Figure 8 ( A ) Mouse forebrain Aβ40 levels in homozygous male and female APOE4 - TR mice with and without CNP520 treatment , 4 hours after dosing , shown are mean ± SEM , n = 6 / group . ( B ) Distribution of APOE4 genotypes in the Phase IIa study , vs their baseline Aβ42 / 40 ratios . The dashed line at Aβ42 / 40 ratio of 0 . 09 represents a cutoff , which separates a normal ratio from an abnormally low ratio . ( C ) Comparison of CSF Aβ40 reduction in APOE4 non - carriers and APOE4 carriers . ( D ) Comparison of CSFAβ40 reduction in participants with Aβ42 / 40 ≥ 0 . 09 and Aβ42 / 40 < 0 . 09 . ( E ) Comparison of Aβ42 / 40 ratio at baseline and at day 91 for all participants in the placebo , 35 mg , and 85 mg dose group . ( F ) Change of Aβ42 / 40 ratio for all participants with Aβ42 / 40 ≤ 0 . 09 at baseline ; bars represent mean ± SD . Full circles : APOE4 carrier , open circles : APOE4 non - carrier .

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Figure 8(A) Mouse forebrain Aβ40 levels in homozygous male and female APOE4-TR mice with and without CNP520 treatment, 4 hours after dosing, shown are mean ± SEM, n = 6/group.(B) Distribution of APOE4 genotypes in the Phase IIa study, vs their baseline Aβ42/40 ratios. The dashed line at Aβ42/40 ratio of 0.09 represents a cutoff, which separates a normal ratio from an abnormally low ratio.(C) Comparison of CSF Aβ40 reduction in APOE4 non-carriers and APOE4 carriers.(D) Comparison of CSFAβ40 reduction in participants with Aβ42/40 ≥ 0.09 and Aβ42/40 < 0.09.(E) Comparison of Aβ42/40 ratio at baseline and at day 91 for all participants in the placebo, 35 mg, and 85 mg dose group.(F) Change of Aβ42/40 ratio for all participants with Aβ42/40 ≤ 0.09 at baseline; bars represent mean ± SD. Full circles: APOE4 carrier, open circles: APOE4 non-carrier.


Figure 1293T cells were transfected with a plasmid encoding FLAG - Stx17 wild type ( WT ) or the indicated constructs . At 24 h after transfection , cell lysates were immunoprecipitated ( IP ) with anti - FLAG M2 beads , and analyzed by IB using antibodies against PGAM5 and FLAG . Five percent of lysates was analyzed as input . HeLa cells stably expressing FLAG - Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with WT ( paired Student ' s t - test ) . MBP or the MBP - Stx17 constructs attached to amylose resin were mixed with GST - PGAM5 , and the proteins bound to the resin were separated by SDS - PAGE and blotted onto PVDF membranes . The blots were detected by an anti - GST antibody ( upper panels ) or stained with Coomassie Brilliant Blue R - 250 ( lower panels ) . Ten percent of the proteins used for each experiment was analyzed as input . Asterisks and double asterisk may represent MBP dimers and degradation products . 293T cells were cotransfected with plasmids encoding FLAG - Stx17 WT and the indicated PGAM5 - GFP constructs , and analyzed293T cells were cotransfected with plasmids encoding FLAG - Stx17 or Stx18 and the C - terminally GFP - tagged transmembrane domain of PGAM5 （ amino acids 1 - 35 ） constructs , and

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O 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 1293T cells were transfected with a plasmid encoding FLAG-Stx17 wild type (WT) or the indicated constructs. At 24 h after transfection, cell lysates were immunoprecipitated (IP) with anti-FLAG M2 beads, and analyzed by IB using antibodies against PGAM5 and FLAG. Five percent of lysates was analyzed as input.HeLa cells stably expressing FLAG-Stx17 WT or the K254C mutant were fixed and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with WT (paired Student's t-test).MBP or the MBP-Stx17 constructs attached to amylose resin were mixed with GST-PGAM5, and the proteins bound to the resin were separated by SDS-PAGE and blotted onto PVDF membranes. The blots were detected by an anti-GST antibody (upper panels) or stained with Coomassie Brilliant Blue R-250 (lower panels). Ten percent of the proteins used for each experiment was analyzed as input. Asterisks and double asterisk may represent MBP dimers and degradation products.293T cells were cotransfected with plasmids encoding FLAG-Stx17 WT and the indicated PGAM5-GFP constructs, and analyzed293T cells were cotransfected with plasmids encoding FLAG-Stx17 or Stx18 and the C-terminally GFP-tagged transmembrane domain of PGAM5（amino acids 1-35）constructs, and analyzed


Figure 2HeLa cells were treated with DMSO ( Vehicle ) or 0 . 03 mg / ml digitonin ( + Digitonin ) , fixed and then double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PGAM5 and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired Student ' sHeLa cells stably expressing FLAG - Stx17 wild type ( WT ) were transfected with a plasmid encoding Su9 - GFP ( mitochondria ) or Sec61β - GFP ( ER ) . At 24 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of PLA dots and Su9 - GFP or Sec61β - GFP . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 ( paired Student ' s t - test ) . HeLa cells were treated with DMSO ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and subjected to Percoll - based fractionation . Equal amounts of proteins were analyzed by IB using the indicated antibodies . PNS , postnuclear supernatant ; MS , microsomes ; Mt , mitochondria . The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment , respectively : PNS ( 6 . 6 mg and 5 . 5 mg ) , cytosol ( 4 . 8 mg and 4 . 9 mg ) , MS ( 2 . 0 mg and 2 . 2 mg ) , MAM ( 0 . 55 mg and 0 . 46 mg ) and Mt ( 0 . 30 mg and 0 . 28 mg ) . Electron microscopic analysis of HeLa cells expressing PGAM5 - GFP and APEX2 - GFP - binding peptide . Arrows indicate the position of 3 , 3 ' - diaminobenzidine reaction at the ER - mitochondria interface . Scale bar , 500 nm . HeLa cells stably expressing FLAG - Stx17 WT were mock - transfected or transfected with siRNA for Mfn1 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) . HeLa cells were mock - transfected or transfected with siRNA for Mfn1 , Stx17 , Mfn2 or PACS - 2 . At 72 h after transfection , the cells were subjected to PLA using antibodies against Drp1 and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) .

