figure 1 ( A ) Blood ammonia in 8 - 9 - week - old C57BL / 6J wild - type ( WT ) mice ( n = 8 ) and Becn1F121A mice with constitutive activation of autophagy ( n = 8 ) at baseline and 30 min after i . p . injection of NH4Cl ( 10 mmol / kg ) . * * p < 0 . 01 ( Unpaired t - test ) . ns : not statistically significant difference . ( B ) Urinary orotic acid of 12 - week - old spf - ash mice treated with TB - 1 ( 15 mg / kg , i . p ) or vehicle at various times as indicated by the arrows ( n = 5 mice / group ) . * p < 0 . 05 ( Two - way ANOVA ) . ( C ; D ) Western blotting and densitometric quantifications of autophagy markers ( LC3II : autophagosomes ; p62 and NBR1 : cargo receptors ) in livers of spf - ash mice harvested after ten days of treatment with TB - 1 or vehicle . β - actin was used as loading control . n = 5 mice / group . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) . ( E ) Survival curves of spf - ash mice fed with a high protein diet ( HPD ) for ten days and treated with TB - 1 alone or combined with scavenger drug and L - Arginine , or treated with scavenger drug and L - Arginine , or left untreated . WT control were included ( n = 5 / group ) . * * * p < 0 . 001 ( Log - rank Mantel - Cox test ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M . ( F ) Blood ammonia levels determined after 4 days under HPD ( n = 5 mice / group ) ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : Treatments Scavenger ( Na - benzoate 250 mg / kg / day , i . p . ) and L - arginine ( L - Arg , 250 mg / kg / day , i . p . ) ; TB - 1 ( 15 mg / kg every 2 days , i . p ) . WT mice were age - , gender - and strain ( C3H ) - matched . All values are shown as averages ± S . E . M .

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 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 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 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 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

Figure 1(A) Blood ammonia in 8-9-week-old C57BL/6J wild-type (WT) mice (n=8) and Becn1F121A mice with constitutive activation of autophagy (n=8) at baseline and 30 min after i.p. injection of NH4Cl (10 mmol/kg). **p<0.01 (Unpaired t-test). ns: not statistically significant difference.(B) Urinary orotic acid of 12-week-old spf-ash mice treated with TB-1 (15 mg/kg, i.p) or vehicle at various times as indicated by the arrows (n=5 mice/group). *p<0.05 (Two-way ANOVA).(C; D) Western blotting and densitometric quantifications of autophagy markers (LC3II: autophagosomes; p62 and NBR1: cargo receptors) in livers of spf-ash mice harvested after ten days of treatment with TB-1 or vehicle. β-actin was used as loading control. n=5 mice/group. **p<0.01, *p<0.05 (Unpaired t-test).(E) Survival curves of spf-ash mice fed with a high protein diet (HPD) for ten days and treated with TB-1 alone or combined with scavenger drug and L-Arginine, or treated with scavenger drug and L-Arginine, or left untreated. WT control were included (n=5/group). ***p<0.001 (Log-rank Mantel-Cox test). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.(F) Blood ammonia levels determined after 4 days under HPD (n=5 mice/group); ***p<0.001, **p<0.01, *p<0.05 (One-way ANOVA). Data information: Treatments Scavenger (Na-benzoate 250 mg/kg/day, i.p.) and L-arginine (L-Arg, 250 mg/kg/day, i.p.); TB-1 (15 mg/kg every 2 days, i.p). WT mice were age-, gender- and strain (C3H)-matched. All values are shown as averages ± S.E.M.


Figure 2 ( A ) Survival curves of AslNeo / Neo mice and age - matched wild - type ( WT ) controls treated with TB - 1 ( 15 mg / kg , i . p . , every 48 hours starting at day 10 of age ) or vehicle . WT + Vehicle n = 8 ; WT + TB - 1 n = 8 ; AslNeo / Neo + Vehicle n = 20 ; AslNeo / Neo + TB - 1 n = 16 . * p < 0 . 05 ( Log - rank Mantel - Cox test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference . ( B ) Isotopic enrichment of 15N - labeled urea in blood , 20 min after i . p . injection of 15NH4Cl tracer ( 4 mmol / kg ) in AslNeo / Neo mice treated with TB - 1 ( n = 15 ) or vehicle ( n = 9 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference . ( C ) Representative Western blotting bands of LC3 , p62 and NBR1 in livers of AslNeo / Neo mice treated with TB - 1 or vehicle . GAPDH and β - actin were used as loading controls . ( D ) Densitometric quantifications . AslNeo / Neo + Vehicle n = 5 ; AslNeo / Neo + TB - 1 n = 9 . * * p < 0 . 01 , * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference . ( E ) Argininosuccinate in dried blood spots ( DBS ) of WT and AslNeo / Neo mice injected with TB - 1 or vehicle ( n = 4 - 14 mice / group ) . * * * p < 0 . 001 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference . ( F ) Isotopic enrichment of 15N - labeled argininosuccinate in livers of AslNeo / Neo mice treated with TB - 1 ( n = 11 ) or vehicle ( n = 5 ) . * p < 0 . 05 ( Unpaired t - test ) . Data information : All values are shown as averages ± S . E . M . ns : not statistically significant difference .

