( F ) Body temperature loss upon cold exposure of AdRiKO and control mice ( n = 20 ( control ) , n = 17 ( AdRiKO ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Body temperature loss upon cold exposure of AdRiKO and control mice (n=20 (control), n=17 (AdRiKO)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is not required for cold - induced lipid droplet mobilization . ( A ) Representative H & amp ; amp ; E staining of sWAT sections from AdRiKO and control mice ( n = 5 / group ) .

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

mTORC2 in adipose tissue is not required for cold-induced lipid droplet mobilization. (A) Representative H&amp;E staining of sWAT sections from AdRiKO and control mice (n=5/group).


( B ) Non - esterified fatty acids ( NEFAs ) in plasma of AdRiKO and control mice ( n = 6 / group ) .

O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O

(B) Non-esterified fatty acids (NEFAs) in plasma of AdRiKO and control mice (n=6/group).


( C ) Glycerol in plasma of AdRiKO and control mice ( n = 6 / group ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O

(C) Glycerol in plasma of AdRiKO and control mice (n=6/group).


( D ) Representative H & amp ; amp ; E staining of BAT sections from AdRiKO and control mice ( n = 5 / group ) .

O O O O 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) Representative H&amp;E staining of BAT sections from AdRiKO and control mice (n=5/group).


( E ) Triglycerides ( TGs ) in BAT of AdRiKO and control mice housed at 22 ° C or 4 ° C for 8h ( n = 6 / group ) .

O O O B-MEASURED_VAR O O O O O 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) Triglycerides (TGs) in BAT of AdRiKO and control mice housed at 22°C or 4°C for 8h (n=6/group).


( F ) NEFAs in BAT of AdRiKO and control mice ( n = 6 / group ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NEFAs in BAT of AdRiKO and control mice (n=6/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is not required for cold - induced mitochondrial uncoupling and β - oxidation . ( A ) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group ) .

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

mTORC2 in adipose tissue is not required for cold-induced mitochondrial uncoupling and β-oxidation. (A) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).


( B ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( C ) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group , each lane represents a mix of 3 mice ) .

(C) mRNA levels of the indicated genes in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group, each lane represents a mix of 3 mice).


( D ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(D) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( E ) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 6 / group ) .

O O O O O O O O O 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) Mitochondrial DNA content of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=6/group).


( F ) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 3 / group ) .

O O O O O O O O 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) Representative electronmicrographs of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=3/group).


( G ) Oxygen consumption rate ( OCR ) of BAT explants from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / group ) .

O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Oxygen consumption rate (OCR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).


H ) Maximal respiration ( VO2 max ) of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h ( n = 9 ( control 22 ° C ) , n = 7 ( AdRiKO 22 ° C ) , n = 8 ( control 4 ° C ) , n = 8 ( AdRiKO 4 ° C ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximal respiration (VO2 max) of AdRiKO and control mice housed at 22°C or at 4°C for 8h (n=9 (control 22°C), n=7 (AdRiKO 22°C), n=8 (control 4°C), n=8 (AdRiKO 4°C).


( I ) Respiration ( VO2 ) of AdRiKO and control mice upon cold exposure ( n = 8 / group ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

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

(I) Respiration (VO2) of AdRiKO and control mice upon cold exposure (n=8/group). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


mTORC2 in adipose tissue is required for cold - induced glucose uptake and glycolysis ( A ) 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 6 / group ) . (

O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

mTORC2 in adipose tissue is required for cold-induced glucose uptake and glycolysis (A) 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=6/group). (


( B ) Extracellular acidification rate ( ECAR ) of BAT explants from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 7 / group ) .

O O O O O O O O O O O O O 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) Extracellular acidification rate (ECAR) of BAT explants from AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=7/group).


( C ) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( D ) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 8h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of isolated plasma membranes from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 8h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( E ) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h for the indicated proteins ( n = 6 / group , each lane represents a mix of 3 mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot analysis of mitochondrial and cytosolic fractions from BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( F ) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 5 ( control 22 ° C ) , n = 5 ( AdRiKO 22 ° C ) , n = 7 ( control 4 ° C ) , n = 7 AdRiKO 4 ° C ) ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cytosolic hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)).