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Figure 2HeLa cells were treated with DMSO (Vehicle) or 0.03 mg/ml digitonin (+Digitonin), fixed and then double immunostained for PGAM5 and Tom20. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PGAM5 and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student'sHeLa cells stably expressing FLAG-Stx17 wild type (WT) were transfected with a plasmid encoding Su9-GFP (mitochondria) or Sec61β-GFP (ER). At 24 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of PLA dots and Su9-GFP or Sec61β-GFP. Values are means ± SEM (n = 3). ***P<0.001 (paired Student's t-test).HeLa cells were treated with DMSO (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and subjected to Percoll-based fractionation. Equal amounts of proteins were analyzed by IB using the indicated antibodies. PNS, postnuclear supernatant; MS, microsomes; Mt, mitochondria. The amounts of proteins recovered on fractionation were as follows for vehicle and CCCP treatment, respectively: PNS (6.6 mg and 5.5 mg), cytosol (4.8 mg and 4.9 mg), MS (2.0 mg and 2.2 mg), MAM (0.55 mg and 0.46 mg) and Mt (0.30 mg and 0.28 mg).Electron microscopic analysis of HeLa cells expressing PGAM5-GFP and APEX2-GFP-binding peptide. Arrows indicate the position of 3,3'-diaminobenzidine reaction at the ER-mitochondria interface. Scale bar, 500 nm.HeLa cells stably expressing FLAG-Stx17 WT were mock-transfected or transfected with siRNA for Mfn1, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).HeLa cells were mock-transfected or transfected with siRNA for Mfn1, Stx17, Mfn2 or PACS-2. At 72 h after transfection, the cells were subjected to PLA using antibodies against Drp1 and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).


Figure 3HeLa cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were fixed and double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm . 293T cells with mock treatment or depleted of Stx17 were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed and then analyzed by IB using the indicated antibodies . 293T cells transiently expressing FLAG - Stx17 wild type ( WT ) or the K254C mutant were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , lysed , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies . HeLa cells stably expressing FLAG - Stx17 WT were incubated with ethanol ( Vehicle ) or 20 μM CCCP ( + CCCP ) for 2 h , and subjected to PLA using antibodies against FLAG and PGAM5 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Vehicle ( paired 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3HeLa cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were fixed and double immunostained for PGAM5 and Tom20. Scale bar, 5 μm.293T cells with mock treatment or depleted of Stx17 were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed and then analyzed by IB using the indicated antibodies.293T cells transiently expressing FLAG-Stx17 wild type (WT) or the K254C mutant were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, lysed, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies.HeLa cells stably expressing FLAG-Stx17 WT were incubated with ethanol (Vehicle) or 20 μM CCCP (+CCCP) for 2 h, and subjected to PLA using antibodies against FLAG and PGAM5. Scale bar, 5 μm. Values are means ± SEM (n = 3). ***P<0.001 as compared with Vehicle (paired Student's t-test).


Figure 4HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted ( KD ) of Stx17 , PGAM5 or Drp1 were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies . HeLa cells stably expressing GFP - Parkin with mock treatment ( Mock ) or depleted of Stx17 , PGAM5 or Drp1 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by immunofluorescence microscopy . Scale bar , 5 μm . The bar graph on the right shows the Manders ' coefficients for the colocalization of GFP - Parkin and Tom20 . Values are means ± SEM ( n = 3 ) . * * * P < 0 . 001 as compared with Mock ( paired Student ' s t - test ) . PINK1 - FLAG and GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( ethanol ) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies . F , full - length PINK1 ; C , cleaved PINK1 .

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Figure 4HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted (KD) of Stx17, PGAM5 or Drp1 were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.HeLa cells stably expressing GFP-Parkin with mock treatment (Mock) or depleted of Stx17, PGAM5 or Drp1 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by immunofluorescence microscopy. Scale bar, 5 μm. The bar graph on the right shows the Manders' coefficients for the colocalization of GFP-Parkin and Tom20. Values are means ± SEM (n = 3). ***P<0.001 as compared with Mock (paired Student's t-test).PINK1-FLAG and GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (ethanol) or presence of 20 μM CCCP for 2 h and analyzed by IB using the indicated antibodies. F, full-length PINK1; C, cleaved PINK1.


Figure 5GFP - Parkin stably expressing HeLa cells were mock - transfected ( Mock ) or transfected with siRNA ( KD ) for Stx17 or PGAM5 . At 48 h after siRNA transfection , the cells were transfected with a plasmid encoding FLAG - DFCP1 , incubated for 24 h , treated with 20 μM CCCP for 2 h and then immunostained for FLAG . Scale bars , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and FLAG - DFCP1 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) . GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3 . Scale bar , 5 μm . The bar graph below shows the Manders ' coefficients for the colocalization of GFP - Parkin and LC3 . Values are means ± SEM ( n = 3 ) . * * P < 0 . 01 as compared with Mock ( paired Student ' s t - test ) . GFP - Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h and analyzed by IB using the indicated antibodies .

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Figure 5GFP-Parkin stably expressing HeLa cells were mock-transfected (Mock) or transfected with siRNA (KD) for Stx17 or PGAM5. At 48 h after siRNA transfection, the cells were transfected with a plasmid encoding FLAG-DFCP1, incubated for 24 h, treated with 20 μM CCCP for 2 h and then immunostained for FLAG. Scale bars, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and FLAG-DFCP1. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated with 20 μM CCCP for 2 h and immunostained for LC3. Scale bar, 5 μm. The bar graph below shows the Manders' coefficients for the colocalization of GFP-Parkin and LC3. Values are means ± SEM (n = 3). **P<0.01 as compared with Mock (paired Student's t-test).GFP-Parkin stably expressing HeLa cells with mock treatment or depleted of Stx17 or PGAM5 were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h and analyzed by IB using the indicated antibodies.


Figure 6HeLa cells stably expressing GFP - Parkin with mock treatment ( Control ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated for 16 h with ethanol ( Vehicle ) or 10 μM CCCP ( + CCCP ) , and analyzed by IB using the indicated antibodies . GFP - Parkin stably expressing HeLa cells with mock treatment ( Mock ) or depleted of FUNDC1 ( FUNDC1 KD ) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20 . Scale bar , 5 μm . 293T cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were incubated in the absence ( Vehicle ) or presence of 20 μM CCCP ( + CCCP ) for 2 h , immunoprecipitated with anti - FLAG M2 beads and then analyzed by IB using the indicated antibodies . Three percent of lysates was analyzed as input . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 . GFP - Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5 . During Stx17 knockdown , cells were transfected with a plasmid encoding FLAG - FUNDC1 . Scale bar , 5 μm . Values are means ± SEM ( n = 3 ) . * P < 0 . 05 and * * P < 0 . 01 as compared with Mock ( + CCCP ) by paired Student ' s t - test .

