Fig 5 ( A - C ) Time course of effect of temperature shift on aggregates , autophagosomes , and larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a animals starved 6 hrs prior to analysis . ( A ) The number of ubiquitin aggregates peaks at 18 - 24 hrs PS , and decreases significantly from 18 to 48 hrs PS ( n = 10 and p - values shown relative to 18 hrs PS ) . ( B ) The number of GFP - Atg8a punctae peaks at 12 - 24 hrs PS and decreases significantly from 18 to 48 hrs PS ( n = 10 and p - values shown relative to 18 hrs PS ) . ( C ) Larval locomotion , measured as the time to crawl 3cm , gradually and significantly worsens from 18 to 48 hrs PS ( n = 6 and p - values shown relative to 0 hrs PS . ( D - E ) tyramine ( Tyr ) treatment reduces GFP - Atg8a punctae in larval muscle . UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a animals were placed on 100mg / ml Tyr ( D ) or control food ( E ) , shifted to restrictive temperature for 18hrs , and starved 6 hrs prior to dissection . ( F - H ) Effect of Tyr treatment on aggregates , autophagosomes , and larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a and Dmef2 - Gal4 , UAS - GFP - Atg8a animals , shifted to restrictive temperature for 18hrs , and starved 6 hrs prior to dissection . Aggregate ( F ) and GFP - Atg8a punctae ( G ) numbers in muscles are significantly reduced with 100 mg / ml Tyr treatment in DTS but not control animals 18 hrs post - shift ( PS ) . ( H ) 100 mg / ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS . n = 12 and p - values shown relative to non - Tyr treated animals . For all measurements , SEM is shown with * p < . 05 ; * * p < . 01 ; * * * p < . 001 . ( I ) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS . 6 hrs starvation induces lipidated Atg8a - II in Dmef2 - G4 control with or without 100 mg / ml Tyr . After 6hrs starvation , Atg8a - II levels are reduced in DTS + 100 mg / ml Tyr relative to untreated DTS .

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

Fig 5(A-C) Time course of effect of temperature shift on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals starved 6 hrs prior to analysis. (A) The number of ubiquitin aggregates peaks at 18-24 hrs PS, and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (B) The number of GFP-Atg8a punctae peaks at 12-24 hrs PS and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (C) Larval locomotion, measured as the time to crawl 3cm, gradually and significantly worsens from 18 to 48 hrs PS (n = 6 and p-values shown relative to 0 hrs PS.(D-E) tyramine (Tyr) treatment reduces GFP-Atg8a punctae in larval muscle. UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals were placed on 100mg/ml Tyr (D) or control food (E), shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection.(F-H) Effect of Tyr treatment on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and Dmef2-Gal4, UAS-GFP-Atg8a animals, shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. Aggregate (F) and GFP-Atg8a punctae (G) numbers in muscles are significantly reduced with 100 mg/ml Tyr treatment in DTS but not control animals 18 hrs post-shift (PS). (H) 100 mg/ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS. n = 12 and p-values shown relative to non-Tyr treated animals. For all measurements, SEM is shown with *p<.05; **p<.01; ***p<.001.(I) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-G4 control with or without 100 mg/ml Tyr. After 6hrs starvation, Atg8a-II levels are reduced in DTS + 100 mg/ml Tyr relative to untreated DTS.


Fig 6 ( D - H ) Dmef2 - Gal4 drives expression of UAS - GFP - Atg8a in animals starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . RNAi knockdown of CG41099 ( E ) , CG7112 ( F ) , CG31935 ( G ) , and Rab3 - GAP ( H ) , blocks the formation of GFP - Atg8a labeled autophagosomes compared to knockdown of white ( D ) . ( I - M ) Dmef2 - Gal4 drives expression of UAS - myc - Atg8a in animals starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . The effect of the RAB3GAPs is not via Rab3 or Rab5 function , as a Rab3 null mutation ( J ) , expression of a constitutive active form of Rab3 ( Q80L ) ( K ) or Rab5 ( Q88L ) ( L ) has no effect on muscle autophagy compared to control ( I ) . Expression of a constitutive active form of Rab39 ( Q69L ) ( M ) reduces myc - Atg8a labeled vesicles . ( N ) Quantification of autophagy phenotypes from panels D - M . SEM is indicated , with n = 8 ventral longitudinal muscles from individual animals and * * p < . 01 ; * * * p < . 001 . ( N ) Quantification of autophagy phenotypes from panels D - M . SEM is indicated , with n = 8 ventral longitudinal muscles from individual animals and * * p < . 01 ; * * * p < . 001 . ( O ) Atg8a and Rab3 - GAP colocalize to autophagosomes . Dmef2 - Gal4 drives expression of UAS - myc - Atg8a and UAS - CFP - Rab3 - GAP in animals starved and treated with CQ for 6 hrs . In all panels green = GFP - Atg8a and red = myc - Atg8a , and blue = CFP - Rab3Gap2 . ( P ) Anti - Atg8a Western blot of third instar muscle lysate from Dmef2 - Gal4 driving the RNAi or overexpression constructs shown in D - N . Total levels of endogenous Atg8a are variable , but only knockdown of CG41099 , CG7112 , CG31935 , Rab3 - GAP , or overexpression of Rab39 ( Q69L ) block induction of lipidated Atg8a - II following 6 hrs starvation .

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 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 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 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

Fig 6(D-H)Dmef2-Gal4 drives expression of UAS-GFP-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. RNAi knockdown of CG41099 (E), CG7112 (F), CG31935 (G), and Rab3-GAP (H), blocks the formation of GFP-Atg8a labeled autophagosomes compared to knockdown of white (D).(I-M) Dmef2-Gal4 drives expression of UAS-myc-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. The effect of the RAB3GAPs is not via Rab3 or Rab5 function, as a Rab3 null mutation (J), expression of a constitutive active form of Rab3 (Q80L) (K) or Rab5 (Q88L) (L) has no effect on muscle autophagy compared to control (I). Expression of a constitutive active form of Rab39 (Q69L) (M) reduces myc-Atg8a labeled vesicles. (N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.(N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.(O) Atg8a and Rab3-GAP colocalize to autophagosomes. Dmef2-Gal4 drives expression of UAS-myc-Atg8a and UAS-CFP-Rab3-GAP in animals starved and treated with CQ for 6 hrs. In all panels green = GFP-Atg8a and red = myc-Atg8a, and blue = CFP-Rab3Gap2.(P) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4 driving the RNAi or overexpression constructs shown in D-N. Total levels of endogenous Atg8a are variable, but only knockdown of CG41099, CG7112, CG31935, Rab3-GAP, or overexpression of Rab39 (Q69L) block induction of lipidated Atg8a-II following 6 hrs starvation.


Fig 7 ( A ) Primary cultured muscles with control lacZ RNAi , treated for 12 hrs with Rap + CQ . Note the formation of large GFP - Atg8a vesicles throughout the muscles . ( B ) GFP - Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi , treated for 12 hrs with Rap + CQ . Knockdown of Pfk ( C ) or CG9485 ( D ) causes increased accumulation of GFP - Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap + CQ . ( E ) Quantification for A - D of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . SEM is indicated with * p < . 05 ; * * p < . 01 . ( F ) Control white RNAi muscles accumulate large autophagosomes at 1 mg / ml CQ . ( G ) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle . ( H ) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle . ( I ) Quantification of the GFP - Atg8a punctae area in muscles from white , CG9485 , and AGBE knockdowns . n = 6 larval muscles with SEM indicated and * p < . 05 . ( J ) Western blot analysis of endogenous Atg8a from third instar muscle lysate . Dmef2 - Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation , indicating reduced autophagy levels .