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

Figure 2(A) Survival curves of AslNeo/Neo mice and age-matched wild-type (WT) controls treated with TB-1 (15 mg/kg, i.p., every 48 hours starting at day 10 of age) or vehicle. WT + Vehicle n=8; WT + TB-1 n=8; AslNeo/Neo + Vehicle n=20; AslNeo/Neo + TB-1 n=16. *p<0.05 (Log-rank Mantel-Cox test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.(B) Isotopic enrichment of 15N-labeled urea in blood, 20 min after i.p. injection of 15NH4Cl tracer (4 mmol/kg) in AslNeo/Neo mice treated with TB-1 (n=15) or vehicle (n=9). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.(C) Representative Western blotting bands of LC3, p62 and NBR1 in livers of AslNeo/Neo mice treated with TB-1 or vehicle. GAPDH and β-actin were used as loading controls. (D) Densitometric quantifications. AslNeo/Neo + Vehicle n=5; AslNeo/Neo + TB-1 n=9. **p<0.01, *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.(E) Argininosuccinate in dried blood spots (DBS) of WT and AslNeo/Neo mice injected with TB-1 or vehicle (n=4-14 mice/group). ***p<0.001, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.(F) Isotopic enrichment of 15N-labeled argininosuccinate in livers of AslNeo/Neo mice treated with TB-1 (n=11) or vehicle (n=5). *p<0.05 (Unpaired t-test). Data information: All values are shown as averages ± S.E.M. ns: not statistically significant difference.


Figure 3 ( A ) Hematoxylin and eosin ( H & E , upper panels ) and periodic acid Schiff ( PAS , lower panels ) staining of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bars : 500 µm . ( B ) Computational analysis of PAS staining ( n ≥ 4 mice / group ) . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ( C ) Quantification of hepatic glycogen in vehicle ( n = 6 ) - and TB - 1 - treated AslNeo / Neo mice ( n = 12 ) compared to WT ( n = 5 ) controls . * * p < 0 . 01 , * p < 0 . 05 ( One - way ANOVA ) . Data information : All values are shown as averages ± S . E . M . ( D ) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo / Neo mice treated with either with TB - 1 or vehicle ( n = 4 mice / group ) . * p < 0 . 05 ( One - way ANOVA ) . GAPDH was used as loading control . Data information : All values are shown as averages ± S . E . M . ( E ) Representative electron microscopy images of liver samples harvested from WT and AslNeo / Neo mice treated with TB - 1 or vehicle . Scale bar : 900 nm .

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 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 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

Figure 3(A) Hematoxylin and eosin (H&E, upper panels) and periodic acid Schiff (PAS, lower panels) staining of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bars: 500 µm.(B) Computational analysis of PAS staining (n≥4 mice/group). **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.(C) Quantification of hepatic glycogen in vehicle (n=6)- and TB-1-treated AslNeo/Neo mice (n=12) compared to WT (n=5) controls. **p<0.01, *p<0.05 (One-way ANOVA). Data information: All values are shown as averages ± S.E.M.(D) Representative Western blotting bands and densitometric quantification of PYGL in livers of WT and AslNeo/Neo mice treated with either with TB-1 or vehicle (n=4 mice/group). *p<0.05 (One-way ANOVA). GAPDH was used as loading control. Data information: All values are shown as averages ± S.E.M.(E) Representative electron microscopy images of liver samples harvested from WT and AslNeo/Neo mice treated with TB-1 or vehicle. Scale bar: 900 nm.