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Figure 6HeLa cells stably expressing GFP-Parkin with mock treatment (Control) or depleted of FUNDC1 (FUNDC1 KD) were incubated for 16 h with ethanol (Vehicle) or 10 μM CCCP (+CCCP), and analyzed by IB using the indicated antibodies.GFP-Parkin stably expressing HeLa cells with mock treatment (Mock) or depleted of FUNDC1 (FUNDC1 KD) were incubated with 20 μM CCCP for 2 h and immunostained for Tom20. Scale bar, 5 μm.293T cells with mock treatment (Mock) or depleted of Stx17 (Stx17 KD) were incubated in the absence (Vehicle) or presence of 20 μM CCCP (+CCCP) for 2 h, immunoprecipitated with anti-FLAG M2 beads and then analyzed by IB using the indicated antibodies. Three percent of lysates was analyzed as input. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1.GFP-Parkin stably expressing HeLa cells were treated and then subjected to PLA using antibodies against FLAG and PGAM5. During Stx17 knockdown, cells were transfected with a plasmid encoding FLAG-FUNDC1. Scale bar, 5 μm. Values are means ± SEM (n = 3). *P<0.05 and **P<0.01 as compared with Mock (+CCCP) by paired Student's t-test.


Figure 7A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss . Transmission electron microscope images of the indirect flight muscle in the indicated genotypes ( a , Control ; b , Stx17 - / - ; c , Stx17 - / - and Stx17 overexpression ; d , Stx17 - / - and PGAM5 overexpression ) of 7 - day - old adult flies are shown . The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages ( mean ± SEM ) using the scoring system : Class 0 , normal ; Class 1 , fuzzy or dilated cristae ; Class 2 , fragmented cristae and loss of electron density . * p < 0 . 05 , # p < 0 . 001 vs . the same class of control ( Dunnett ' s test ) . n = 50 - 119 mitochondria from three or four independent samples . Genotypes used were : + / y ; Act5c - GAL4 / + ( a ) , + / y ; Act5c - GAL4 / + ; Stx17LL06330 / Stx17LL06330 ( b ) , + / y ; Act5c - GAL4 / UAS - FLAG - Stx17 ; Stx17LL06330 / Stx17LL06330 ( c ) , + / y ; Act5c - GAL4 / UAS - PGAM5 ; Stx17LL06330 / Stx17LL06330 ( d ) . Scale bars , 200 nm . B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration . a , Stx17 + / - ; b , PGAM5 - / - ; c , Stx17 + / - , PGAM5 - / - . The bar graph on the right represents the mitochondrial phenotypes classified p < 0 . 05 , * * p < 0 . 01 vs . the same class of Stx17 + / - ( Dunnett ' s test ) . n = 62 - 195 mitochondria from three independent samples . Scale bars = 1 µm . Genotypes used were : + / y ; Act5c - GAL4 / + ; Stx17LL06330 / + ( a ) , PGAM51 / y ; Act5c - GAL4 / + ( b ) , PGAM51 / y ; Act5c - GAL4 / UAS - Stx17 - FLAG ; Stx17LL06330 / + ( c ) .

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

Figure 7A PGAM5 overexpression partially rescues the mitochondrial defects by Stx17 loss. Transmission electron microscope images of the indirect flight muscle in the indicated genotypes (a, Control; b, Stx17-/-; c, Stx17-/- and Stx17 overexpression; d, Stx17-/- and PGAM5 overexpression) of 7-day-old adult flies are shown. The bar graph on the right shows frequency of healthy and abnormal mitochondria presented as percentages (mean ± SEM) using the scoring system : Class 0, normal; Class 1, fuzzy or dilated cristae; Class 2, fragmented cristae and loss of electron density. *p < 0.05, #p < 0.001 vs. the same class of control (Dunnett's test). n = 50-119 mitochondria from three or four independent samples. Genotypes used were: +/y; Act5c-GAL4/+ (a), +/y; Act5c-GAL4/+; Stx17LL06330/Stx17LL06330 (b), +/y; Act5c-GAL4/UAS-FLAG-Stx17; Stx17LL06330/Stx17LL06330 (c), +/y; Act5c-GAL4/UAS-PGAM5; Stx17LL06330/Stx17LL06330 (d). Scale bars, 200 nm.B Simultaneous reduction of Stx17 and PGAM5 results in mitochondrial degeneration. a, Stx17+/-; b, PGAM5-/-; c, Stx17+/-, PGAM5-/-. The bar graph on the right represents the mitochondrial phenotypes classified p < 0.05, **p < 0.01 vs. the same class of Stx17+/- (Dunnett's test). n = 62-195 mitochondria from three independent samples. Scale bars = 1 µm. Genotypes used were: +/y; Act5c-GAL4/+; Stx17LL06330/+ (a), PGAM51/y; Act5c-GAL4/+ (b), PGAM51/y; Act5c-GAL4/UAS-Stx17-FLAG; Stx17LL06330/+ (c).


Figure 1A ) Single dose AR - 42 plasma and tissue pharmacokinetic study . Tumor - free CD2F1 mice were administered a single dose of 10 , 20 or 50 mg / kg AR - 42 ( n = 3 ) and plasma ( dashed ) and gastrocnemius ( solid ) tissue were analyzed for AR - 42 content at different times using LC - MS / MS analyses according to the Materials and Methods ( Mean ± SD ) . B ) AR - 42 dose - response . Starting six days after C - 26 cell injection , animals received vehicle or AR - 42 orally at 1 , 3 , 10 or 20 mg / kg daily or 50 mg / kg every other day for 13 days . Individual animal terminal ( Day 18 post - injection ) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor - free controls . Groups : 1mg / kg ( n = 8 ) , 3mg / kg ( n = 8 ) , 10mg / kg ( n = 13 ) , 20mg / kg ( n = 6 ) , 50mg / kg ( n = 5 ) . Dashed reference line ( 100 % ) represents the mean tumor - free control value , solid line represents non - linear fit of dose - response data . Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) and tumor - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . C ) : Terminal ( Day 18 post - injection ) body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . D ) Terminal tumor volumes ( mean ± SD ) . Study 1 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 5 ) , AR - 42 ( 10 mg / kg ; n = 5 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 5 ) or Vehicle ( n = 5 ) and tumor - free mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . E ) Terminal gastrocnemius ( mean ± SD ) and F ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Single dose AR-42 plasma and tissue pharmacokinetic study. Tumor-free CD2F1 mice were administered a single dose of 10, 20 or 50 mg/kg AR-42 (n=3) and plasma (dashed) and gastrocnemius (solid) tissue were analyzed for AR-42 content at different times using LC-MS/MS analyses according to the Materials and Methods (Mean±SD).B) AR-42 dose-response. Starting six days after C-26 cell injection, animals received vehicle or AR-42 orally at 1, 3, 10 or 20 mg/kg daily or 50 mg/kg every other day for 13 days. Individual animal terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods are compared to tumor-free controls. Groups: 1mg/kg (n=8), 3mg/kg (n=8), 10mg/kg (n=13), 20mg/kg (n=6), 50mg/kg (n=5). Dashed reference line (100%) represents the mean tumor-free control value, solid line represents non-linear fit of dose-response data.Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. C): Terminal (Day 18 post-injection) body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) Terminal tumor volumes (mean±SD).Study 1, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=5), AR-42 (10 mg/kg; n=5 ), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=5 ) or Vehicle (n=5) and tumor-free mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. E) Terminal gastrocnemius (mean±SD) and F) quadriceps muscle mass (mean±SD) compared to tumor-free controls.