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 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

Fig 7(A) Primary cultured muscles with control lacZ RNAi, treated for 12 hrs with Rap+CQ. Note the formation of large GFP-Atg8a vesicles throughout the muscles. (B) GFP-Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi, treated for 12 hrs with Rap+CQ. Knockdown of Pfk (C) or CG9485 (D) causes increased accumulation of GFP-Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap+CQ. (E) Quantification for A-D of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). SEM is indicated with *p<.05; **p<.01.(F) Control white RNAi muscles accumulate large autophagosomes at 1 mg/ml CQ. (G) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle. (H) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle. (I) Quantification of the GFP-Atg8a punctae area in muscles from white, CG9485, and AGBE knockdowns.n = 6 larval muscles with SEM indicated and *p<.05.(J) Western blot analysis of endogenous Atg8a from third instar muscle lysate. Dmef2-Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation, indicating reduced autophagy levels.


Figure 1A H & E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID - 19 patients who presented with gastrointestinal ( GI ) symptoms . The inflammatory infiltrates were indicated by a yellow arrow . The edema area was indicated by a red star . Scale bars , 100 µm . B The bar chart shows the degrees of intestinal inflammation in patients with COVID - 19 . Number of samples for each group as indicated . Data information : All data are shown as mean ± SD . C Cytokine levels in the plasma of COVID - 19 patients with ( n = 12 ) or without ( n = 6 ) GI symptoms by cytokine assay . Data information : All data are shown as mean ± SD . P values are determined by Student ' s t - test . D Levels of plasma VEGF in COVID - 19 patients with ( n = 8 ) or without ( n = 10 ) disease progression by cytokine assay . Data information : All data are shown as mean ± SD . P values are determined by Student ' s t - test . E Temporal course of plasma VEGF at the early and late stage of COVID - 19 infection by cytokine assay . Data shown are the levels of plasma VEGF in patients with ( n = 8 ) and without ( n = 10 ) disease progression at the early stage ( one to three days after laboratory - confirmed for COVID - 19 ) and late stage ( more than three days after laboratory - confirmed for COVID - 19 ) . F Levels of plasma VEGF in COVID - 19 patients with ( n = 7 ) or without ( n = 6 ) interstitial vasodilation by cytokine assay . G Levels of plasma VEGF in COVID - 19 patients with ( n = 8 ) or without ( n = 5 ) interstitial edema by cytokine assay . Data information : All data are shown as mean ± SD . P values are determined by Student ' s t - test .

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

Figure 1A H&E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID-19 patients who presented with gastrointestinal (GI) symptoms. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bars, 100 µm. B The bar chart shows the degrees of intestinal inflammation in patients with COVID-19. Number of samples for each group as indicated. Data information: All data are shown as mean ± SD.C Cytokine levels in the plasma of COVID-19 patients with (n=12) or without (n=6) GI symptoms by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.D Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=10) disease progression by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.E Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by cytokine assay. Data shown are the levels of plasma VEGF in patients with (n=8) and without (n=10) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19).F Levels of plasma VEGF in COVID-19 patients with (n=7) or without (n=6) interstitial vasodilation by cytokine assay. G Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=5) interstitial edema by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


Figure 2A Levels of VEGF - A mRNA in the intestinal tissues from COVID - 19 patients ( n = 5 ) or healthy controls ( n = 5 ) by RNA - seq . Data information : All data are shown as mean ± SD . For ( A ) , P values are determined by Student ' s t - testC , D Representative H & E images ( C ) and the quantitative analysis of inflammation ( D ) of the intestinal tissues from animals treated by the protocol shown in ( B ) . The inflammatory infiltrates were indicated by a yellow arrow . Scale bar , 100 µm . Control - Fc , n = 5 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . E qRT - PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control - Fc or Spike RBD - Fc . Control - Fc , n = 5 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . ; for ( E ) , P values are determined by Mann - Whitney test . F ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control - Fc or Spike RBD - Fc . Control - Fc , n = 4 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . For ( F ) P values are determined by Student ' s t - testG ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control - Fc or Spike RBD - Fc , or seven days after treated with Control - Fc or Spike RBD - Fc daily . Control - Fc , n = 4 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . H Evaluation of murine peritoneal permeability six hours after treatment with Control - Fc or Spike RBD - Fc , or seven days after treated with Control - Fc or Spike RBD - Fc daily by TRITC - dextran dye extravasation assay . For each group , n = 8 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . For ( H ) , P values are determined by Student ' s t -

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

Figure 2A Levels of VEGF-A mRNA in the intestinal tissues from COVID-19 patients (n=5) or healthy controls (n=5) by RNA-seq. Data information: All data are shown as mean ± SD. For (A), P values are determined by Student's t-testC, D Representative H&E images (C) and the quantitative analysis of inflammation (D) of the intestinal tissues from animals treated by the protocol shown in (B). The inflammatory infiltrates were indicated by a yellow arrow. Scale bar, 100 µm. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).E qRT-PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test.F ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (F) P values are determined by Student's t-testG ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).H Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (H), P values are determined by Student's t-test


Figure 3A qRT - PCR analysis of levels of VEGF mRNA in HUVEC treated with Control - Fc or Spike RBD - Fc . For each group , n = 3 . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( A ) P values are determined by Paired Student ' s t - testB ELISA analysis of VEGF concentration in the supernatant from Caco - 2 cells treated with Control - Fc or Spike - Fc . For each group , n = 4 . Data are from 4 independent biological experiments . Data information : All data are shown as mean ± SD . For ( B ) , P values are determined by Paired Student ' s t - test ; C Immunofluorescence analysis of VEGF , cytokeratin ( CK ) and CD34 protein in the intestinal tissues of COVID - 19 patients . CK and CD34 staining in green , VEGF staining in red and nuclear staining in blue . Scale bars , 50 µm . D Levels of ERK and pERK in Caco - 2 cells treated with Control - Fc or Spike RBD - Fc by Western blot . Protein expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( D ) , P values are determined by Paired Student ' s t - testE ELISA analysis of VEGF concentration in the supernatant from Caco - 2 cells treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 . For each group , n = 4 . Data are from 4 independent biological experiments . Data information : All data are shown as mean ± SD . ; for ( E ) , P values are determined by Mann - Whitney test ; F , G Immunohistochemical staining shows ERK ( E ) and pERK ( F ) expression in the duodenum of COVID - 19 patients and healthy controls . Scale bars , 100 μm . For each group , n = 7 . n , biologically independent samples ( human specimen ) . Data information : All data are shown as mean ± SD . For ( F ) and ( G ) , P values are determined by Student ' s t - testH Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control - Fc , Spike RBD - Fc or Spike RBD - Fc combined with SCH772984 . Scale bar , 100 µm . For each group , n = 4 . Data information : All data are shown as mean ± SD . for ( H ) P values are determined by one - way ANOVA . I The levels of Ras , c - Raf , pMEK , ERK , pERK and p - P90RSK in Caco - 2 cells treated with Control - Fc , Spike RBD - Fc or Spike RBD - Fc combined with SCH772984 by Western blot . Protein expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . for ( I ) , P values are determined by one - way ANOVA .

