HeLa 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 '

B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa 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's


HeLa 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 ) .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa 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 ) .

B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O

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 ) .

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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 ) .

B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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).


HeLa cells with mock treatment ( Mock ) or depleted of Stx17 ( Stx17 KD ) were fixed and double immunostained for PGAM5 and Tom20 . Scale bar , 5 μm .

B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O

HeLa 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 .

B-CELL_LINE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY O O O O O

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 .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O

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 ) .

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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).


HeLa 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 .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O

HeLa 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 ) .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O

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.


GFP - 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 ) .

B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

GFP-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 ) .

B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O

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.


HeLa 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 .

B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O

HeLa 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 .

B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O

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 .

B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O

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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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.


A 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 .

O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O

A 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 O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O

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).


( D ) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 ( n = 3 ) or WT EAAT2 ( n = 3 ) . Cells were intracellularly dialyzed with a 115mM KGluc - based solution and held at 0 mV .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-CELL_LINE O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O

(D) Mean current responses to rapid switching from 140 mM CholGluc to 140 mM KGluc of HEK293T cells expressing WT EAAT1 (n=3) or WT EAAT2 (n=3). Cells were intracellularly dialyzed with a 115mM KGluc-based solution and held at 0 mV.


( B ) Representative WT and mutant whole - cell current responses to voltage jumps before and after removal of external K + . Cells were dialyzed with a KNO3 - based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3 .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

(B) Representative WT and mutant whole-cell current responses to voltage jumps before and after removal of external K+. Cells were dialyzed with a KNO3-based pipette solution and the external solution contained either 140 mM CholineNO3 or 140 mM KNO3.


( C ) Relative changes in fluorescence for WT , D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [ K + ] . Solid lines represent non - parametric fits , and shaded area show bootstrapped 99 % confidence intervals for those fits . Non - overlapping confidence intervals indicate significant difference between WT and mutant binding curves . Values are given as mean ± SD ( n ≥ 3 ) .

O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Relative changes in fluorescence for WT, D312N and D405N GltPh upon a temperature jump in MST experiments as a function of [K+]. Solid lines represent non-parametric fits, and shaded area show bootstrapped 99% confidence intervals for those fits. Non-overlapping confidence intervals indicate significant difference between WT and mutant binding curves. Values are given as mean ± SD (n≥3).


( D ) Representative whole - cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline - NO3 - based external solutions or in NaNO3 - based solutions supplemented with 1 mM L - glutamate . Glutamate - induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 ( GltPh D312 and D405 ) are K + - independent , but are functionally expressed in the plasma membrane .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

(D) Representative whole-cell current recordings from cells internally dialyzed with NaNO3 and glutamate in choline-NO3-based external solutions or in NaNO3-based solutions supplemented with 1 mM L-glutamate. Glutamate-induced changes in anion current amplitudes indicate that D399N and D486N EAAT2 (GltPh D312 and D405) are K+-independent, but are functionally expressed in the plasma membrane.


( E ) Current - voltage relationships of steady - state currents from cells dialyzed with a KNO3 - based pipette solution . Currents were measured at external 140 mM KNO3 and normalized to the current at - 170 mV from a consecutive recording of the same cell in K + - free external solutions . Values are given as mean ± SD ( n ≥ 3 )

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(E) Current-voltage relationships of steady-state currents from cells dialyzed with a KNO3-based pipette solution. Currents were measured at external 140 mM KNO3 and normalized to the current at -170 mV from a consecutive recording of the same cell in K+-free external solutions. Values are given as mean ± SD (n≥3)


( E ) Representative transport current recordings from cells expressing L448A or L448T ( GltPh A360 ) or WT EAAT1 upon voltage jumps to ˗ 140 mV before and after superfusion with 1 mM l - glutamate . Cells were intracellularly dialyzed with a K + - free solution without permeating anions to isolate K + - independent transport currents . Transient capacitive currents during the first 5 ms after a voltage jump were blanked .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O

(E) Representative transport current recordings from cells expressing L448A or L448T (GltPh A360) or WT EAAT1 upon voltage jumps to ˗140 mV before and after superfusion with 1 mM l-glutamate. Cells were intracellularly dialyzed with a K+-free solution without permeating anions to isolate K+-independent transport currents. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.


( F ) Current - voltage relationship for net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for choline - based pipette solutions . Values are given as mean ± SD with indicated numbers of experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(F) Current-voltage relationship for net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for choline-based pipette solutions. Values are given as mean ± SD with indicated numbers of experiments.


( G ) Time - dependent changes in L448A EAAT1 ( GltPh A360 ) uptake currents during two repetitive glutamate applications and subsequent removals . Current amplitudes were measured at the end of voltage jumps to - 140mV and plotted versus the time . Glutamate application is indicated by green bars . Values are given as mean ± SD of the last 100 data points in the current trace .

O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(G) Time-dependent changes in L448A EAAT1 (GltPh A360) uptake currents during two repetitive glutamate applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus the time. Glutamate application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.


( H ) Net transport current amplitudes for WT and L448A / T EAAT1 ( GltPh A360 ) for different pipette solutions , Values are given as mean ± SD with indicated numbers of experiments ( * * * , p < 0 . 001 Student ' s t - test ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Net transport current amplitudes for WT and L448A/T EAAT1 (GltPh A360) for different pipette solutions, Values are given as mean ± SD with indicated numbers of experiments (***, p<0.001 Student's t-test).


( E ) Representative transport current recordings from a cell expressing R478A EAAT2 ( GltPh R397 ) in the absence of internal K + . Transport currents were elicited by voltage jumps to - 140mV before and after superfusion with 5 mM l - serine . Transient capacitive currents during the first 5 ms after a voltage jump were blanked .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O

(E) Representative transport current recordings from a cell expressing R478A EAAT2 (GltPh R397) in the absence of internal K+. Transport currents were elicited by voltage jumps to -140mV before and after superfusion with 5 mM l-serine. Transient capacitive currents during the first 5 ms after a voltage jump were blanked.