Figure 2Study 2 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) and tumor - free male mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 cells . A ) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 2 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) and tumor - free male mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 cells . B ) Terminal gastrocnemius ( mean ± SD ) and C ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls . Study 2 , tumor - bearing male mice receiving GTx - 024 ( 15 mg / kg ; n = 10 ) , AR - 42 ( 10 mg / kg ; n = 9 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 9 ) or Vehicle ( n = 7 ) and tumor - free male mice receiving GTx - 024 ( 15mg / kg ; n = 6 ) or Vehicle ( n = 6 ) were treated daily by oral gavage for 12 days starting 6 days post - injection of C - 26 cells . D ) grip strength ( mean ± SD ) compared to tumor - free controls . Statistics : V , G , A indicate significant differences versus tumor - bearing vehicle - treated , tumor - bearing GTx - 024 - treated and tumor - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S2A - D , one - way ANOVA followed by Tukey ' s multiple comparison test . E ) Survival analyses of Study 2 animals performed as outlined in the Materials and Methods . Statistics : V , G indicate significant differences versus tumor - bearing vehicle - treated and tumor - bearing GTx - 024 - treated groups , respectively . p = 0 . 0005 for Combo vs . V , p = 0 . 0064 for Combo vs . G , Logrank ( Mantel - Cox ) test using Bonferroni corrected threshold for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 2, tumor-bearing male mice receiving GTx-024 (15 mg/kg; n=10), AR-42 (10 mg/kg; n=9), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=9) or Vehicle (n=7) and tumor-free male mice receiving GTx-024 (15mg/kg; n=6) or Vehicle (n=6) were treated daily by oral gavage for 12 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S2A-D, one-way ANOVA followed by Tukey's multiple comparison test.E) Survival analyses of Study 2 animals performed as outlined in the Materials and Methods. Statistics: V, G indicate significant differences versus tumor-bearing vehicle-treated and tumor-bearing GTx-024-treated groups, respectively. p=0.0005 for Combo vs. V, p=0.0064 for Combo vs. G, Logrank (Mantel-Cox) test using Bonferroni corrected threshold for multiple comparisons.


Figure 3Study 3 , tumor - bearing female mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) and tumor - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . A ) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 3 , tumor - bearing female mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) and tumor - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . B ) Terminal gastrocnemius ( mean ± SD ) and C ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls . Study 3 , tumor - bearing female mice receiving GTx - 024 ( 15 mg / kg ; n = 12 ) , AR - 42 ( 10 mg / kg ; n = 12 ) , Combination ( 15 mg / kg GTx - 024 and 10 mg / kg AR - 42 ; n = 12 ) or Vehicle ( n = 12 ) and tumor - free mice receiving Vehicle ( n = - 6 ) were treated daily by oral gavage for 13 days starting 6 days post - injection of C - 26 cells . D ) grip strength ( mean ± SD ) compared to tumor - free controls . Statistics : V , G , A indicate significant differences versus tumor - bearing vehicle - treated , tumor - bearing GTx - 024 - treated and tumor - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S3A - D , one - way ANOVA followed by Tukey ' s multiple comparison test .

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

Figure 3Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD) .Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 3, tumor-bearing female mice receiving GTx-024 (15 mg/kg; n=12), AR-42 (10 mg/kg; n=12), Combination (15 mg/kg GTx-024 and 10 mg/kg AR-42; n=12) or Vehicle (n=12) and tumor-free mice receiving Vehicle (n=-6) were treated daily by oral gavage for 13 days starting 6 days post-injection of C-26 cells. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, G, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing GTx-024-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S3A-D, one-way ANOVA followed by Tukey's multiple comparison test.


Figure 4Study 4 , tumor - bearing male mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 and DHT ( 10 mg / kg oral gavage and 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 and TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) and tumor free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after cell injection . A ) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor - free controls ( mean ± SD ) . Study 4 , tumor - bearing male mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 and DHT ( 10 mg / kg oral gavage and 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 and TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) and tumor free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after cell injection . B ) Terminal gastrocnemius ( mean ± SD ) and C ) quadriceps muscle mass ( mean ± SD ) compared to tumor - free controls . Study 4 , tumor - bearing male mice receiving AR - 42 ( 10 mg / kg , oral gavage ; n = 9 ) , TFM - 4AS - 1 ( 10 mg / kg , subcutaneous ; n = 10 ) , Combination of AR - 42 and DHT ( 10 mg / kg oral gavage and 3 mg / kg subcutaneous , respectively ; n = 10 ) , Combination of AR - 42 and TFM - 4AS - 1 ( 10 mg / kg , both , n = 9 ) or Vehicle ( n = 6 ) and tumor free mice receiving Vehicle ( n = 6 ) were treated daily for 12 days starting 6 days after cell injection . D ) grip strength ( mean ± SD ) compared to tumor - free controls . Statistics : V , T , A indicate significant differences versus tumor - bearing vehicle - treated , tumor - bearing TFM - 4AS - 1 - treated and tumor - bearing AR - 42 - treated groups , respectively . p values provided in Appendix Table S4A - D , Tukey ' s multiple comparison test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. A) Terminal body weights corrected for tumor mass according to the Materials and Methods compared to tumor-free controls (mean±SD).Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. B) Terminal gastrocnemius (mean±SD) and C) quadriceps muscle mass (mean±SD) compared to tumor-free controls.Study 4, tumor-bearing male mice receiving AR-42 (10 mg/kg, oral gavage; n=9), TFM-4AS-1 (10 mg/kg, subcutaneous; n=10), Combination of AR-42 and DHT (10 mg/kg oral gavage and 3 mg/kg subcutaneous, respectively; n=10), Combination of AR-42 and TFM-4AS-1 (10 mg/kg, both, n=9) or Vehicle (n=6) and tumor free mice receiving Vehicle (n=6) were treated daily for 12 days starting 6 days after cell injection. D) grip strength (mean±SD) compared to tumor-free controls. Statistics: V, T, A indicate significant differences versus tumor-bearing vehicle-treated, tumor-bearing TFM-4AS-1-treated and tumor-bearing AR-42-treated groups, respectively. p values provided in Appendix Table S4A-D, Tukey's multiple comparison test.