( G ) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22 ° C or at 4 ° C for 4h ( n = 5 ( control 22 ° C ) , n = 5 ( AdRiKO 22 ° C ) , n = 7 ( control 4 ° C ) , n = 7 AdRiKO 4 ° C ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between temperatures were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Mitochondrial hexokinase activity in BAT of AdRiKO and control mice housed at 22°C or at 4°C for 4h (n=5 (control 22°C), n=5 (AdRiKO 22°C), n=7 (control 4°C), n=7 AdRiKO 4°C)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between temperatures were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice . ( A ) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty ( n = 8 / group ) .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O

Restoration of glucoseuptake or Akt signaling suppresses the thermogenic defect in AdRiKO mice. (A) HKII mRNA expression level in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty (n=8/group).


( B ) Cold - induced 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty housed at 4 ° C for 4h ( n = 8 / group ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

(B) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 4°C for 4h (n=8/group).


( C ) Body temperature of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty housed at 22 ° C ( n = 8 / group ) .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

(C) Body temperature of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty housed at 22°C (n=8/group).


( D ) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9 - HKII or AAV9 - empty . The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point ( n = 8 / group ) . a = significant difference between AdRiKO and control mice infected with AAV9 - empty , b = significant difference between AdRiKO and control mice infected with AAV9 - HKII , d = significant difference between AdRiKO mice infected with AAV9 - empty and AAV9 - HKII .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

(D) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV9-HKII or AAV9-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=8/group). a=significant difference between AdRiKO and control mice infected with AAV9-empty, b= significant difference between AdRiKO and control mice infected with AAV9-HKII, d= significant difference between AdRiKO mice infected with AAV9-empty and AAV9-HKII.


( E ) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty ( n = 6 / group , each lane represents a mix of 3 mice ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(E) Immunoblot analysis of BAT from AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty (n=6/group, each lane represents a mix of 3 mice).


( F ) Body temperature of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty housed at 22 ° C ( n = 11 / group ) .

(F) Body temperature of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 22°C (n=11/group).


( G ) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty . The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point ( n = 11 / group ) . a = significant difference between AdRiKO and control mice infected with AAV8 - empty , b = significant difference between AdRiKO and control mice infected with AAV8 - Akt2S474D , d = significant difference between AdRiKO mice infected with AAV8 - empty and AAV8 - Akt2S474D .

(G) Body temperature upon cold exposure of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty. The left panel represents body temperature after each hour of cold exposure while the right panel represents body temperature as a bar graph for the 3h cold exposure time point (n=11/group). a=significant difference between AdRiKO and control mice infected with AAV8-empty, b= significant difference between AdRiKO and control mice infected with AAV8-Akt2S474D, d= significant difference between AdRiKO mice infected with AAV8-empty and AAV8-Akt2S474D.


( H ) Cold - induced 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO and control mice infected with either AAV8 - Akt2S474D or AAV8 - empty housed at 4 ° C for 4h ( n = 7 ( control AAV8 - null ) , n = 6 ( AdRiKO AAV8 - null ) , n = 6 ( control AAV8 - AktS474D ) , n = 6 ( AdRiKO AAV8 - AktS474D ) ) . Data represent mean ± SEM . Statistically significant differences between AdRiKO and control mice were determined with unpaired Student ' s t - test and are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between viruses were determined with unpaired Student ' s t - test and are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . The exact p - value for each significant difference can be found in Appendix Table S2 .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cold-induced 2-Deoxyglucose-6-phosphate (2DG6P) accumulation in BAT of AdRiKO and control mice infected with either AAV8-Akt2S474D or AAV8-empty housed at 4°C for 4h (n=7 (control AAV8-null), n=6 (AdRiKO AAV8-null), n=6 (control AAV8-AktS474D), n=6 (AdRiKO AAV8-AktS474D)). Data represent mean ± SEM. Statistically significant differences between AdRiKO and control mice were determined with unpaired Student's t-test and are indicated with asterisks (*=p<0.05; **=p<0.01, ***=p<0.001). Statistically significant differences between viruses were determined with unpaired Student's t-test and are indicated with a number sign (#=p<0.05; ##=p<0.01; ###=p<0.001). The exact p-value for each significant difference can be found in Appendix Table S2.