( F ) Time - dependent changes in steady - state R478A EAAT2 ( GltPh R397 ) uptake currents during two consecutive serine applications and subsequent removals . Current amplitudes were measured at the end of voltage jumps to - 140mV and plotted versus time . Serine application is indicated by green bars . Values are given as mean ± SD of the last 100 data points in the current trace .

O O O O O O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(F) Time-dependent changes in steady-state R478A EAAT2 (GltPh R397) uptake currents during two consecutive serine applications and subsequent removals. Current amplitudes were measured at the end of voltage jumps to -140mV and plotted versus time. Serine application is indicated by green bars. Values are given as mean ± SD of the last 100 data points in the current trace.


( G ) Current - voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l - serine for different intracellular solutions . Values are given as mean ± SD for the indicated number of experiments .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(G) Current-voltage relationship for mean net transport currents for R478A EAAT2 induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.


( H ) Mean net transport currents for WT and R478A EAAT2 ( GltPh R397 ) induced by 5 mM l - serine for different intracellular solutions . Values are given as mean ± SD for the indicated number of experiments .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(H) Mean net transport currents for WT and R478A EAAT2 (GltPh R397) induced by 5 mM l-serine for different intracellular solutions. Values are given as mean ± SD for the indicated number of experiments.


( A - B ) Western - Blotting ( WB ) of p53 - negative lung cancer H1299 cells stably expressing ( A ) or transiently expressing ( B ) the indicated constructs . Post - translationally modified Δ133p53 and Δ160p53 isoforms are indicated with * .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) Western-Blotting (WB) of p53-negative lung cancer H1299 cells stably expressing (A) or transiently expressing (B) the indicated constructs. Post-translationally modified Δ133p53 and Δ160p53 isoforms are indicated with *.


( C ) The same WB membrane containing lysates from R273Hp53 - expressing A431cells was incubated with rabbit polyclonal CM - 1 antibody and mouse monoclonal 1801 antibody against the N - terminus of p53 . Detection using anti - rabbit IRDye 680LT ( red ) and anti - mouse IRDye 800CW ( green ) secondary antibodies confirmed that the Δ160p53 band corresponds to a C - terminal isoform of p53 .

O O O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O

(C) The same WB membrane containing lysates from R273Hp53-expressing A431cells was incubated with rabbit polyclonal CM-1 antibody and mouse monoclonal 1801 antibody against the N-terminus of p53. Detection using anti-rabbit IRDye 680LT (red) and anti-mouse IRDye 800CW (green) secondary antibodies confirmed that the Δ160p53 band corresponds to a C-terminal isoform of p53.


( D ) WB of H1299 cells transiently expressing the indicated constructs .

O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O

(D) WB of H1299 cells transiently expressing the indicated constructs.


( E ) WB of cell lines expressing wild - type ( HCT116 , U2OS , A549 ) or mutant ( SW480 [ R273H / P309S ] , MDAMB231 [ R280K ] , A431 [ R273H ] , HT29 [ R273H ] and SKBR3 [ R175H ] ) p53 .

O O O B-EXP_ASSAY O O O O O O O O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O O O O B-CELL_LINE O O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O B-GENEPROD O

(E) WB of cell lines expressing wild-type (HCT116, U2OS, A549) or mutant (SW480 [R273H/P309S], MDAMB231 [R280K], A431 [R273H], HT29 [R273H] and SKBR3 [R175H]) p53.


( F ) Cell lines harbouring endogenous wild - type ( A549 ) or R273H mutant ( A431 ) p53 were treated or not with control siRNA ( ctl ) or siRNA targeting both p53 transcripts ( ex6 and ex7 ) , full - length mRNA only ( ex2 / 3 ) or Δ133p53 mRNA only ( in4 ) before lysis and WB .

O O O O O O O O O O O B-CELL_LINE O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O

(F) Cell lines harbouring endogenous wild-type (A549) or R273H mutant (A431) p53 were treated or not with control siRNA (ctl) or siRNA targeting both p53 transcripts (ex6 and ex7), full-length mRNA only (ex2/3) or Δ133p53 mRNA only (in4) before lysis and WB.


( G ) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO , proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide ( CHX ) . Shown are representative data of three independent experiments . The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α - tubulin or GAPDH and relative to the value indicated in bold and set to 1 . 0 . In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells .

O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O

(G) WB of H1299 cells stably expressing R273Hp53 and treated with DMSO, proteasome inhibitor MG132 or mRNA translation inhibitor cycloheximide (CHX). Shown are representative data of three independent experiments. The numbers in parenthesis indicate the amounts of protein for the indicated bands according to WB quantifications and normalization against α-tubulin or GAPDH and relative to the value indicated in bold and set to 1.0. In WBs for endogenous p53 cell lines the Δ160 lane is used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.


( A ) Cell lines endogenously expressing mutant R273Hp53 ( A431 and HT29 ) were treated with control siRNA ( ctl ) or siRNA targeting exon 7 of p53 , submitted to endoplasmic reticulum ( ER ) stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . Data was normalized against si ctl condition , which was set to 100 .

O O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O

(A) Cell lines endogenously expressing mutant R273Hp53 (A431 and HT29) were treated with control siRNA (ctl) or siRNA targeting exon 7 of p53, submitted to endoplasmic reticulum (ER) stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. Data was normalized against si ctl condition, which was set to 100.


( B and C ) A549 ( B ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( over - confluency ( O - C ) for A549 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) . Also shown is the WB of A549 cells used in ( B ) and stably expressing the indicated constructs .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O

(B and C) A549 (B) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (over-confluency (O-C) for A549). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53). Also shown is the WB of A549 cells used in (B) and stably expressing the indicated constructs.