Figure 4 ( A ) Representative electron microscopy images of liver samples harvested from wild - type ( WT ) and AslNeo / Neo mice treated with TB - 1 or vehicle . False color on the images indicates glycogen within the nuclei . Scale bar : 1 μm . ( B ) Quantification of nuclei containing glycogen ( approx . 180 nuclei were analyzed in total per condition , n = 3 mice / 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 O O O O O O O O O O O O O O O O O O O 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) Representative electron microscopy images of liver samples harvested from wild-type (WT) and AslNeo/Neo mice treated with TB-1 or vehicle. False color on the images indicates glycogen within the nuclei. Scale bar: 1 μm. (B) Quantification of nuclei containing glycogen (approx. 180 nuclei were analyzed in total per condition, n=3 mice/group).


Figure 1A . Tryptophan - fluorescence time course associated to Na + ( blue ) and K + ( black ) binding , respectively , to detergent - purified apo EAAT1CRYST at pH 7 . 4 . Horizontal and vertical scale bars represent time and ∆ F / F0 , respectively . B . Tryptophan - fluorescence time course associated to L - asp binding in presence of 1 mM ( blue ) and absence ( black ) of Na + , respectively , at pH 9 . CC . Na + binding isotherms of EAAT1CRYST at pH 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Na + binding ( green ) . Solid lines represent fits of Hill equation .

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 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

Figure 1A. Tryptophan-fluorescence time course associated to Na+ (blue) and K+ (black) binding, respectively, to detergent-purified apo EAAT1CRYST at pH 7.4. Horizontal and vertical scale bars represent time and ∆F/F0, respectively. B. Tryptophan-fluorescence time course associated to L-asp binding in presence of 1 mM (blue) and absence (black) of Na+, respectively, at pH 9. CC. Na+ binding isotherms of EAAT1CRYST at pH 7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Na+ binding (green). Solid lines represent fits of Hill equation.


Figure 3A . Na + - induced L - glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K + . Choline ( Ch + ) condition was subtracted to the Na + condition . B . Log - log plots of Na + ( red , n = 19 ) and L - asp ( blue , n = 27 ) KD , as a function of pH at [ Na + ] = 0 . 5 mM . Na + ( n = 10 ) and L - asp ( n = 7 ) titration data at pH 7 . 4 and 9 . 0 , respectively , are also reported in Fig . 1 . Solid lines are fits of straight lines . C . Log - log plots of L - asp KD for EAAT1CRYST - E406386Q ( light blue , n = 24 ) and EAAT1CRYST - Y405385F ( dark blue , n = 18 ) , as a function of pH at [ Na + ] = 0 . 5 mM . .

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 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 O O O O O O O O O

Figure 3A. Na+-induced L-glutamate uptake by purified EAAT1CRYST reconstituted in liposomes loaded with K+. Choline (Ch+) condition was subtracted to the Na+ condition.B. Log-log plots of Na+ (red, n=19) and L-asp (blue, n=27) KD, as a function of pH at [Na+]=0.5 mM. Na+ (n=10) and L-asp (n=7) titration data at pH 7.4 and 9.0, respectively, are also reported in Fig. 1. Solid lines are fits of straight lines.C. Log-log plots of L-asp KD for EAAT1CRYST-E406386Q (light blue, n=24) and EAAT1CRYST-Y405385F (dark blue, n=18), as a function of pH at [Na+]=0.5 mM..


Figure 4A . Purified EAAT1CRYST takes up L - glutamate in liposomes loaded with K + or Rb + , but not with choline ( Ch + ) , or with Ch + and Ca2 + . Plots depict data as in Fig . 3A .

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Figure 4A. Purified EAAT1CRYST takes up L-glutamate in liposomes loaded with K+ or Rb+, but not with choline (Ch+), or with Ch+ and Ca2+. Plots depict data as in Fig. 3A.


Figure 5B . Ca2 + binding isotherms of EAAT1CRYST at pH = 7 . 4 in the presence ( pink ) and absence ( blue ) of UPCH101 . Mutation D400380N at Na3 strongly impairs Ca2 + binding ( green ) . Symbols represent average and s . e . m . values of at least three independent experiments . Solid lines represent fits of Hill equation and dashed line is the fit in Fig . 1C ( blue line ) corresponding to Na + binding . C . Log - log plot of L - asp KD as a function of Ca2 + ( n = 13 ) .

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Figure 5B. Ca2+ binding isotherms of EAAT1CRYST at pH=7.4 in the presence (pink) and absence (blue) of UPCH101. Mutation D400380N at Na3 strongly impairs Ca2+ binding (green). Symbols represent average and s.e.m. values of at least three independent experiments. Solid lines represent fits of Hill equation and dashed line is the fit in Fig. 1C (blue line) corresponding to Na+ binding.C. Log-log plot of L-asp KD as a function of Ca2+ (n=13).