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

Figure 3A qRT-PCR analysis of levels of VEGF mRNA in HUVEC treated with Control-Fc or Spike RBD-Fc. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-testB ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc or Spike-Fc. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-test;C Immunofluorescence analysis of VEGF, cytokeratin (CK) and CD34 protein in the intestinal tissues of COVID-19 patients. CK and CD34 staining in green, VEGF staining in red and nuclear staining in blue. Scale bars, 50 µm.D Levels of ERK and pERK in Caco-2 cells treated with Control-Fc or Spike RBD-Fc by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (D), P values are determined by Paired Student's t-testE ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test;F, G Immunohistochemical staining shows ERK (E) and pERK (F) expression in the duodenum of COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. For (F) and (G), P values are determined by Student's t-testH Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. Data information: All data are shown as mean ± SD. for (H) P values are determined by one-way ANOVA.I The levels of Ras, c-Raf, pMEK, ERK, pERK and p-P90RSK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. for (I), P values are determined by one-way ANOVA.


Figure 4A The normalized fluorescence intensity of TRITC - Dextran dye from coculture systems in which Caco - 2 cells were incubated with Control - Fc or Spike RBD - Fc after 24 hours . For each group , n = 3 . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( A ) P values are determined by Paired Student ' s t - testB The expression of junctional proteins ZO - 1 , VE - cadherin ( VE - cad ) , phosphorylated VE - cad ( pVE - cad ; 658 ) and pVE - cad ( 731 ) in HUVECs co - cultured with the supernatant of Caco - 2 cells that were treated with either Control - Fc or Spike RBD - Fc detected by Western blot . Protein expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( B ) , P values are determined by Paired Student ' s t - testC , D Immunohistochemical staining shows levels of VE - cad ( C ) and pVE - Cad ( 731 ) ( D ) in the duodenum tissue from COVID - 19 patients and healthy controls . Scale bars , 100 μm . For each group , n = 7 . n , biologically independent samples ( human specimen ) . Data information : All data are shown as mean ± SD . for ( C ) and ( D ) , P values are determined by Student ' s t - testE Immunofluorescence analysis of VE - cad expression and subcellular localization in Caco - 2 cells that were treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab by confocal microscopy . VE - cad staining in green and nuclear staining in blue . Scale bar , 20 µm . F Fluorescence intensity of TRITC - Dextran dye from coculture systems in which Caco - 2 cells were incubated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab . Data are from five technical replicates with similar results from three biological replicates . Data information : All data are shown as mean ± SD . ; for ( F ) , P values are determined by one - way ANOVA . G Immunohistochemical staining shows levels of VE - cad and pVE - Cad ( 731 ) in the intestinal tissues of mice treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab . Scale bar , 100 µm . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . for ( G ) , P values are determined by one - way ANOVA .

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

Figure 4A The normalized fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc or Spike RBD-Fc after 24 hours. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-testB The expression of junctional proteins ZO-1, VE-cadherin (VE-cad), phosphorylated VE-cad (pVE-cad; 658) and pVE-cad (731) in HUVECs co-cultured with the supernatant of Caco-2 cells that were treated with either Control-Fc or Spike RBD-Fc detected by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-testC, D Immunohistochemical staining shows levels of VE-cad (C) and pVE-Cad (731) (D) in the duodenum tissue from COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. for (C) and (D), P values are determined by Student's t-testE Immunofluorescence analysis of VE-cad expression and subcellular localization in Caco-2 cells that were treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab by confocal microscopy. VE-cad staining in green and nuclear staining in blue. Scale bar, 20 µm.F Fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Data are from five technical replicates with similar results from three biological replicates. Data information: All data are shown as mean ± SD. ; for (F) , P values are determined by one-way ANOVA.G Immunohistochemical staining shows levels of VE-cad and pVE-Cad (731) in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. for (G), P values are determined by one-way ANOVA.


Figure 5A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti - VEGF antibody . SCH772984 , ERK inhibitor ; Bevacizumab , anti - VEGF antibody . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . P values are determined by one - way ANOVA . B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay . For each group , n = 6 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . P values are determined by one - way ANOVA . C , D Representative H & E images ( C ) and the degrees of inflammation ( D ) of intestinal tissues from animals treated with SCH772984 or Bevacizumab . The inflammatory infiltrates were indicated by a yellow arrow . The edema area was indicated by a red star . Scale bar , 100 µm . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . P values are determined by one - way ANOVA .

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 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 5A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.C, D Representative H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


Fig 1 . ( A ) Electron micrograph of an area with abundant double - membrane spherules ( DMSs ) . DMVs ( asterisks ) are interspersed and surrounding the DMS cluster . ( B ) Slice through a tomogram ( left ) and corresponding surface - rendered model ( right ) of a representative area containing the different types of MERS - CoV - induced membrane modifications : DMSs ( orange ) , convoluted membranes ( CM , blue ) and DMVs ( yellow and lilac , outer and inner membranes ) . The model also highlights ER membranes ( green ) and a vesicle ( silver ) containing new virions ( pink ) . ( See also S1 Video ) . ( C ) Comparison of DMSs and virions ( arrowheads in left and right panels , respectively ) in enlarged views of tomographic slices from the regions boxed in ( B ) . The DMSs are similar in size but distinct in appearance from newly - formed MERS - CoV particles . ( D ) Whisker plots of the size distribution of DMSs ( n = 58 ) , virions ( n = 28 ) and DMVs ( n = 109 ) , as measured from the tomograms . DMSs and virions have a comparable size ( median diameter , 80 nm ) , while the median diameter of the DMVs is 247 nm . ( E ) Models and tomographic slices through an open ( left ) and closed ( right ) DMS . Both types of DMSs are connected with the CM . In open DMSs , both the inner and outer membranes ( dark blue and orange , respectively ) are continuous with CM . Two ~ 8 - nm apart slices through the reconstruction are shown . For closed DMSs only the outer membrane is connected to CM , while the inner membrane seems to define a closed compartment . ( F ) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS - CoV RO , and of these with the ER . These include CM - ER ( black arrowheads ) , DMV - ER ( white arrowheads ) , CM - DMV ( dark grey arrowheads ) , and CM - DMS ( light grey arrowhead ) connections . Scale bars , 250 nm ( A , B ) , and 100 nm ( C - F ) .

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

Fig 1.(A) Electron micrograph of an area with abundant double-membrane spherules (DMSs). DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two ~8-nm apart slices through the reconstruction are shown. For closed DMSs only the outer membrane is connected to CM, while the inner membrane seems to define a closed compartment.(F) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO, and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (dark grey arrowheads), and CM-DMS (light grey arrowhead) connections. Scale bars, 250 nm (A, B), and 100 nm (C -F).


Fig 2 . alpha - CoVs . 2D - EM images from 100 - nm thick sections of different mammal cells infected with ( A ) SARS - CoV ( MOI 10 , 9 hpi ) , ( B ) MHV ( MOI 10 , 8 hpi ) and ( C ) HCoV - 229E ( MOI 5 , 24 hpi ) . Both beta - CoVs ( A , B ) and the alpha - CoV ( C ) induce membrane modifications that include not only DMVs ( asterisks ) and CM , but also DMSs ( white arrowheads ) . Scale bars , 250 nm .

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

Fig 2.alpha-CoVs.2D-EM images from 100-nm thick sections of different mammal cells infected with (A) SARS-CoV (MOI 10, 9 hpi), (B) MHV (MOI 10, 8 hpi) and (C) HCoV-229E (MOI 5, 24 hpi). Both beta-CoVs (A,B) and the alpha-CoV (C) induce membrane modifications that include not only DMVs (asterisks) and CM, but also DMSs (white arrowheads). Scale bars, 250 nm.