( B and C ) H1299 ( C ) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli ( thapsigargin ( Th ) for H1299 ) . Data was normalized against 1st day values . Δ160p53 shows similar pro - proliferative capacities as mutant R273Hp53 . On the other hand R273Hp53 lost pro - proliferative functions when deficient for Δ160p53 expression ( M160AR273Hp53 ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B and C) H1299 (C) lung cancer cell lines stably expressing the indicated constructs were counted with Trypan blue for several days following stress stimuli (thapsigargin (Th) for H1299). Data was normalized against 1st day values. Δ160p53 shows similar pro-proliferative capacities as mutant R273Hp53. On the other hand R273Hp53 lost pro-proliferative functions when deficient for Δ160p53 expression (M160AR273Hp53).


( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) or cultured under regular conditions ( D ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G) or cultured under regular conditions (D). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).


( D to G ) H1299 cells transfected with the indicated constructs were submitted to ER stress ( E and G ) , DNA - damage by 36h 3 μM etoposide ( Eto ) treatment ( F ) . Data was normalized against empty vector ( E ) or p53 ( D ) condition , which were set to 100 . Cells were then analysed for apoptosis as in ( A ) . Shown are representative data or averages + s . d . of three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 and ^ P > 0 . 05 compared to control ( " Th + si ctl " or " - " ) or as indicated ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D to G) H1299 cells transfected with the indicated constructs were submitted to ER stress (E and G), DNA-damage by 36h 3 μM etoposide (Eto) treatment (F). Data was normalized against empty vector (E) or p53 (D) condition, which were set to 100. Cells were then analysed for apoptosis as in (A). Shown are representative data or averages + s.d. of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.005 and ^P > 0.05 compared to control ("Th + si ctl" or "-" ) or as indicated).


( A ) Adhesion assay shows that MCF10A cells stably expressing Δ133p53 , Δ160p53 , R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells . Excluding Δ160p53 expression from the mutant background ( M160A / R273Hp53 ) rescued control - cell phenotype . Scale bar represents 200 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Adhesion assay shows that MCF10A cells stably expressing Δ133p53, Δ160p53, R273HΔ160p53 or missense mutant R273Hp53 adhere more strongly than control cells. Excluding Δ160p53 expression from the mutant background (M160A/R273Hp53) rescued control-cell phenotype. Scale bar represents 200 μm.


( B ) WB of MCF10A cells used in ( A ) and stably expressing the indicated constructs . Shown are representative data of three independent experiments and quantifications .

O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O

(B) WB of MCF10A cells used in (A) and stably expressing the indicated constructs. Shown are representative data of three independent experiments and quantifications.


( C and D ) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel . Control - and Δ133p53 - expressing cells formed regular three - dimensional ( 3D ) mammary acinar structures with hollow lumina but Δ160p53 - , R273HΔ160p53 and to a smaller extent mutant R273Hp53 - expressing cells formed acini with filled lumen and long invasive structures ( n > 30 acini per experiment per condition ) .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O

(C and D) MCF10A human breast epithelial cells stably expressing the indicated constructs were cultured in matrigel. Control- and Δ133p53-expressing cells formed regular three-dimensional (3D) mammary acinar structures with hollow lumina but Δ160p53-, R273HΔ160p53 and to a smaller extent mutant R273Hp53-expressing cells formed acini with filled lumen and long invasive structures (n > 30 acini per experiment per condition).


O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O


( E ) H1299 cells stably expressing empty vector ( control ) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) H1299 cells stably expressing empty vector (control) or the indicated constructs were assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


( A ) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos ( Ctl - 1 or Ctl - 2 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation ( MO )

O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) WB of H1299 cells stably expressing Δ133p53 and treated with control morpholino oligos (Ctl-1 or Ctl-2) or antisense morpholino oligo targeting Δ160p53's translation initiation (MO)


( B ) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos ( Ctl - 2 or Ctl - 1 ) or antisense morpholino oligo targeting Δ160p53 ' s translation initiation site ( MO ) before lysis and WB . wtp53 - expressing A549 cells were used as reference for endogenous wtp53 expression levels . Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53 - null H1299 cells .

O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_LINE O O

(B) A431 and HT29 cells expressing endogenous mutant R273Hp53 were treated with control morpholinos (Ctl-2 or Ctl-1) or antisense morpholino oligo targeting Δ160p53's translation initiation site (MO) before lysis and WB. wtp53-expressing A549 cells were used as reference for endogenous wtp53 expression levels. Δ160 lane was used as a marker lane showing Δ160p53 as transiently expressed in p53-null H1299 cells.


( C ) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino ( Ctl - 1 ) or morpholino targeting 160p53 ' s translation initiation site ( MO ) . MO efficiently targets 160p53 in endogenous R273Hp53 cell lines . See also figure EV3 for the raw data .

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

(C) Shown are the average Δ160p53 protein levels expressed in R273Hp53 versus other mutant p53cell lines that were treated with control morpholino (Ctl-1) or morpholino targeting 160p53's translation initiation site (MO). MO efficiently targets 160p53 in endogenous R273Hp53 cell lines. See also figure EV3 for the raw data.


( D - E ) A431 ( D ) and HT29 ( E ) cells endogenously expressing mutant R273Hp53 were treated with control siRNA ( si Ctl ) or control MO ( MO Ctl - 2 or MO Ctl - 1 ) or siRNA targeting exon 7 of p53 ( si ) or MO targeting Δ160p53 ' s translation initiation site ( MO ) , as indicated , submitted to endoplasmic reticulum stress by exposure to thapsigargin ( Th ) and then analysed for apoptosis by incubation with propidium - iodide ( PI ) and FACS analysis . wtp53 - expressing A549 cells were similarly control - treated and used as comparison . Data was normalized against A549 cell condition , which was set to 100 .