Figure 5A ) Gene expression of multiple cachexia - associated markers in gastrocnemius muscles of individual animals from Study 1 ( n = 5 for tumor - bearing groups ; n = 6 for tumor - free groups ) . Expression was determined by qRT - PCR and presented as described in the Materials and Methods ( Geometric Mean ± Geometric STD ) . B ) Androgen receptor ( AR ) mRNA expression in gastrocnemius muscles from Study 1 . Statistics : p values provided in Appendix Table S5A - B . Dunnett ' s multiple comparison test . CEBPδ , n = 4 , insufficient sample to analyze all tumor - bearing GTx - 024 - treated animals .

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Figure 5A) Gene expression of multiple cachexia-associated markers in gastrocnemius muscles of individual animals from Study 1 (n=5 for tumor-bearing groups; n=6 for tumor-free groups). Expression was determined by qRT-PCR and presented as described in the Materials and Methods (Geometric Mean ± Geometric STD). B) Androgen receptor (AR) mRNA expression in gastrocnemius muscles from Study 1. Statistics: p values provided in Appendix Table S5A-B. Dunnett's multiple comparison test. CEBPδ, n=4, insufficient sample to analyze all tumor-bearing GTx-024-treated animals.


Figure 6A ) ELISA analysis of serum IL - 6 levels in terminal samples from Study 2 . Tumor - free groups ( n = 6 ) , tumor - bearing receiving Vehicle ( n = 7 ) , GTx - 024 ( n = 10 ) , AR - 42 ( n = 8 ) or Combination ( n = 9 ) . ND , not detected . B and C ) Phospho ( p ) STAT3 ( Y705 ) / total ( t ) STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 ( B ) and Study 3 ( C ) . Black circle - tumor - free , blue square - tumor - free / GTx - 024 , black triangle - tumor - bearing , blue diamond - tumor / GTx - 024 , red triangle - tumor / AR - 42 , green circle - tumor / combo . D ) Spleen weights normalized to tumor - corrected terminal body weights of mice from Study 2 . Tumor - free groups ( n = 6 ) , tumor - bearing Vehicle ( n = 7 ) , tumor - bearing receiving GTx - 024 ( n = 10 ) , AR - 42 ( n = 9 ) or Combination ( n = 9 ) . E ) ELISA analysis of pSTAT3 ( Y705 ) / tSTAT3 within C - 26 tumors from Study 2 . Tumor - bearing Vehicle - treated ( n = 7 ) , tumor - bearing receiving GTx - 024 , AR - 42 or Combination ( n = 8 ) .

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Figure 6A) ELISA analysis of serum IL-6 levels in terminal samples from Study 2. Tumor-free groups (n=6), tumor-bearing receiving Vehicle (n=7), GTx-024 (n=10), AR-42 (n=8) or Combination (n=9). ND, not detected.B and C) Phospho(p)STAT3 (Y705)/total (t)STAT3 western blot analysis of gastrocnemius tissues from individual animals treated in Study 1 (B) and Study 3 (C). Black circle - tumor-free, blue square - tumor-free/GTx-024, black triangle - tumor-bearing, blue diamond - tumor/GTx-024, red triangle - tumor/AR-42, green circle - tumor/combo.D) Spleen weights normalized to tumor-corrected terminal body weights of mice from Study 2. Tumor-free groups (n=6), tumor-bearing Vehicle (n=7), tumor-bearing receiving GTx-024 (n=10), AR-42 (n=9) or Combination (n=9).E) ELISA analysis of pSTAT3(Y705)/tSTAT3 within C-26 tumors from Study 2. Tumor-bearing Vehicle-treated (n=7), tumor-bearing receiving GTx-024, AR-42 or Combination (n=8).


Figure 7A ) Effect of GTx - 024 and AR - 42 monotherapies on cachexia - related differentially regulated genes ( DEGs ) from RNA - seq analyses of Study 1 gastrocnemius muscles . All three panels consist of individual genes plotted with respect to their log2 fold change and - log10 Benjamini - Hochburg adjusted p - values from the comparison of cachexia vs tumor - free controls . Colors of the points reflect the DEG status of each gene for the given comparison . C ) Significance values from GSEA using combined STAT3 gene sets identified in B . Each treatment group is compared to tumor - bearing control ( cachexia ) transcriptomes . D ) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data . Tumor - free control ( black checkered ) , GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . E ) Results of GSEA using combined ATF - 1 gene sets identified in B across treatment groups versus tumor - bearing control ( cachexia ) transcriptomes . Tumor - free control ( black checkered ) , GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . F ) Heat map of DEGs within the combined ATF - 1 gene sets ( mean z score ) . Treatment groups are as in D . G ) mRNA expression of mediators of IL - 6 signaling upstream of STAT3 . Data presented as mean ± SD of per animal log - transformed fold change ( log2FC ) values versus tumor - free controls . Groups : Tumor - free control ( n = 6 ) , tumor - free GTx - 024 treated ( n = 5 ) , tumor - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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Figure 7A) Effect of GTx-024 and AR-42 monotherapies on cachexia-related differentially regulated genes (DEGs) from RNA-seq analyses of Study 1 gastrocnemius muscles. All three panels consist of individual genes plotted with respect to their log2 fold change and -log10 Benjamini-Hochburg adjusted p-values from the comparison of cachexia vs tumor-free controls. Colors of the points reflect the DEG status of each gene for the given comparison.C) Significance values from GSEA using combined STAT3 gene sets identified in B. Each treatment group is compared to tumor-bearing control (cachexia) transcriptomes.D) Heat map of DEGs within the combined STAT3 gene sets representing mean z scores calculated from normalized RNAseq count data. Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes.Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D.G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


Figure 8A ) Volcano plot from RNA - seq analyses of Study 1 gastrocnemius muscles for tumor - bearing combination - treated mice versus tumor - bearing controls . Genes not differentially expressed in this comparison are indicated with blue . The remaining genes ( red and green ) are DEGs in the combination - treated versus tumor - bearing control comparison . The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR - 42 - treated tumor - bearing mice versus tumor - bearing controls , suggesting these genes are responsive to only the combination therapy . Log2 - transformed fold change ( FC ) in expression is plotted on the x - axis and - log10 transformed Benjamini - Hochburg adjusted p - values are plotted on the y - axis . C ) Significance values from GSEA using combined CTNNB1 gene sets . Each treatment group was compared to tumor - free control transcriptomes . * FDR < 1e - 5 determined , set to 1e - 5 for plot . D ) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor - free control comparisons . GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . E ) mRNA expression of WNT effectors upstream of β - catenin . Data are presented as mean ± SD of log - transformed fold change ( log2FC ) values versus tumor - free controls . Groups : Tumor - free control ( n = 6 ) , tumor - free GTx - 024 treated ( n = 5 ) , tumor - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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Figure 8A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis.C) Significance values from GSEA using combined CTNNB1 gene sets. Each treatment group was compared to tumor-free control transcriptomes. *FDR < 1e-5 determined, set to 1e-5 for plot.D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


Figure 1A ) Neutrophils and lymphocytes count . B ) Levels of creatine kinase ( CK ) and creatinine . C ) Levels of procalcitonin ( PCT ) and C - reactive protein ( CRP ) . D ) Levels of erythrocyte sedimentation rate ( ESR ) and ferritin . E ) Levels of D - dimer and lactate dehydrogenase ( LDH ) . F ) Proportion of total CD3 + and CD4 + T cells . G ) Proportion of CD8 + T cells and CD4 + / CD8 + T cell ratio . H ) Proportion of B and natural killer ( NK ) cells . cytokine profile of COVID - 19 patients for IL - 2 , IL - 4 ( I ) cytokine profile of COVID - 19 patients for , IL - 6 , IL - 10 ( J ) and TNF - α and IFN - γ ( K ) .