Fig 4 . ( A ) Overview of an infected cell in which regions with different virus - induced modifications are annotated in yellow ( DMVs ) , blue ( CM ) and orange ( DMSs ) . Several densely - labelled regions containing DMVs , but not the other virus - induced structures are apparent . A close - up of one of these regions ( boxed area ) is shown in ( B ) . ( C , D ) Distribution of the autoradiography signal around DMVs ( nDMVs = 36 , see Material and Methods for selection criteria and details ) . The data is plotted ( C ) as a histogram , or ( D ) normalized by the radius to the DMV centre to account for the increase in the screened area . Scale bars , ( A ) 5 μm , ( B ) 500 nm .

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

Fig 4.(A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B).(C, D) Distribution of the autoradiography signal around DMVs (nDMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.


Fig . 3 . ( A ) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS - CoV - infected Vero E6 cells ( MOI 10 ) , as measured by scintillation counting on the RNA isolated from the cells . The label was provided simultaneously to all the samples at 6 hpi . ( B - D ) EM detection by autoradiography . ( B ) Overview of a SARS - CoV - infected Vero E6 cell ( MOI 10 ) were labelled for 20 min just before fixation at 7 hpi , and then processed for EM autoradiography . Autoradiography grains accumulate in the RO regions . N , nucleus ; m , mitochondria ; VCR , virion - containing regions ; LD , lipid droplets . Scale bar , 1 μm . ( C , D ) Quantification of the autoradiography signal per subcellular structure ( see also S1 Table ) . Labelling densities and relative labelling indexes ( RLI ) in different subcellular regions of ( C ) Vero E6 cells infected with SARS - CoV ( MOI 10 ) or ( D ) Huh7 cells infected with MERS - CoV ( MOI 5 ) . Radioactively - labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi , respectively . Control mock - infected cells are excluded from the RLI plots , as RLI comparisons between conditions require the same number of classes ( subcellular regions ) and these cells lack ROs and virions .

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

Fig. 3.(A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi.(B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10) were labelled for 20 min just before fixation at 7 hpi, and then processed for EM autoradiography. Autoradiography grains accumulate in the RO regions. N, nucleus; m, mitochondria; VCR, virion-containing regions; LD, lipid droplets. Scale bar, 1 μm.(C, D) Quantification of the autoradiography signal per subcellular structure (see also S1 Table). Labelling densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions.


Fig . 5 . ( A ) Overview of a cluster of MERS - CoV - induced membrane modifications in Huh7 cells prepared as described in Fig . 4 . Some DMSs are boxed in orange while regions with CM are encircled in blue . In comparison with the densely - labelled surrounding DMVs , these regions are relatively devoid of autoradiography signal . ( B ) The distribution of autoradiography grains on CM was not homogeneous ( nCM = 9 ) , and label was predominantly found close to the boundaries of the CM , as expected if the signal arises from the surrounding DMVs . ( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . ( C ) Enlargements of the DMS areas boxed in ( A ) . ( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . Most DMSs were devoid of signal and those who contained label were close to labelled DMVs ( D ) ( nDMS = 105 ) . ( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . ( E ) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre , as expected from a random distribution ( nDMSs = 54 ) . Scale bars , ( A ) 500 nm , ( C ) 100 nm .

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

Fig. 5.(A) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig. 4. Some DMSs are boxed in orange while regions with CM are encircled in blue. In comparison with the densely-labelled surrounding DMVs, these regions are relatively devoid of autoradiography signal.(B) The distribution of autoradiography grains on CM was not homogeneous (nCM=9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs.(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A).(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). Most DMSs were devoid of signal and those who contained label were close to labelled DMVs (D) (nDMS = 105).(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre, as expected from a random distribution (nDMSs = 54). Scale bars, (A) 500 nm, (C) 100 nm.


Fig . 6 . IEM detection of viral markers in MERS - CoV - infected cells . ( A - G ) Immunogold labeling of thawed cryo - sections of MERS - CoV - infected Huh7 cells ( 12 hpi ) for the detection of the indicated viral proteins . ( A - C ) Structural proteins were detected on virions ( black arrowheads ) and , for the M and S proteins , also on Golgi cisterna . While regions containing DMS ( white arrowheads ) and CM labelled for the N protein ( D ) and nsp3 ( G ) , the M and S protein were not detected in these areas . ( H - I ) Immunogold labeling of dsRNA in HPF - FS samples of MERS - CoV - infected Huh7 cells ( 13 hpi ) . The label accumulated on DMVs , which could be easily detected in this type of samples ( grey arrowheads ) , while the regions with CM and DMSs , which appeared as dark areas among the DMV clusters , where devoid of dsRNA signal . G , Golgi complex ; m , mitochondria . Scale bars , 250 nm .

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Fig. 6. IEM detection of viral markers in MERS-CoV-infected cells.(A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.


Figure 1FLMycER cells , primed or not with OHT ( 48h ) , were treated with 135 nm IACS - 010759 ( IACS ) for 48 hours ( B ) H2O2 quantification , expressed as fold - increase of the 405 / 488 nm fluorescence ratio in treated vs . untreated FLMycER cells , expressing either the cytoplasmic ( left ) or mitochondrial ( right ) roGFP2 - ORP1 biosensor . Data information : * p ≤ 0 . 05 ( one - way ANOVA ) . Each point in the graphs in B from an independent biological replicate , each representing the average of thousands of events ( single cells ) in a distinct cell population , normalized to the untreated condition . FLMycER cells , primed or not with OHT ( 48h ) , were treated with 135 nm IACS - 010759 ( IACS ) for 48 hours ( C ) Superoxide anion O2 • - production in treated vs . untreated FLMycER cells , based on dihydroethidium staining . Data information : * p ≤ 0 . 05 ( one - way ANOVA ) . Each point in the graphs in B and C is from an independent biological replicate , each representing the average of thousands of events ( single cells ) in a distinct cell population , normalized to the untreated condition .

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

Figure 1FLMycER cells, primed or not with OHT (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (B) H2O2 quantification, expressed as fold-increase of the 405/488 nm fluorescence ratio in treated vs. untreated FLMycER cells, expressing either the cytoplasmic (left) or mitochondrial (right) roGFP2-ORP1 biosensor. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.FLMycER cells, primed or not with OHT (48h), were treated with 135 nm IACS-010759 (IACS) for 48 hours (C) Superoxide anion O2•- production in treated vs. untreated FLMycER cells, based on dihydroethidium staining. Data information: * p ≤ 0.05 (one-way ANOVA). Each point in the graphs in B and C is from an independent biological replicate, each representing the average of thousands of events (single cells) in a distinct cell population, normalized to the untreated condition.


Figure 2FLMycER cells were primed with100 nM OHT ( 48h ) and / or treated with 135nM IACS - 010759 , as indicated . ( B ) glutathione quantification in FLMycER cells , measured after 40 hours of IACS - 010759 treatment . Data information : n = 3 biological replicates ; error bars : SD . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ( one - way ANOVA ) . ( C ) Cell viability , as determined by PI staining after 48 hours of IACS - 010759 treatment , with or without addition of 10 mM N - acetyl - cysteine ( NAC ) . ( D ) Same as ( C ) , with 50 µM buthionine - sulfoximine ( BSO ) . Data information : n = 3 biological replicates ; error bars : SD . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ( one - way ANOVA ) . ( F ) Immunoblot on lysates from FLMycER cells treated as indicated , with the addition of 10 mM NAC concomitantly with IACS - 010759 .