O O O O O B-CELL_LINE O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O

(D-E) A431 (D) and HT29 (E) cells endogenously expressing mutant R273Hp53 were treated with control siRNA (si Ctl) or control MO (MO Ctl-2 or MO Ctl-1) or siRNA targeting exon 7 of p53 (si) or MO targeting Δ160p53's translation initiation site (MO), as indicated, submitted to endoplasmic reticulum stress by exposure to thapsigargin (Th) and then analysed for apoptosis by incubation with propidium-iodide (PI) and FACS analysis. wtp53-expressing A549 cells were similarly control-treated and used as comparison. Data was normalized against A549 cell condition, which was set to 100.


( F ) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl - 1 or morpholino targeting Δ160p53 ' s translation initiation site ( MO ) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel - fibronectin matrix towards EGF - supplemented growth media . The 30 μm plane is indicated and scale bars represent 100 μm . Quantifications are shown in the right panel .

O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) A431 and HT29 cells endogenously expressing mutant R273Hp53 were treated with control morpholino Ctl-1 or morpholino targeting Δ160p53's translation initiation site (MO) and then assessed by confocal microscopy for invasion further than 30 μm through a Matrigel-fibronectin matrix towards EGF-supplemented growth media. The 30 μm plane is indicated and scale bars represent 100 μm. Quantifications are shown in the right panel.


Immunoblot analysis of LC3B and GABARAP lipidation in wild - type ( WT ) and ATG16L1 - KO HEK293A cells , with or without stimulation for 2h as indicated . BafA1 stands for Bafilomycin A1 . Gamma - Tubulin ( γ - Tubulin ) is loading control . Densitometry analysis of lipidated LC3B ( LC3B - II ) to γ - tubulin ( normalized to untreated WT cells ) from immunoblots in ( A ) . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of LC3B and GABARAP lipidation in wild-type (WT) and ATG16L1-KO HEK293A cells, with or without stimulation for 2h as indicated. BafA1 stands for Bafilomycin A1. Gamma-Tubulin (γ-Tubulin) is loading control. Densitometry analysis of lipidated LC3B (LC3B-II) to γ-tubulin (normalized to untreated WT cells) from immunoblots in (A). Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.


LC3B / GABARAP lipidation in ATG16L1 - and ATG5 - KO HEK293A cells treated for 2h as indicated . Vinculin is loading control . Cell lysates were immunoblotted for the specified proteins . Ratio of LC3B - II to Vinculin quantified from immunoblots in ( C ) and normalized to MC3 treated ATG16L1 KO cells . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3B/GABARAP lipidation in ATG16L1- and ATG5-KO HEK293A cells treated for 2h as indicated. Vinculin is loading control. Cell lysates were immunoblotted for the specified proteins. Ratio of LC3B-II to Vinculin quantified from immunoblots in (C) and normalized to MC3 treated ATG16L1 KO cells. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.


Immunoblot analysis of LC3B and GABARAP lipidation from WT , ATG16L1 - KO ( L1 - KO ) , ATG16L2 - KO ( L2 - KO ) , TECPR1 - KO ( TEC - KO ) , ATG16L1 / L2 - DKO ( L1 / L2 - DKO ) , ATG16L1 / TECPR1 - DKO ( L1 / TEC DKO ) , and ATG16L1 / L2 / TECPR1 - TKO ( L1 / L2 / TEC - TKO ) PC - 3 cells , treated as indicated for 2h , were immunoblotted for the indicated proteins . Vinculin is loading control . MRT68921 and SAR405 are ULK and VPS34 inhibitors , respectively . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of LC3B and GABARAP lipidation from WT, ATG16L1-KO (L1-KO), ATG16L2-KO (L2-KO), TECPR1-KO (TEC-KO), ATG16L1/L2-DKO (L1/L2-DKO), ATG16L1/TECPR1-DKO (L1/TEC DKO), and ATG16L1/L2/TECPR1-TKO (L1/L2/TEC-TKO) PC-3 cells, treated as indicated for 2h, were immunoblotted for the indicated proteins. Vinculin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.


LC3B / GABARAP lipidation in ATG16L1 - KO and ATG16L1 / TECPR1 - DKO HEK293A cells with or without TECPR1 rescue , treated as indicated for 2h . Cell lysates were immunoblotted against the indicated proteins . Vinculin is loading control . Ratio of LC3B - II to Vinculin quantified from immunoblots in ( G ) and normalized to ATG16L1 - KO cells treated with MC3 . Data information : All data presented as mean + SEM , from n = 3 independent experiments . P - value determined using two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3B/GABARAP lipidation in ATG16L1-KO and ATG16L1/TECPR1-DKO HEK293A cells with or without TECPR1 rescue, treated as indicated for 2h. Cell lysates were immunoblotted against the indicated proteins. Vinculin is loading control. Ratio of LC3B-II to Vinculin quantified from immunoblots in (G) and normalized to ATG16L1-KO cells treated with MC3. Data information: All data presented as mean + SEM, from n = 3 independent experiments. P-value determined using two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant.


LC3B / GABARAP lipidation in WT , ATG16L1 / TECPR1 - DKO HeLa cells with and without EGFP - TECPR1 or EGFP - ATG16L1β rescue , treated for 2h as indicated . Cell lysates were immunoblotted for the indicated proteins . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( A ) and normalized to untreated WT cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO HeLa cells with and without EGFP-TECPR1 or EGFP-ATG16L1β rescue, treated for 2h as indicated. Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (A) and normalized to untreated WT cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


Confocal images of ATG16L1 / TECPR1 - DKO HeLa cells expressing mScarlet - LC3B , with or without EGFP - TECPR1 , treated for 2h with Cy5 - labeled MC3 particles ( left ) or LLOMe ( right ) as indicated . Scale bars : 10 µm .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing mScarlet-LC3B, with or without EGFP-TECPR1, treated for 2h with Cy5-labeled MC3 particles (left) or LLOMe (right) as indicated. Scale bars: 10 µm.


Confocal time lapse images of ATG16L1 / TECPR1 - DKO HeLa cells , showing localization of mScarlet - LC3B and EGFP - TECPR1 , after treatment with 400 μM LLOMe . Scale bar : 10 µm .