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Figure 1A) Neutrophils and lymphocytes count.B) Levels of creatine kinase (CK) and creatinine.C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).D) Levels of erythrocyte sedimentation rate (ESR) and ferritin.E) Levels of D-dimer and lactate dehydrogenase (LDH).F) Proportion of total CD3+ and CD4+ T cells.G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio.H) Proportion of B and natural killer (NK) cells.cytokine profile of COVID-19 patients for IL-2, IL-4 (I)cytokine profile of COVID-19 patients for , IL-6, IL-10 (J) and TNF-α and IFN-γ (K).


Figure 2Time from symptom onset to initial diagnosis ( TFSD ) . Time from symptom onset to development of pneumonia ( TFSP ) . Time from symptom onset to treatment ( TFST ) . Time from development of pneumonia to recovery ( TFPR ) .

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Figure 2Time from symptom onset to initial diagnosis (TFSD).   Time from symptom onset to development of pneumonia (TFSP).   Time from symptom onset to treatment (TFST).Time from development of pneumonia to recovery (TFPR).


Figure 3Correlation between IL - 6 and maximal body temperature during hospitalization . Correlation between IL - 6 and C - reactive protein ( CRP ) . Correlation between IL - 6 and lactate dehydrogenase ( LDH ) . Correlation between IL - 6 and D - dimer . Correlation between IL - 6 and ferritin . Levels of baseline IL - 6 in patients who received glucocorticoids , human immunoglobulin , high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not . Error bars , SEM .

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Figure 3Correlation between IL-6 and maximal body temperature during hospitalization.Correlation between IL-6 and C-reactive protein (CRP).Correlation between IL-6 and lactate dehydrogenase (LDH).Correlation between IL-6 and D-dimer.Correlation between IL-6 and ferritin.Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.


Figure 4IL - 6 levels before and after treatment in three patient groups classified according to baseline IL - 6 levels of ≥ 40 pg / mL ( n = 11 ) , ≥ 20 pg / mL ( n = 6 ) or < 20 pg / mL ( n = 9 ) ( left ) . Two representative chest computed tomography ( CT ) scans before and after treatment from each group ( right ) . IL - 6 levels at diagnosis and after disease progression in three exacerbated patients ( left ) . Progressed radiological findings were recorded in two patients ( right ) , while radiological assessment after treatment was not performed in the third patient due to poor general condition . The baseline IL - 6 level was 197 . 39 pg / mL in a 69 - year - old female patient who showed high fever and dyspnea . IL - 6 decreased to 9 . 47 pg / mL after treatment ( day 8 ) , while the symptoms were not relieved . The C - reactive protein ( CRP ) rebounded and procalcitonin ( PCT ) increased together with disease exacerbation . Follow - up chest computed tomography ( CT ) assessment was not performed due to poor general condition , whereas chest X - ray showed aggravated pneumonia . Follow - up sputum culture confirmed the exacerbation was caused by bacterial infection .

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Figure 4IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right).IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition.The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.


Figure 5A ) The chest computed tomography ( CT ) was normal five days after symptom onset . Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities . Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset . B ) Procalcitonin ( PCT ) levels stayed within normal range throughout the course of disease . C ) Lymphocyte count was normal five days after symptom onset , while firstly decreasing then returning to normal range during the course of disease . Both abnormal C - reactive protein ( CRP ) ( D ) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal . In association with changes in chest CT scans , CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset . IL - 6 ( E ) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal . In association with changes in chest CT scans , IL - 6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset . F ) The body temperature was elevated when CRP and IL - 6 levels were rising , but returned to normal when CRP and IL - 6 . Levels were declining .

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Figure 5A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset.B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease.Both abnormal C-reactive protein (CRP) (D) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.


Figure 1RAW 264 . 7 mouse macrophages were treated with either oxLDL ( 50 µg / ml ) or TG ( 300 nM ) for 6 hours . Protein lysates were treated with λ Phosphatase ( PPase ) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 6 biological replicates ) . Control - or IRE1 - siRNA transfected HEK293T cells were stimulated by either PA ( 500 µM ) or TG ( 600 nM ) for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) . MEF cells were transfected with either empty vector , EGFP - FMRP or 3xFLAG - IRE1 plasmids then pre - treated either with vehicle ( dimethyl sulfoxide , DMSO ) or AMG - 18 ( 25 µM ; 1 hour ) followed by TG ( 600 nM ) stimulation for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) . Purified FMRP and IRE1 kinase ( activated ) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP , IRE1 and FMRP ( n = 3 biological replicates ) and with LC - MS / MS . Identified IRE1 kinase - mediated FMRP phosphorylation sites ( bottom ) .

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Figure 1RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 hours. Protein lysates were treated with λ Phosphatase (PPase) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=6 biological replicates).Control- or IRE1-siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).MEF cells were transfected with either empty vector, EGFP-FMRP or 3xFLAG-IRE1 plasmids then pre-treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG-18 (25 µM; 1 hour) followed by TG (600 nM) stimulation for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP, IRE1 and FMRP (n=3 biological replicates) and with LC-MS/MS. Identified IRE1 kinase-mediated FMRP phosphorylation sites (bottom).


Figure 2Fmr1 + / + and Fmr1 - / - mice were injected with AAV _ PCSK9 and fed with 16 weeks of WD . Residential PM were stained with Oil Red O ( ORO ) and imaged ( n = 7 mice per group ; Scale bar = 50 µm ) . Apoe - / - mice were fed with WD ( 12 weeks ) and injected with vehicle ( DMSO ) or AMG - 18 ( 30 mg / kg / day ) in the last 4 weeks of WD . Residential PM were stained with ORO and imaged ( n = 5 mice per group ; Scale bar = 50 µm ) . RCT experiment ( I ) plasma cholesterol levels after 24 and 48 hours , ( J ) liver cholesterol levels after 48 hours , and ( K ) feces cholesterol levels after 48 hours ( n = 12 mice per group ) .

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Figure 2Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).    Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).  RCT experiment (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).