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Figure 2FLMycER cells were primed with100 nM OHT (48h) and/or treated with 135nM IACS-010759, as indicated. (B) glutathione quantification in FLMycER cells, measured after 40 hours of IACS-010759 treatment. Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).(C) Cell viability, as determined by PI staining after 48 hours of IACS-010759 treatment, with or without addition of 10 mM N-acetyl-cysteine (NAC). (D) Same as (C), with 50 µM buthionine-sulfoximine (BSO). Data information: n=3 biological replicates; error bars: SD. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 (one-way ANOVA).(F) Immunoblot on lysates from FLMycER cells treated as indicated, with the addition of 10 mM NAC concomitantly with IACS-010759.


Figure 3FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS - 010759 , as indicated . ( B ) NADPH / NADP redox state in FLMycER cells , assayed after 40 hours of IACS - 010759 treatment . n = 3 biological replicates . * * p ≤ 0 . 01 ; * * * * p ≤ 0 . 0001 ( one - way ANOVA ) . FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS - 010759 , as indicated . ( E - G ) Cell viability after 40 hours of IACS - 010759 treatment in the presence of ( E ) 50 µM dehydroepiandrosterone ( DHEA ) , ( F ) 10 µM 6 - aminonicotinamide ( 6AN ) or ( G ) 1 mM 2 - deoxyglucose ( 2DG ) . See Supplemental Figure3F for detailed statistical analysis . Data information : In E , G ) n = 6 ; in ( F ) n = 9 ; all biological replicates ; error bars : SD .

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Figure 3FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS-010759, as indicated. (B) NADPH/NADP redox state in FLMycER cells, assayed after 40 hours of IACS-010759 treatment. n=3 biological replicates. ** p ≤ 0.01; **** p ≤ 0.0001 (one-way ANOVA).FLMycER cells were primed with 100 nM OHT and treated with 135 nM IACS-010759, as indicated. (E-G) Cell viability after 40 hours of IACS-010759 treatment in the presence of (E) 50 µM dehydroepiandrosterone (DHEA), (F) 10 µM 6- aminonicotinamide (6AN) or (G) 1 mM 2-deoxyglucose (2DG). See Supplemental Figure3F for detailed statistical analysis. Data information: In E,G) n=6; in (F) n=9; all biological replicates; error bars: SD.


Figure 4 ( A ) Viability of FLMycER and BaFMycER cells primed or not with 100 nM OHT and treated with 135 nM IACS - 010759 for 48h and / or with ascorbate at the indicated concentration for 6h . n = 3 biological replicates ; error bars : SD . ( C ) Cell viability of FL5 . 12cells at the end of treatment ( 48h IACS - 010759 , 6h Asc ) , in the presence or absence of 50 µM deferoxamine ( DFX , added 1 hour before Asc ) . n = 3 biological replicates ; error bars : SD . ( D ) Quantification of lipid peroxides in FL5 . 12 cells treated with 135 nM IACS - 010759 for 24h and / or 400 µM Asc for 3 hours . * * p ≤ 0 . 01 ; * p ≤ 0 . 05 ( one - way ANOVA ) .

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Figure 4(A) Viability of FLMycER and BaFMycER cells primed or not with 100 nM OHT and treated with 135 nM IACS-010759 for 48h and/or with ascorbate at the indicated concentration for 6h. n=3 biological replicates; error bars: SD.(C) Cell viability of FL5.12cells at the end of treatment (48h IACS-010759,6h Asc), in the presence or absence of 50 µM deferoxamine (DFX, added 1 hour before Asc). n=3 biological replicates; error bars: SD.(D) Quantification of lipid peroxides in FL5.12 cells treated with 135 nM IACS-010759 for 24h and/ or 400 µM Asc for 3 hours. ** p ≤ 0.01; * p ≤ 0.05 (one-way ANOVA).


Figure 5The DLBCL cell lines DoHH2 , SU - DHL - 6 , Karpas 422 and HBL - 1 were treated with the indicated concentrations of IACS - 010759 and ascorbate , and cell viability determined after 24 hours . ( A ) Live cell counts . n = 3 biological replicates ; error bars : SD .

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

Figure 5The DLBCL cell lines DoHH2, SU-DHL-6, Karpas 422 and HBL-1 were treated with the indicated concentrations of IACS-010759 and ascorbate, and cell viability determined after 24 hours. (A) Live cell counts. n=3 biological replicates; error bars: SD.


Figure 6The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS - 010759 ( IACS ) by oral gavage and / or ascorbate ( Asc ) by intraperitoneal injection , as indicated ( see Materials and methods ) . Daily doses and the number of animals per group ( n ) are indicated in parenthesis . ( A , B ) Tumor progression ( mm3 ) in CD1 nude mice bearing subcutaneous Ramos ( A ) or DoHH2 tumors ( B ) . Data information : In each panel , the plot on the right shows the data for individual groups on the indicated day . Error bars : SD ; n = animals per group . Right : one - way between - group ANOVA . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 . The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS - 010759 ( IACS ) by oral gavage and / or ascorbate ( Asc ) by intraperitoneal injection , as indicated ( see Materials and methods ) . Daily doses and the number of animals per group ( n ) are indicated in parenthesis . ( C , D ) Tumor progression in NSG mice injected with the luciferase - positive DHL - derived PDX lines DFBL - 20954 ( C ) or DFBL - 69487 ( D ) , as determined by in vivo imaging and quantification of bilateral femur radiance efficiency . Examples from the control and double - treated groups are shown at the bottom . Data information : In each panel , the plot on the right shows the data for individual groups on the indicated day . Error bars : SD ; n = animals per group . Right : one - way between - group ANOVA . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (A, B) Tumor progression (mm3) in CD1 nude mice bearing subcutaneous Ramos (A) or DoHH2 tumors (B). Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.The indicated lymphoma cell lines and PDX samples were xenografted in recipient animals and tumors allowed to develop to detectable sizes prior to randomization and treatment with IACS-010759 (IACS) by oral gavage and/or ascorbate (Asc) by intraperitoneal injection, as indicated (see Materials and methods). Daily doses and the number of animals per group (n) are indicated in parenthesis. (C, D) Tumor progression in NSG mice injected with the luciferase-positive DHL-derived PDX lines DFBL-20954 (C) or DFBL-69487 (D), as determined by in vivo imaging and quantification of bilateral femur radiance efficiency. Examples from the control and double-treated groups are shown at the bottom. Data information: In each panel, the plot on the right shows the data for individual groups on the indicated day. Error bars: SD; n = animals per group. Right: one-way between-group ANOVA. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.