B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O

Confocal time lapse images of ATG16L1/TECPR1-DKO HeLa cells, showing localization of mScarlet-LC3B and EGFP-TECPR1, after treatment with 400 μM LLOMe. Scale bar: 10 µm.


Immunoblot analysis of LC3B / GABARAP lipidation in ATG16L1 - KO HEK293A cells , treated with 400µM LLOMe for indicated periods of time . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( E ) and normalized to 2hr ( 120min ) LLOMe treatment . ( Data presented as mean + SEM , n = 4 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ) .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO HEK293A cells, treated with 400µM LLOMe for indicated periods of time. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (E) and normalized to 2hr (120min) LLOMe treatment. (Data presented as mean + SEM, n = 4 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test).


Time series of confocal images showing redistribution of EqtSM - mCherry ( sphingomyelin ( SM ) probe ) and EGFP - TECPR1 in ATG16L1 / TECPR1 - DKO cells with doxycycline - inducible ( dox , 2μg / ml ) expression of EqtSM - mCherry cells following treatment with 400µM LLOMe . Scale bar : 10 µm .

O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O

Time series of confocal images showing redistribution of EqtSM-mCherry (sphingomyelin (SM) probe) and EGFP-TECPR1 in ATG16L1/TECPR1-DKO cells with doxycycline-inducible (dox, 2μg/ml) expression of EqtSM-mCherry cells following treatment with 400µM LLOMe. Scale bar: 10 µm.


LC3B / GABARAP lipidation in ATG16L1 - and TECPR1 - KO HEK293A cells with or without dox - inducible ( 2μg / ml ) expression of nSMase2 - mCherry , treated for 2h as indicated . Cell lysates were immunoblotted against the indicated proteins . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( B ) and normalized to untreated ATG16L1 - KO cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3B/GABARAP lipidation in ATG16L1- and TECPR1-KO HEK293A cells with or without dox-inducible (2μg/ml) expression of nSMase2-mCherry, treated for 2h as indicated. Cell lysates were immunoblotted against the indicated proteins. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (B) and normalized to untreated ATG16L1-KO cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


Confocal images of ATG16L1 / TECPR1 - DKO HeLa cells expressing EGFP - TECPR1 , with or without dox - inducible ( 2μg / ml ) nSMase2 overexpression , treated for 2h as indicated . Scale bars : 10 µm . Beeswarm - Superplot displaying mean number of EGFP - TECPR1 dots per cell per image of data represented in ( E ) quantified by automated analysis using CellProfiler . Mean ± SEM from each independent experiment ( 3 / group ) presented as large data points , with the black line corresponding to the mean of means . Individual data points ( small ) corresponding to single images are color - coded and superimposed according to the biological replicate they originate from . Total cells per condition / images per condition : 952 / 30 ( LLOMe treated - nSMase2 ) and 1056 / 30 ( LLOMe treated + nSMase2 ) were analyzed . ( P - values determined using Student ' s unpaired t test ) .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O

Confocal images of ATG16L1/TECPR1-DKO HeLa cells expressing EGFP-TECPR1, with or without dox-inducible (2μg/ml) nSMase2 overexpression, treated for 2h as indicated. Scale bars: 10 µm. Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they originate from. Total cells per condition/images per condition: 952/30 (LLOMe treated - nSMase2) and 1056/30 (LLOMe treated + nSMase2) were analyzed. (P-values determined using Student's unpaired t test).


Liposome co - sedimentation assay of the indicated recombinant proteins and liposomes with or without SM . S : supernatant ; P : pellet . Liposome binding in ( C ) was quantified as percentage of total protein in the pellet . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S: supernatant; P: pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


Immunoblot analysis of LC3B / GABARAP lipidation in ATG16L1 - KO or ATG16L1 / TECPR1 - DKO HEK293A cells with or without rescue with TECPR1 ( WT ) , TECPR1 W77A , TECPR1 W154A , TECPR1 W829A or TECPR1 F908A , and treated with 400 µM LLOMe for 2h . γ - Tubulin is loading control . Ratio of LC3B - II to γ - Tubulin quantified from immunoblots in ( C ) and normalized to ATG16L1 - KO cells . Data information : All data presented as mean + SEM ( in ( D ) n = 3 independent experiments . P - values determined using one - way ANOVA followed by Tukey ' s multiple comparisons test in D and H ; ns = not significant .

B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot analysis of LC3B/GABARAP lipidation in ATG16L1-KO or ATG16L1/TECPR1-DKO HEK293A cells with or without rescue with TECPR1 (WT), TECPR1 W77A, TECPR1 W154A, TECPR1 W829A or TECPR1 F908A, and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control. Ratio of LC3B-II to γ-Tubulin quantified from immunoblots in (C) and normalized to ATG16L1-KO cells. Data information: All data presented as mean + SEM (in (D) n = 3 independent experiments. P-values determined using one-way ANOVA followed by Tukey's multiple comparisons test in D and H; ns= not significant.


LC3B / GABARAP lipidation in ATG16L1 / TECPR1 - DKO HEK293A cells rescued with TECPR1 ( WT ) , TECPR1 W77A , TECPR1 W829A or TECPR1 W77A / W829A ( WAWA ) , and treated with 400 µM LLOMe for 2h . γ - Tubulin is loading control .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

LC3B/GABARAP lipidation in ATG16L1/TECPR1-DKO HEK293A cells rescued with TECPR1 (WT), TECPR1 W77A, TECPR1 W829A or TECPR1 W77A/W829A (WAWA), and treated with 400 µM LLOMe for 2h. γ-Tubulin is loading control.