Figure 3A - E In vitro and in vivo efferocytosis experiments , where percentage of macrophages F4 / 80 + ( red ) that ingested apoptotic cells ( AC ) labeled with carboxyfluorescein succinimidyl ester ( CFSE ) + ( green ) were reported as % efferocytosis . ( A ) BMDMs were transfected with Fmr1 - or control - siRNA and incubated CFSE - labeled AC for the indicated hours ( n = 4 biological replicates ) . ( B ) Fmr1 + / + and Fmr1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and then incubated with CFSE - labeled ACs for 4 hours ( n = 4 biological replicates ) . ( C ) Fmr1 + / + and Fmr1 - / - mice were fed WD ( 16 weeks ) and injected intraperitoneally with CFSE - labeled AC ( 1 . 5 hours ) , followed by PM elicitation ( n = 4 - 5 mice per group ) . ( D ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour then incubated with CFSE - labeled ACs for 4 hours ( n = 3 biological replicates ) . ( E ) C57BL / 6 mice were injected with AMG - 18 ( 30 mg / kg ) or vehicle ( DMSO ) for 8 hours , followed by intraperitoneal injection with CFSE - labeled ACs for 1 . 5 hours and PM elicitation ( n = 4 mice per group ) . F - G In vitro continuous efferocytosis experiments , where macrophages were stained for F4 / 80 + ( red ) , AC were labeled with CFSE ( AC - 1 ; green ) or Violet ( AC2 ; violet ) . % continuous efferocytosis was determined by the ratio of F4 / 80 + , CFSE + and Violet + ( triple positive ) cells to total F4 / 80 + and CFSE + ( double positive ) cells . ( F ) Fmr1 + / + and Fmr1 - / - BMDM were incubated with AC - 1 for 2 hours , and after 2 hours interval , incubated with AC - 2 for 2 more hours ( n = 4 - 3 biological replicates ) . ( G ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour , incubated with CFSE - labeled AC - 1 for 2 hours , followed by incubation with Violet - labeled AC - 2 for 2 hours and PM collection ( n = 4 biological replicates ) .

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Figure 3A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).


Figure 4RNA lysates from Fmr1 + / + and Fmr1 - / - BMDM that were treated with PA ( 500 µM ; 6 hours ) were fractionated using a 10 % - 50 % sucrose gradient and separated to polysome , monosome / NTR fractions . The absorbance ( 260 nm ) of RNA was measured and plotted as a function of time ( n = 3 biological replicates ) . ( B ) The ratio of the Abca1 , Abcg1 , Mertk , Lrp1 , Cd36 , Cd47 and Rac1 mRNA in polysome to NTR fraction ( n = 3 biological replicates ) . D Fmr1 - / - MEF cells were transfected with EV , WT - FMRP or STSA - FMRP plasmids followed by PA treatment ( 500 µM ; 6 hours ) . Protein lysates were analyzed by western blotting using specific antibodies for ABCA1 , MerTK , LRP1 , pFMRP , FMRP and β - Actin and fold inductions relative to β - Actin are depicted above the blots ( n = 5 biological replicates ) .

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Figure 4RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).


Figure 5Lesion area calculated from en face aorta , stained with ORO ( n = 12 - 13 mice per group ; Scale bar = 5 mm ) . Lesion area calculated from en face aorta , stained with ORO ( n = 9 mice per group ; Scale bar = 5 mm ) .

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Figure 5Lesion area calculated from en face aorta, stained with ORO (n=12-13 mice per group; Scale bar = 5 mm).Lesion area calculated from en face aorta, stained with ORO (n=9 mice per group; Scale bar = 5 mm).


Figure 6Lesion area calculated from en face aorta , stained with ORO ( n = 5 mice per group ; Scale bar = 5 mm ) . Lesion area calculated from en face aorta , stained with ORO ( n = 6 mice per group ; Scale bar = 5 mm ) .

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Figure 6Lesion area calculated from en face aorta, stained with ORO (n=5 mice per group; Scale bar = 5 mm).Lesion area calculated from en face aorta, stained with ORO (n=6 mice per group; Scale bar = 5 mm).


Figure 1 ( C ) Competition of sybodies and ACE2 for spike protein binding , investigated by GCI . Biotinylated spike protein was immobilized on the GCI sensor and then Sb # 15 ( 200 nM , left ) , or Sb # 68 ( 200 nM , right ) were injected alone or premixed with human ACE2 ( 100 nM ) . Sb # 0 represents a non - randomized control sybody .

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Figure 1(C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.


Figure 2Neutralization assays using VSVΔG pseudotyped with wildtype SARS - CoV - 2 spike protein . Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs . ( A ) Sb # 15 , Sb # 68 , or an equimolar mixure of both sybodies . ( B ) Sb # 14 , Sb # 16 , Sb # 45 , or , as a positive control , the previously - described sybody MR3 . ( C ) Neutralization by bivalent sybody - Fc fusions . Error bars correspond to standard deviations of three biological replicates .

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Figure 2Neutralization assays using VSVΔG pseudotyped with wildtype SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. (A) Sb#15, Sb#68, or an equimolar mixure of both sybodies. (B) Sb#14, Sb#16, Sb#45, or, as a positive control, the previously-described sybody MR3. (C) Neutralization by bivalent sybody-Fc fusions. Error bars correspond to standard deviations of three biological replicates.


Figure 4 ( C ) Neutralization assay using VSVΔG pseudotyped with wildtype Spike - protein . Relative infectivity was determined in response to increasing GS4 or Tripod - GS4r concentration . The corresponding neutralization data for isolated Sb # 15 and Sb # 68 are provided as reference . ( D ) Control experiment using VSVΔG pseudotyped with VSV - G . Infectivity was not affected by Tripod - GS4r .

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Figure 4(C) Neutralization assay using VSVΔG pseudotyped with wildtype Spike-protein. Relative infectivity was determined in response to increasing GS4 or Tripod-GS4r concentration. The corresponding neutralization data for isolated Sb#15 and Sb#68 are provided as reference. (D) Control experiment using VSVΔG pseudotyped with VSV-G. Infectivity was not affected by Tripod-GS4r.


Figure 5 ( C ) Neutralization assay using VSVΔG pseudotyped with Spike - protein containing the Q493R or P384H mutation , respectively . Relative infectivity in response to increasing binder concentrations was determined .

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Figure 5(C) Neutralization assay using VSVΔG pseudotyped with Spike-protein containing the Q493R or P384H mutation, respectively. Relative infectivity in response to increasing binder concentrations was determined.


Figure 6 ( A ) GCI - based kinetic analysis of interactions between immobilized sybodies ( indicated above each plot ) and wild - type ( WT ) or mutant RBDs carrying the individual K417N , E484K or N501Y mutations ( left panels ) , the combined triple KEN ( Beta ) mutations ( middle panels ) , or the double L452R / T478K ( Delta ) mutations ( right panels ) . Sb # 15 , Sb # 68 and GS4 were immobilized on independent flow - cells via biotinylated Avi - tags , and the RBD variants were sequentially injected at a concentration of 200 nM .