Figure 1A Immunoblot of PPAR - α and Tubulin ( control ) ( top ) in WAT from WT and FADD - D mice with relative quantification ( bottom ) . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0035 ( Student ' s t - test ) . B Immunostaining of PPAR - α expression in WAT from WT and FADD - D mice ( Scale bars = 50 μm ) . Shown are typical results from four different fields and three different experiments . C Quantitative real time PCR for PPAR - α and PPAR - α target genes , using RNA from epididymalfat from 10 - week - old male WT and FADD - D mice fed a SD . Data are expressed as mean ± SEM from two independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0022 , * * P = 0 . 0083 , * * * P = 0 . 0107 , # P = 0 . 0011 , # # P = 0 . 0037 ( Student ' s t - test ) . D ChIP assay of CPT1b promoter in adipocytes isolated from WT , FADD - D and ad - FADDmice . Soluble chromatin from adipocytes was immunoprecipitated with control mouse IgG or antibodies against PPAR - α . The experiments were performed in triplicate . E ChIP assay of CPT1b promoter in adipocytes isolated from WT and FADD - D mice . Soluble chromatin from adipocytes was immunoprecipitated with control mouse IgG or antibodies against FADD . The experiments were performed in triplicate . F PPAR - α and RIP140coimmunoprecipitate in WT but not FADD knockout or FADD - D MEFs . PPAR - α was immunoprecipitated ( IP ) from the cell lysates of WT or FADD knockout MEFs . Data shown are representative of three independent experiments having similar results . G PPAR - α and RIP140coimmunoprecipitate in WT but not FADD knockout MEFs upon WY - 14 , 643 stimulation . Ligand - dependent interactions were examined in the presence of 100 μM WY - 14 , 643 where indicated . Data shown are representative of three independent experiments having similar results . H Coimmunoprecipitation analysis of FADD and RIP140 in 293T cells . As indicated , lysates from the cells transfected with plasmids encoding Flag - RIP140 , HA - FADD - A or HA - FADD - D were subjected to immunoprecipitation with an anti - Flag antibody . FADD - A and FADD - D were detected using an anti - HA antibody . Data shown are representative of three independent experiments having similar results . I Coimmunoprecipitation analysis of FADD and PPAR - α in 293T cells . As indicated , lysates from the cells transfected with plasmids encoding Flag - FADD - A , Flag - FADD - D or HA - PPAR - α were subjected to immunoprecipitation with an anti - Flag antibody . PPAR - α was visualized by anti - HA . Data shown are representative of three independent experiments having similar results .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 1A Immunoblot of PPAR-α and Tubulin (control) (top) in WAT from WT and FADD-D mice with relative quantification (bottom). Data are expressed as mean ± SEM from three independent experiments. *P = 0.0035 (Student's t-test).B Immunostaining of PPAR-α expression in WAT from WT and FADD-D mice (Scale bars = 50 μm). Shown are typical results from four different fields and three different experiments.C Quantitative real time PCR for PPAR-α and PPAR-α target genes, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice fed a SD. Data are expressed as mean ± SEM from two independent experiments (n = 6 total for each genotype). *P = 0.0022, **P = 0.0083, ***P = 0.0107, #P = 0.0011, ##P = 0.0037 (Student's t-test).D ChIP assay of CPT1b promoter in adipocytes isolated from WT, FADD-D and ad-FADDmice. Soluble chromatin from adipocytes was immunoprecipitated with control mouse IgG or antibodies against PPAR-α. The experiments were performed in triplicate.E ChIP assay of CPT1b promoter in adipocytes isolated from WT and FADD-D mice. Soluble chromatin from adipocytes was immunoprecipitated with control mouse IgG or antibodies against FADD. The experiments were performed in triplicate.F PPAR-α and RIP140coimmunoprecipitate in WT but not FADD knockout or FADD-D MEFs. PPAR-α was immunoprecipitated (IP) from the cell lysates of WT or FADD knockout MEFs. Data shown are representative of three independent experiments having similar results.G PPAR-α and RIP140coimmunoprecipitate in WT but not FADD knockout MEFs upon WY-14,643 stimulation. Ligand-dependent interactions were examined in the presence of 100 μM WY-14,643 where indicated. Data shown are representative of three independent experiments having similar results.H Coimmunoprecipitation analysis of FADD and RIP140 in 293T cells. As indicated, lysates from the cells transfected with plasmids encoding Flag-RIP140, HA-FADD-A or HA-FADD-D were subjected to immunoprecipitation with an anti-Flag antibody. FADD-A and FADD-D were detected using an anti-HA antibody. Data shown are representative of three independent experiments having similar results.I Coimmunoprecipitation analysis of FADD and PPAR-α in 293T cells. As indicated, lysates from the cells transfected with plasmids encoding Flag-FADD-A, Flag-FADD-D or HA-PPAR-α were subjected to immunoprecipitation with an anti-Flag antibody. PPAR-α was visualized by anti-HA. Data shown are representative of three independent experiments having similar results.


Figure 2A Left , Body weights of male WT and FADD - D mice on either a SD ( n = 8 for each genotype ) or a HFD ( n = 6 for each genotype ) . Right , Food intake per mouse fed a HFD measured over 20 days normalized by body weight ( n = 10 for each genotype ) . Results are means ± SEM . * P = 0 . 0007 ( Student ' s t - test ) . B Representative photographs of fat pads and organs of 10 - week - old male FADD - D and WT mice . C Left , fat pad weights as a percentage of body weight ( BW ) and in absolute amounts ( inset ) from male FADD - D and WT mice . Mice were fed a SD or a HFD until 15 weeks of age ( n = 6 for each genotype ) . Results are means ± SEM . * P = 0 . 0012 , * * P = 0 . 0002 , * * * P = 0 . 0027 , * * * * P = 0 . 0014 , ^ P = 0 . 0038 , ^ ^ P = 0 . 0019 , # P = 0 . 0045 , # # P = 0 . 0007 , * ^ P = 0 . 0252 , * ^ ^ P = 0 . 0082 , # # # P = 0 . 0275 , # # # # P = 0 . 0071 ( one - way ANOVA ) . Middle , fat pad weights of male FADD - D and WT mice on a HFD at 30 weeks of age ( n = 6 for each genotype ) . Inset , triacylglycerol ( TAG ) content in epididymal WAT . Data are expressed as means ± SEM . * P = 0 . 0003 , * * P = 0 . 0005 , * * * P = 0 . 0019 , * * * * P = 0 . 0011 ( Student ' s t - test ) . Right , weight of liver normalized by body weight for male FADD - D and WT littermates at the age of 15 weeks ( n = 6 for each genotype ) . Data are expressed as means ± SEM . NS , not statistically significant ( Student ' s t - test ) . Epi , epididymal fat ; Ren , renal fat ; Ing , inguinal fat . C Left , fat pad weights as a percentage of body weight ( BW ) and in absolute amounts ( inset ) from male FADD - D and WT mice . Mice were fed a SD or a HFD until 15 weeks of age ( n = 6 for each genotype ) . Results are means ± SEM . * P = 0 . 0012 , * * P = 0 . 0002 , * * * P = 0 . 0027 , * * * * P = 0 . 0014 , ^ P = 0 . 0038 , ^ ^ P = 0 . 0019 , # P = 0 . 0045 , # # P = 0 . 0007 , * ^ P = 0 . 0252 , * ^ ^ P = 0 . 0082 , # # # P = 0 . 0275 , # # # # P = 0 . 0071 ( one - way ANOVA ) . Middle , fat pad weights of male FADD - D and WT mice on a HFD at 30 weeks of age ( n = 6 for each genotype ) . Inset , triacylglycerol ( TAG ) content in epididymal WAT . Data are expressed as means ± SEM . * P = 0 . 0003 , * * P = 0 . 0005 , * * * P = 0 . 0019 , * * * * P = 0 . 0011 ( Student ' s t - test ) . Right , weight of liver normalized by body weight for male FADD - D and WT littermates at the age of 15 weeks ( n = 6 for each genotype ) . Data are expressed as means ± SEM . NS , not statistically significant ( Student ' s t - test ) . Epi , epididymal fat ; Ren , renal fat ; Ing , inguinal fat . D Weight of brown adipose tissue normalized by body weight ( n = 5 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0008 ( Student ' s t - test ) . E MRI of body fat content : standard non - fat - suppressed ( left ) , fat - suppressed ( middle ) and fat - only ( right ) . Representative images of three independent experiments ( n = 6 total for each genotype ) showing similar results are shown . F Paraffin - embedded sections of WAT and BAT from WT and FADD - D mice were stained with H & amp ; amp ; E . Scale bar = 50 μm . Shown are typical results from four different fields and three different experiments . G Left , relative amounts of liver and muscle triacylglycerol contents in 10 - month - old WT and FADD - D mice ( n = 6 for each genotype ) . Right , relative amounts of cholesterol in the liver and muscle of the same group of WT and FADD - D mice ( n = 6 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0015 , * * P = 0 . 0027 , * * * P = 0 . 0318 ( Student ' s t - test ) . NS , not statistically significant . H Left , plasma triacylglycerol levels in 10 - month - old WT and FADD - D mice following fasting for 20 h ( n = 5 for each genotype ) . Right , plasma cholesterol levels in the same group of WT and FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0124 ( Student ' s t - test ) . NS , not statistically significant .