Maximum intensity projection of confocal images of HeLaK cells expressing EGFP - TECPR1 ( WT ) or W77A / W829A ( WAWA ) mutant , with or without 400µM LLOMe for 30min . Nuclei were counterstained with Hoechst 33342 ( in blue ) . Scale bar : 10μm Beeswarm - Superplot displaying mean number of EGFP - TECPR1 dots per cell per image of data represented in ( G ) quantified by automated analysis using CellProfiler . Mean ± SEM from each independent experiment ( 3 / group ) presented as large data points , with the black line corresponding to the mean of means . Individual data points ( small ) corresponding to single images are color - coded and superimposed according to the biological replicate they came from . Total cells per condition / images per condition : 2502 / 59 ( WT untreated ) , 2549 / 48 ( WT LLOMe ) , 2256 / 59 ( WAWA untreated ) and 2816 / 59 ( WAWA LLOMe ) were analyzed .

O O O O B-EXP_ASSAY O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O

Maximum intensity projection of confocal images of HeLaK cells expressing EGFP-TECPR1 (WT) or W77A/W829A (WAWA) mutant, with or without 400µM LLOMe for 30min. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying mean number of EGFP-TECPR1 dots per cell per image of data represented in (G) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single images are color-coded and superimposed according to the biological replicate they came from. Total cells per condition/images per condition: 2502/59 (WT untreated), 2549/48 (WT LLOMe), 2256/59 (WAWA untreated) and 2816/59 (WAWA LLOMe) were analyzed.


Coomassie - stained gel depicting in vitro LC3B lipidation reactions using the indicated combinations of proteins and liposomes ( with or without SM ) , after being subjected to a liposome co - sedimentation assay . LC3B - AMP is denoted with an asterix ( * ) . The extent of LC3B - I to - II conversion in ( A ) was determined and plotted as percentage of total LC3B . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Coomassie-stained gel depicting in vitro LC3B lipidation reactions using the indicated combinations of proteins and liposomes (with or without SM), after being subjected to a liposome co-sedimentation assay. LC3B-AMP is denoted with an asterix (*). The extent of LC3B-I to -II conversion in (A) was determined and plotted as percentage of total LC3B. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


Liposome co - sedimentation assay of the indicated recombinant proteins and liposomes with or without SM . S , supernatant ; P , pellet . Liposome binding in ( C ) was quantified as percentage of total protein in the pellet . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) .

Liposome co-sedimentation assay of the indicated recombinant proteins and liposomes with or without SM. S, supernatant; P, pellet. Liposome binding in (C) was quantified as percentage of total protein in the pellet. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant).


LC3B / GABARAP lipidation in WT , ATG16L1 / TECPR1 - DKO , ATG16L1 - KO and TECPR1 - KO HeLa cells with or without dox - inducible ( 2μg / ml ) expression of mCherry - SopF , treated as indicated for 2h , were immunoblotted for the indicated proteins . γ - Tubulin is loading control . MRT68921 and SAR405 are ULK and VPS34 inhibitors , respectively . Ratio of LC3B - II to γ - Tubulin or Vinculin quantified from immunoblots in ( A ) and normalized to WT monensin treated cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . ( Data has skewed distribution . P - values were determined with Kruskal - Wallis followed by Dunn ' s multiple comparisons test ) .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).


LC3B / GABARAP lipidation in WT , ATG16L1 / TECPR1 - DKO , ATG16L1 - KO and TECPR1 - KO HeLa cells with or without dox - inducible ( 2μg / ml ) expression of mCherry - SopF , treated as indicated for 2h , Cell lysates were immunoblotted for the indicated proteins . γ - Tubulin is loading control . MRT68921 and SAR405 are ULK and VPS34 inhibitors , respectively . Ratio of LC3B - II to γ - Tubulin or Vinculin quantified from immunoblots in ( A ) and normalized to WT monensin treated cells . ( Data presented as mean + SEM , n = 3 independent experiments , P - value from two - way ANOVA followed by Tukey ' s multiple comparisons test ; ns = not significant ) . ( Data has skewed distribution . P - values were determined with Kruskal - Wallis followed by Dunn ' s multiple comparisons test ) .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LC3B/GABARAP lipidation in WT, ATG16L1/TECPR1-DKO, ATG16L1-KO and TECPR1-KO HeLa cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h, Cell lysates were immunoblotted for the indicated proteins. γ-Tubulin is loading control. MRT68921 and SAR405 are ULK and VPS34 inhibitors, respectively. Ratio of LC3B-II to γ-Tubulin or Vinculin quantified from immunoblots in (A) and normalized to WT monensin treated cells. (Data presented as mean + SEM, n = 3 independent experiments, P-value from two-way ANOVA followed by Tukey's multiple comparisons test; ns= not significant). (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).


Confocal images of WT and TECPR1 - KO cells with or without dox - inducible ( 2μg / ml ) expression of mCherry - SopF , treated as indicated for 2h . Nuclei were counterstained with Hoechst 33342 ( in blue ) . Scale bar : 10μm Beeswarm - Superplot displaying number of LC3 puncta per cell in the images represented in ( E ) quantified by automated analysis using CellProfiler . Mean ± SEM from each independent experiment ( 3 / group ) presented as large data points , with the black line corresponding to the mean of means . Individual data points ( small ) corresponding to single cells are color - coded and superimposed according to the biological replicate they originated from . Total cells per condition / images per condition : 1169 / 30 ( WT LLOMe ) , 1064 / 29 ( WT - mcherrySopF LLOMe ) , 692 / 23 ( TECKO LLOMe ) and 983 / 30 ( TECKO - mcherrySopF untreated ) were analyzed . ( Data has skewed distribution . P - values were determined with Kruskal - Wallis followed by Dunn ' s multiple comparisons test ) .

B-EXP_ASSAY O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Confocal images of WT and TECPR1-KO cells with or without dox-inducible (2μg/ml) expression of mCherry-SopF, treated as indicated for 2h. Nuclei were counterstained with Hoechst 33342 (in blue). Scale bar: 10μm Beeswarm-Superplot displaying number of LC3 puncta per cell in the images represented in (E) quantified by automated analysis using CellProfiler. Mean ± SEM from each independent experiment (3/group) presented as large data points, with the black line corresponding to the mean of means. Individual data points (small) corresponding to single cells are color-coded and superimposed according to the biological replicate they originated from. Total cells per condition/images per condition: 1169/30 (WT LLOMe), 1064/29 (WT-mcherrySopF LLOMe), 692/23 (TECKO LLOMe) and 983/30 (TECKO-mcherrySopF untreated) were analyzed. (Data has skewed distribution. P-values were determined with Kruskal-Wallis followed by Dunn's multiple comparisons test).