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Figure 6(A) GCI-based kinetic analysis of interactions between immobilized sybodies (indicated above each plot) and wild-type (WT) or mutant RBDs carrying the individual K417N, E484K or N501Y mutations (left panels), the combined triple KEN (Beta) mutations (middle panels), or the double L452R/T478K (Delta) mutations (right panels). Sb#15, Sb#68 and GS4 were immobilized on independent flow-cells via biotinylated Avi-tags, and the RBD variants were sequentially injected at a concentration of 200 nM.


Figure 2A . Growth curves of the library ( black ) compared to two different controls under increasing selective pressures . DSB ( double - stranded break ) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK . n = 3 for each

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Figure 2A. Growth curves of the library (black) compared to two different controls under increasing selective pressures. DSB (double-stranded break) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK. n=3 for each curve


Figure 3B . Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non - Target gRNA . n = 3 . C . Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non - Target gRNA . n =

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Figure 3B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3


Figure 4D . Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants . Quantification was performed using LC - MS , as described in the methods section . n = 3 . A two - sample student t - test assuming unequal variances was performed to calculate statistical significance . Concentrations are reported as fold - change relative to the wild - type control samplesE . DapF assay showing kinetics of the wild type , G210D and M260Y mutants . Assay was performed as described in the methods section . n = 5F . Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT , DapF G210D and DapF M260Y

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Figure 4D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samplesE. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds


Figure 5C . Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control samplesF . Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control

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Figure 5C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samplesF. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples


Figure 1A Representative images of E18 . 5 coronal cortical sections from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmids encoding EGFP , EGFP + Cre ( Cre ) or EGFP + Cre + KCC2FL ( Cre + KCC2FL ) . DAPI staining ( blue ) marks cell nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . B Quantification of the number of EGFP + neurons / ROI from embryos electroporated with constructs in ( A ) . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 . Data are presented as mean ± S . E . M . , n ( EGFP ) = 8 embryos ; n ( Cre ) = 8 embryos ; n ( Cre + KCC2FL ) = 13 embryos . C Representative image of cleaved Caspase 3 ( Cas - 3 , upper panel ) and TUNEL ( lower panel ) staining in coronal sections from Kcc2lox / lox cortex at E16 . 5 electroporated with EGFP or EGFP + Cre are shown . Arrowheads point to neurons expressing Cas - 3 ( upper panel ) and TUNEL ( lower panel ) . DAPI staining ( blue ) marks cell nuclei . CP , cortical plate ; VZ , ventricular zone ; SVZ , subventricular zone ; Sp , subplate ; IZ , intermediate zone . Large scale bar : 50 µm , small scale bar : 20 µm . D Quantification of the percentage of EGFP + neurons expressing apoptotic markers at E16 . 5 from embryos electroporated with EGFP ± Cre . Statistical significance was determined using Mann - Whitney U test ( Cas - 3 ) ; and two - tailed t test ( TUNEL ) , * * * P < 0 . 001 . Data are presented as mean ± S . E . M . , n ( - Cre ) = 6 embryos ; n ( + Cre ) = 6 embryos . E The number of EGFP + Cre neurons as a percentage of neurons expressing EGFP alone . Statistical significance was determined using a two - tailed Student ' s t test . Data are presented as mean ± S . E . M . , n = 6 embryos .

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Figure 1A Representative images of E18.5 coronal cortical sections from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmids encoding EGFP, EGFP+Cre (Cre) or EGFP+Cre+KCC2FL (Cre+KCC2FL). DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. B Quantification of the number of EGFP+ neurons/ROI from embryos electroporated with constructs in (A). Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01. Data are presented as mean ± S.E.M., n (EGFP) = 8 embryos; n (Cre) = 8 embryos; n (Cre+KCC2FL) = 13 embryos. C Representative image of cleaved Caspase 3 (Cas-3, upper panel) and TUNEL (lower panel) staining in coronal sections from Kcc2lox/lox cortex at E16.5 electroporated with EGFP or EGFP+Cre are shown. Arrowheads point to neurons expressing Cas-3 (upper panel) and TUNEL (lower panel). DAPI staining (blue) marks cell nuclei. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; Sp, subplate; IZ, intermediate zone. Large scale bar: 50 µm, small scale bar: 20 µm. D Quantification of the percentage of EGFP+ neurons expressing apoptotic markers at E16.5 from embryos electroporated with EGFP ± Cre. Statistical significance was determined using Mann-Whitney U test (Cas-3); and two-tailed t test (TUNEL), ***P < 0.001. Data are presented as mean ± S.E.M., n (-Cre) = 6 embryos; n (+Cre) = 6 embryos. E The number of EGFP+Cre neurons as a percentage of neurons expressing EGFP alone. Statistical significance was determined using a two-tailed Student's t test. Data are presented as mean ± S.E.M., n = 6 embryos.


Figure 2A Top : Representative images of E18 . 5 coronal cortical brain sections from Kcc2lox / lox embryos electroporated in utero at E14 . 5 together with plasmid constructs encoding Cre , a fluorescent marker ( EGFP or mRFP , both pseudo - colored in green ) and one of the following constructs : KCC2ΔNTD , KCC2CTD , KCC2R952H , or cofilinS3A . DAPI staining ( blue ) marks cell nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . Bottom : schematic representation of the KCC2 constructs . B Quantification of the number of transfected neurons / ROI from embryos electroporated with constructs in ( A ) . The mean number of transfected neurons from embryos electroporated with Cre + KCC2FL taken from Fig 1B shown as dotted line . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 to Cre + KCC2FL . Data are presented as mean ± S . E . M . , n ( KCC2ΔNTD ) = 7 embryos ; n ( Cre + KCC2CTD ) = 9 embryos ; n ( Cre + KCC2R952H ) = 9 embryos ; n ( Cre + cofilinS3A ) = 8 embryos .

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Figure 2A Top: Representative images of E18.5 coronal cortical brain sections from Kcc2lox/lox embryos electroporated in utero at E14.5 together with plasmid constructs encoding Cre, a fluorescent marker (EGFP or mRFP, both pseudo-colored in green) and one of the following constructs: KCC2ΔNTD, KCC2CTD, KCC2R952H, or cofilinS3A. DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. Bottom: schematic representation of the KCC2 constructs. B Quantification of the number of transfected neurons/ROI from embryos electroporated with constructs in (A). The mean number of transfected neurons from embryos electroporated with Cre+KCC2FL taken from Fig 1B shown as dotted line. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Cre+KCC2FL. Data are presented as mean ± S.E.M., n (KCC2ΔNTD) = 7 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos; n (Cre+cofilinS3A) = 8 embryos.