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

Figure 2A Left, Body weights of male WT and FADD-D mice on either a SD (n = 8 for each genotype) or a HFD (n = 6 for each genotype). Right, Food intake per mouse fed a HFD measured over 20 days normalized by body weight (n = 10 for each genotype). Results are means ± SEM. *P = 0.0007 (Student's t-test).B Representative photographs of fat pads and organs of 10-week-old male FADD-D and WT mice.C Left, fat pad weights as a percentage of body weight (BW) and in absolute amounts (inset) from male FADD-D and WT mice. Mice were fed a SD or a HFD until 15 weeks of age (n = 6 for each genotype). Results are means ± SEM. *P = 0.0012, **P = 0.0002, ***P = 0.0027, ****P = 0.0014, ^P = 0.0038, ^^P = 0.0019, #P = 0.0045, ##P = 0.0007, *^P = 0.0252, *^^P = 0.0082, ###P = 0.0275, ####P = 0.0071 (one-way ANOVA). Middle, fat pad weights of male FADD-D and WT mice on a HFD at 30 weeks of age (n = 6 for each genotype). Inset, triacylglycerol (TAG) content in epididymal WAT. Data are expressed as means ± SEM. *P = 0.0003, **P = 0.0005, ***P = 0.0019, ****P = 0.0011 (Student's t-test). Right, weight of liver normalized by body weight for male FADD-D and WT littermates at the age of 15 weeks (n = 6 for each genotype). Data are expressed as means ± SEM. NS, not statistically significant (Student's t-test). Epi, epididymal fat; Ren, renal fat; Ing, inguinal fat.C Left, fat pad weights as a percentage of body weight (BW) and in absolute amounts (inset) from male FADD-D and WT mice. Mice were fed a SD or a HFD until 15 weeks of age (n = 6 for each genotype). Results are means ± SEM. *P = 0.0012, **P = 0.0002, ***P = 0.0027, ****P = 0.0014, ^P = 0.0038, ^^P = 0.0019, #P = 0.0045, ##P = 0.0007, *^P = 0.0252, *^^P = 0.0082, ###P = 0.0275, ####P = 0.0071 (one-way ANOVA). Middle, fat pad weights of male FADD-D and WT mice on a HFD at 30 weeks of age (n = 6 for each genotype). Inset, triacylglycerol (TAG) content in epididymal WAT. Data are expressed as means ± SEM. *P = 0.0003, **P = 0.0005, ***P = 0.0019, ****P = 0.0011 (Student's t-test). Right, weight of liver normalized by body weight for male FADD-D and WT littermates at the age of 15 weeks (n = 6 for each genotype). Data are expressed as means ± SEM. NS, not statistically significant (Student's t-test). Epi, epididymal fat; Ren, renal fat; Ing, inguinal fat.D Weight of brown adipose tissue normalized by body weight (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0008 (Student's t-test).E MRI of body fat content: standard non-fat-suppressed (left), fat-suppressed (middle) and fat-only (right). Representative images of three independent experiments (n = 6 total for each genotype) showing similar results are shown.F Paraffin-embedded sections of WAT and BAT from WT and FADD-D mice were stained with H&amp;E. Scale bar = 50 μm. Shown are typical results from four different fields and three different experiments.G Left, relative amounts of liver and muscle triacylglycerol contents in 10-month-old WT and FADD-D mice (n = 6 for each genotype). Right, relative amounts of cholesterol in the liver and muscle of the same group of WT and FADD-D mice (n = 6 for each genotype). Data are expressed as means ± SEM. *P = 0.0015, **P = 0.0027, ***P = 0.0318 (Student's t-test). NS, not statistically significant.H Left, plasma triacylglycerol levels in 10-month-old WT and FADD-D mice following fasting for 20 h (n = 5 for each genotype). Right, plasma cholesterol levels in the same group of WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0124 (Student's t-test). NS, not statistically significant.


Figure 3A Serum free fatty acid ( FFA ) levels and triacylglycerol levels in WT and FADD - D mice fed a HFD for 20 days ( n = 5 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0059 , * * P = 0 . 0012 ( Student ' s t - test ) . B Serum levels of leptin in WT and FADD - D mice ( n = 4 for each genotype ) fed a SD or a HFD . Data are expressed as means ± SEM . * P = 0 . 0032 , * * P = 0 . 0003 , * * * P = 0 . 0038 ( one - way ANOVA ) . C Morphology of inguinal WAT , BAT , and liver from WT and FADD - D mice fed a HFD for 20 days . WAT and BAT were stained with hematoxylin and eosin . Liver tissues were stained with Oil red O to demonstrate lipid accumulation and counterstained with hematoxylin . Scale bars = 50 μm . Shown are typical results from four different fields and three different experiments . D Comparison of cell size in WAT of WT and FADD - D mice maintained on SD and HFD ( n = 5 for each genotype ) . E FADD - D mice lost more weight as compared with WT on 20 - h fasting ( n = 4 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0085 ( Student ' s t - test ) . F Whole - body oxygen consumption rate ( VO2 ) during 24 hr in WT and FADD - D mice fed a SD ( n = 4 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0055 , * * P = 0 . 0081 ( Student ' s t - test ) . G Average respiratory exchange ratio ( VCO2 / VO2 ) ( RER ) for the dark and light period in WT and FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0042 , * * P = 0 . 0079 ( Student ' s t - test ) . H Rectal temperature ( n = 5 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0217 ( Student ' s t - test ) . I Average values of energy expenditure ( EE ) for the 24 hr period in WT and FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± SEM . * P = 0 . 0054 ( Student ' s t - test ) . J Physical activity for the dark and light period in WT and FADD - D mice ( n = 5 for each genotype ) . Data are expressed as means ± SEM . NS , not statistically significant ( Student ' s t - test ) .

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

Figure 3A Serum free fatty acid (FFA) levels and triacylglycerol levels in WT and FADD-D mice fed a HFD for 20 days (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0059, **P = 0.0012 (Student's t-test).B Serum levels of leptin in WT and FADD-D mice (n = 4 for each genotype) fed a SD or a HFD. Data are expressed as means ± SEM. *P = 0.0032, **P = 0.0003, ***P = 0.0038 (one-way ANOVA).C Morphology of inguinal WAT, BAT, and liver from WT and FADD-D mice fed a HFD for 20 days. WAT and BAT were stained with hematoxylin and eosin. Liver tissues were stained with Oil red O to demonstrate lipid accumulation and counterstained with hematoxylin. Scale bars = 50 μm. Shown are typical results from four different fields and three different experiments.D Comparison of cell size in WAT of WT and FADD-D mice maintained on SD and HFD (n = 5 for each genotype).E FADD-D mice lost more weight as compared with WT on 20-h fasting (n = 4 for each genotype). Data are expressed as means ± SEM. *P = 0.0085 (Student's t-test).F Whole-body oxygen consumption rate (VO2) during 24 hr in WT and FADD-D mice fed a SD (n = 4 for each genotype). Data are expressed as means ± SEM. *P = 0.0055, **P = 0.0081 (Student's t-test).G Average respiratory exchange ratio (VCO2/VO2) (RER) for the dark and light period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0042, **P = 0.0079 (Student's t-test).H Rectal temperature (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0217 (Student's t-test).I Average values of energy expenditure (EE) for the 24 hr period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. *P = 0.0054 (Student's t-test).J Physical activity for the dark and light period in WT and FADD-D mice (n = 5 for each genotype). Data are expressed as means ± SEM. NS, not statistically significant (Student's t-test).