A Analysis of published single - cell RNA sequencing data from lungs of non - infected mice and mice infected with SARS - CoV - 2 MA10 ( GEO accession No . GSE186360 ) . Av Mφ = alveolar macrophages , DC = dendritic cells , infl mo = inflammatory monocytes , non clas mo = non - classical monocytes , trans mo = transitional / intermediate monocytes .

A Analysis of published single-cell RNA sequencing data from lungs of non-infected mice and mice infected with SARS-CoV-2 MA10 (GEO accession No. GSE186360). Av Mφ = alveolar macrophages, DC = dendritic cells, infl mo = inflammatory monocytes, non clas mo = non-classical monocytes, trans mo = transitional/intermediate monocytes.


D Viral titers in lung homogenates at the indicated days post infection from mice infected as in B ( bars / error bars represent mean ± SD , data points represent distinct biological samples , data points at the x axis represent samples below the limit of detection , day 3 data from two independent experiments , day 2 and 5 data are from single experiments , * * p < 0 . 01 , Mann - Whitney test ) .

O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O O O O O B-EXP_ASSAY O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Viral titers in lung homogenates at the indicated days post infection from mice infected as in B (bars/error bars represent mean ± SD, data points represent distinct biological samples, data points at the x axis represent samples below the limit of detection, day 3 data from two independent experiments, day 2 and 5 data are from single experiments, **p < 0.01, Mann-Whitney test).


E IL - 6 levels from lung homogenates and TNFα and IL - 1β levels from serum on day 3 post infection ( bars / error bars represent mean ± SD , data points represent distinct biological samples , data from two independent experiments , * * * p < 0 . 001 , t - test ) .

O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E IL-6 levels from lung homogenates and TNFα and IL-1β levels from serum on day 3 post infection (bars/error bars represent mean ± SD, data points represent distinct biological samples, data from two independent experiments, ***p < 0.001, t-test).


F Viral titers in extrapulmonary organs on day 3 post infection were measured ( bars / error bars represent mean ± SD , data points represent distinct biological samples [ n = 7 for heart , spleen , brain ; n = 5 for kidney and liver ] , data points at the x axis represent samples below the limit of detection , ns , multiple unpaired t - tests ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O

F Viral titers in extrapulmonary organs on day 3 post infection were measured (bars/error bars represent mean ± SD, data points represent distinct biological samples [n=7 for heart, spleen, brain; n=5 for kidney and liver], data points at the x axis represent samples below the limit of detection, ns, multiple unpaired t-tests).


G Representative lung sections from WT or IFITM3 KO animals stained for SARS - CoV - 2 N protein . Scale bars represent 2 mm for whole lung images . Colored boxes overlaying the whole lung images correspond to the displayed magnified regions and scale bars in these images represent 0 . 2 mm . Red triangles , infected airways ; solid black arrow , non - infected blood vessels ; open black arrow , infected blood vessels .

O O B-TISSUE O O O O B-GENEPROD O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O O O O O B-TISSUE O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY B-TISSUE I-TISSUE O O O O O B-EXP_ASSAY B-TISSUE I-TISSUE O

G Representative lung sections from WT or IFITM3 KO animals stained for SARS-CoV-2 N protein. Scale bars represent 2 mm for whole lung images. Colored boxes overlaying the whole lung images correspond to the displayed magnified regions and scale bars in these images represent 0.2 mm. Red triangles, infected airways; solid black arrow, non-infected blood vessels; open black arrow, infected blood vessels.


K18 - hACE2 ( hACE2 ) and K18 - hACE2 / IFITM3 KO mice were infected with 104 TCID SARS - CoV - 2 WA1 . A Weight loss ( data points represent mean and error bars represent SEM , data from two independent experiments [ hACE2 n = 7 , hACE2 / IFITM3 KO n = 8 ] , skull / crossbones indicates that KO mice did not survive beyond this timepoint , * p < 0 . 05 , * * p < 0 . 01 , ANOVA with Bonferoni ' s multiple comparison ' s test ) .

O O O O O O O O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. A Weight loss (data points represent mean and error bars represent SEM, data from two independent experiments [hACE2 n=7, hACE2/IFITM3 KO n=8], skull/crossbones indicates that KO mice did not survive beyond this timepoint, *p < 0.05, ** p < 0.01, ANOVA with Bonferoni's multiple comparison's test).


K18 - hACE2 ( hACE2 ) and K18 - hACE2 / IFITM3 KO mice were infected with 104 TCID SARS - CoV - 2 WA1 . C Viral titers in extrapulmonary organs on the indicated days post infection were measured ( bars / error bars represent mean ± SD , data points represent distinct biological samples , day 3 data for heart , spleen , and brain are from two independent experiments , day 3 data for liver and kidney and day 5 data are from single experiments , data points at the x axis represent samples below the limit of detection , * * p < 0 . 01 , Mann - Whitney test ) .

O O O O O O O O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. C Viral titers in extrapulmonary organs on the indicated days post infection were measured (bars/error bars represent mean ± SD, data points represent distinct biological samples, day 3 data for heart, spleen, and brain are from two independent experiments, day 3 data for liver and kidney and day 5 data are from single experiments, data points at the x axis represent samples below the limit of detection, **p < 0.01, Mann-Whitney test).


K18 - hACE2 ( hACE2 ) and K18 - hACE2 / IFITM3 KO mice were infected with 104 TCID SARS - CoV - 2 WA1 . D Representative lung sections from WT mice infected with SARS - CoV - 2 MA10 ( 105 TCID ) or K18 - hACE2 or K18 - hACE2 / IFITM3 KO mice infected with SARS - CoV - 2 WA1 ( 104 TCID ) stained for SARS - CoV - 2 N protein at day 5 post infection . Scale bars represent 2mm .

O O O O O O O O O O O B-GENEPROD O B-ORGANISM O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O B-TISSUE O O O B-ORGANISM B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-ORGANISM B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O O O O O O

K18-hACE2 (hACE2) and K18-hACE2/IFITM3 KO mice were infected with 104 TCID SARS-CoV-2 WA1. D Representative lung sections from WT mice infected with SARS-CoV-2 MA10 (105 TCID) or K18-hACE2 or K18-hACE2/IFITM3 KO mice infected with SARS-CoV-2 WA1 (104 TCID) stained for SARS-CoV-2 N protein at day 5 post infection. Scale bars represent 2mm.


WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS - CoV - 2 MA10 or mock infected with PBS ( Non - Infected , NI ) . A Representative lung images from mice at the indicated times post infection were stained with hematoxylin and eosin . Scale bars represent 2mm for whole lung images and 500um for zoomed images . B Staining quantifications from multiple mice are shown ( bars / error bars represent mean ± SD , data points represent distinct biological samples , * p < 0 . 05 , ANOVA with Bonferoni ' s multiple comparison ' s test ) .

O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O B-TISSUE O O B-ORGANISM O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with hematoxylin and eosin. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).


WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS - CoV - 2 MA10 or mock infected with PBS ( Non - Infected , NI ) . A Representative lung images from mice at the indicated times post infection were stained with anti - CD45 . Scale bars represent 2mm for whole lung images and 500um for zoomed images . B Staining quantifications from multiple mice are shown ( bars / error bars represent mean ± SD , data points represent distinct biological samples , * p < 0 . 05 , ANOVA with Bonferoni ' s multiple comparison ' s test ) .

O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O B-TISSUE O O B-ORGANISM O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10 or mock infected with PBS (Non-Infected, NI). A Representative lung images from mice at the indicated times post infection were stained with anti-CD45. Scale bars represent 2mm for whole lung images and 500um for zoomed images. B Staining quantifications from multiple mice are shown (bars/error bars represent mean ± SD, data points represent distinct biological samples, *p < 0.05, ANOVA with Bonferoni's multiple comparison's test).


WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS - CoV - 2 MA10 . RNA sequencing analysis was performed on RNA extracted from infected WT and IFITM3 KO lungs on day 2 post infection . D Relative gene expression heat maps for genes from the top two most significant GO Biological Processes as shown in C .

O O B-GENEPROD O B-ORGANISM O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-TISSUE O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O

WT and IFITM3 KO mice were intranasally infected with 105 TCID50 SARS-CoV-2 MA10. RNA sequencing analysis was performed on RNA extracted from infected WT and IFITM3 KO lungs on day 2 post infection. D Relative gene expression heat maps for genes from the top two most significant GO Biological Processes as shown in C.


Primary skin fibroblasts from a normal individual , SLOS patient , ZS patients , X - ALD patient , and NPC patient incubated for 24 h without serum were immunostained with anti - pericentrin ( red ) and anti - acetylated tubulin ( blue ) antibodies . Cholesterol was stained with Filipin III ( green ) . Arrows indicate primary cilia . Scale bar , 5 μm . The intensity of Filipin III signal at primary cilia from ( a ) was remarkably reduced in SLOS and ZS patient cells ( * * p < 0 . 01 : one - way ANOVA with Tukey ' s HSD , n = 3 : 45 - 50 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

O B-TISSUE B-CELL_TYPE O O O O O B-DISEASE O O B-DISEASE O O B-DISEASE I-DISEASE I-DISEASE O O O B-DISEASE O O O O O O B-TISSUE O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O O O O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Primary skin fibroblasts from a normal individual, SLOS patient, ZS patients, X-ALD patient, and NPC patient incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 μm. The intensity of Filipin III signal at primary cilia from (a) was remarkably reduced in SLOS and ZS patient cells (**p<0.01: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.


Immunofluorescent staining of primary skin fibroblasts in quiescent G0 phase treated with 50 nM Shh - N for 24 h using anti - Smo ( green ) , anti - acetylated - tubulin ( blue ) , and anti - γ - tubulin ( red ) antibodies . Arrows indicate primary cilia . Scale bar , 2 . 5 μm . Quantification of proportion of Smo - positive ciliated cells upon Shh - N stimulation from ( C ) . The populations of Smo - positive ciliated fibroblasts from the SLOS and ZS patients were significantly reduced ( mean ± s . d . : * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 100 cells per experiment ) . The Smo intensity at primary cilia in the fibroblasts from ( C ) . The ciliary localization of Smo upon Shh - N stimulation was significantly impaired in fibroblasts from the SLOS and ZS patients ( * * * p < 0 . 001 : one - way ANOVA with Tukey ' s HSD , n = 3 : 90 - 100 cells per experiment ) . In the boxplot , medians , 25th / 75th percentile and min / max were represented by the central lines , the box limits , and the whiskers / error bars , representatively .

B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR B-CELL_TYPE O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O B-CELL_TYPE O O B-DISEASE O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunofluorescent staining of primary skin fibroblasts in quiescent G0 phase treated with 50 nM Shh-N for 24 h using anti-Smo (green), anti-acetylated-tubulin (blue), and anti-γ-tubulin (red) antibodies. Arrows indicate primary cilia. Scale bar, 2.5 μm. Quantification of proportion of Smo-positive ciliated cells upon Shh-N stimulation from (C). The populations of Smo-positive ciliated fibroblasts from the SLOS and ZS patients were significantly reduced (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 100 cells per experiment). The Smo intensity at primary cilia in the fibroblasts from (C). The ciliary localization of Smo upon Shh-N stimulation was significantly impaired in fibroblasts from the SLOS and ZS patients (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 90-100 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.