Figure 4A Left , transmission electron microscopy analysis of adipocytes from WAT and BAT of WT and FADD - D mice . Arrowheads indicate mitochondria and lipid droplets ( L ) ( × 5 , 000 magnification ) . Right , quantification of mitochondria in WAT and BAT of WT and FADD - D mice , respectively . Data are expressed as mean ± SEM from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0007 , * * P = 0 . 0083 ( Student ' s t - test ) . B Quantitative real time PCR for UCP1 , Dio2 , PPARd , UCP3 , CPT1 and PGC1 - α , using RNA from epididymalfat from 10 - week - old male WT and FADD - D mice on a SD . Data are expressed as mean ± SEM from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0014 , * * P = 0 . 0025 , * * * P = 0 . 0006 , * * * * P = 0 . 0007 , # P = 0 . 0003 , # # P = 0 . 0008 ( Student ' s t - test ) . C Quantitative real time PCR for UCP3 , CPT1 , PPARd and PGC1 - α , using RNA from skeletal muscle from 10 - week - old male WT and FADD - D mice on a SD . Data are expressed as mean ± SEM from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0027 , * * P = 0 . 0329 , * * * P = 0 . 0092 , * * * * P = 0 . 0085 ( Student ' s t - test ) . D Quantitative real time PCR for UCP1 , using RNA from BAT from 10 - week - old male WT and FADD - D mice on a SD . Data are expressed as mean ± SEM from three independent experiments ( n = 6 total for each genotype ) . * P = 0 . 0018 ( Student ' s t - test ) . E Immunoblotting of UCP1 and Tubulin ( control ) ( top ) with relative quantification ( bottom ) , using total cell lysates of BAT from 10 - week - old male WT and FADD - D mice on a SD . Data are expressed as mean ± SEM from three independent experiments . * P = 0 . 0217 ( Student ' s t - test ) . F Oxygen consumption in isolated BAT from 10 - week - old male WT and FADD - D mice on a SD ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0025 , * * P = 0 . 0006 ( Student ' s t - test ) . G Quantitative real time PCR for PGC1α , C / EBPα and PPARγ , using RNA from BAT from 10 - week - old male WT and FADD - D mice on a SD . Data are expressed as mean ± SEM from two independent experiments ( n = 4 total for each genotype ) . * P = 0 . 0113 ( Student ' s t - test ) . NS , not statistically significant . H β - oxidation analysis of white adipocytes isolated from WT ( n = 5 ) and FADD - D mice ( n = 4 ) . 1 - 14C labeled oleic acid was added to the medium containing isolated adipocytes ( 5 × 105 ) , and 14CO2 trapped by the filter paper soaked with hyamine hydroxide was measured after a 3 h - incubation by a scintillation counter . Data are expressed as mean ± SEM . * P = 0 . 0022 ( Student ' s t - test ) . I Basal and stimulated ( + 100 nM isoproterenol ) lipolysis , as measured by glycerol ( left ) and fatty acids ( right ) released from explants of epididymal WAT from overnight fasted 10 - week - old male WT and FADD - D mice fed a HFD ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0078 , * * P = 0 . 0065 , * * * P = 0 . 0008 , ^ P = 0 . 0133 , ^ ^ P = 0 . 0053 , ^ ^ ^ P = 0 . 0006 , ^ ^ ^ ^ P = 0 . 0001 , # P = 0 . 0082 , # # P = 0 . 0051 , # # # P = 0 . 0009 , * ^ P = 0 . 0151 , * ^ ^ P = 0 . 0019 , * * ^ ^ P = 0 . 0005 , * * * ^ P = 0 . 0002 ( Student ' s t - test ) . J Molar ratio of FFA to glycerol release from WAT explants ( n = 4 for each genotype ) . Data are expressed as mean ± SEM . * P = 0 . 0412 , * * P = 0 . 0033 ( Student ' s t - test ) .

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

Figure 4A Left, transmission electron microscopy analysis of adipocytes from WAT and BAT of WT and FADD-D mice. Arrowheads indicate mitochondria and lipid droplets (L) (× 5,000 magnification). Right, quantification of mitochondria in WAT and BAT of WT and FADD-D mice, respectively. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0007, **P = 0.0083 (Student's t-test).B Quantitative real time PCR for UCP1, Dio2, PPARd, UCP3, CPT1 and PGC1-α, using RNA from epididymalfat from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0014, **P = 0.0025, ***P = 0.0006, ****P = 0.0007, #P = 0.0003, ##P = 0.0008 (Student's t-test).C Quantitative real time PCR for UCP3, CPT1, PPARd and PGC1-α, using RNA from skeletal muscle from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0027, **P = 0.0329, ***P = 0.0092, ****P = 0.0085 (Student's t-test).D Quantitative real time PCR for UCP1, using RNA from BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments (n = 6 total for each genotype). *P = 0.0018 (Student's t-test).E Immunoblotting of UCP1 and Tubulin (control) (top) with relative quantification (bottom), using total cell lysates of BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from three independent experiments. *P = 0.0217 (Student's t-test).F Oxygen consumption in isolated BAT from 10-week-old male WT and FADD-D mice on a SD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0025, **P = 0.0006 (Student's t-test).G Quantitative real time PCR for PGC1α, C/EBPα and PPARγ, using RNA from BAT from 10-week-old male WT and FADD-D mice on a SD. Data are expressed as mean ± SEM from two independent experiments (n = 4 total for each genotype). *P = 0.0113 (Student's t-test). NS, not statistically significant.H β-oxidation analysis of white adipocytes isolated from WT (n = 5) and FADD-D mice (n = 4). 1-14C labeled oleic acid was added to the medium containing isolated adipocytes (5×105), and 14CO2 trapped by the filter paper soaked with hyamine hydroxide was measured after a 3 h-incubation by a scintillation counter. Data are expressed as mean ± SEM. *P = 0.0022 (Student's t-test).I Basal and stimulated (+ 100 nM isoproterenol) lipolysis, as measured by glycerol (left) and fatty acids (right) released from explants of epididymal WAT from overnight fasted 10-week-old male WT and FADD-D mice fed a HFD (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0078, **P = 0.0065, ***P = 0.0008, ^P = 0.0133, ^^P = 0.0053, ^^^P = 0.0006, ^^^^P = 0.0001, #P = 0.0082, ##P = 0.0051, ###P = 0.0009, *^P = 0.0151, *^^P = 0.0019, **^^P = 0.0005, ***^P = 0.0002 (Student's t-test).J Molar ratio of FFA to glycerol release from WAT explants (n = 4 for each genotype). Data are expressed as mean ± SEM. *P = 0.0412, **P = 0.0033 (Student's t-test).