Blistered samples at P1 . ( B ) Hematoxylin and eosin ( H & E , top ) and alkaline phosphatase ( AP , bottom ) staining . Hair follicles ( HFs ) remaining in the dermis ( indicated by arrows ) . Scale bar : 500 μm . ( C ) α6 Integrin ( ITGA6 , indicated by arrowheads ) and type IV collagen ( COL4 , arrows ) labeling ( left ) . Laminin 332 ( L332 , arrows ) staining ( right ) . Scale bar : 100 μm . ( D ) Ultrastructural findings of blistered skin ( left image : blister roof , right image : blister bottom ) . Hemidesmosomes ( white arrowheads ) and lamina densa ( arrows ) are indicated . Scale bar : 1 μm .

O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O 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-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Blistered samples at P1. (B) Hematoxylin and eosin (H&E, top) and alkaline phosphatase (AP, bottom) staining. Hair follicles (HFs) remaining in the dermis (indicated by arrows). Scale bar: 500 μm. (C) α6 Integrin (ITGA6, indicated by arrowheads) and type IV collagen (COL4, arrows) labeling (left). Laminin 332 (L332, arrows) staining (right). Scale bar: 100 μm. (D) Ultrastructural findings of blistered skin (left image: blister roof, right image: blister bottom) . Hemidesmosomes (white arrowheads) and lamina densa (arrows) are indicated. Scale bar: 1 μm.


Keratin 14 ( K14 ) and keratin 10 ( K10 ) staining of the nonlesional ( intact ) and lesional ( blistered ) skin at P2 ( upper images and inlets : sections , lower images : whole - mount imaging ) . HFs are indicated by arrows in the whole - mount images . Scale bar : 30 μm .

B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Keratin 14 (K14) and keratin 10 (K10) staining of the nonlesional (intact) and lesional (blistered) skin at P2 (upper images and inlets: sections, lower images: whole-mount imaging). HFs are indicated by arrows in the whole-mount images. Scale bar: 30 μm.


H & E ( left ) , PCK ( middle ) , and LOR staining ( right ) at P4 . Scale bar : 200 μm .

O O O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O

H&E (left), PCK (middle), and LOR staining (right) at P4. Scale bar: 200 μm.


Scatter plots of differentially expressed genes in the regenerated epidermis . The red dots represent upregulated genes , and the blue dots represent downregulated genes . The gray dotted lines indicate | logFC | > 1 .

Scatter plots of differentially expressed genes in the regenerated epidermis. The red dots represent upregulated genes, and the blue dots represent downregulated genes. The gray dotted lines indicate |logFC|>1.


Hair canals in the regenerated ( lesional ) and nonlesional epidermis at P4 ( indicated by asterisks ) . Scale bar : 300 μm .

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Hair canals in the regenerated (lesional) and nonlesional epidermis at P4 (indicated by asterisks). Scale bar: 300 μm.


BrdU labeling of blistered samples at P2 . Scale bar : 100 μm . BrdU - positive cells are indicated by arrows . Blisters are indicated by stars . ( Bottom ) Quantification of BrdU - positive cells in the epidermis ( left ) and HFs ( right ) ( n = 4 biological replicates ) . The data are shown as the mean ± SE . * 0 . 01 test , followed by Tukey ' s test . NS , no significance .

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BrdU labeling of blistered samples at P2. Scale bar: 100 μm. BrdU-positive cells are indicated by arrows. Blisters are indicated by stars. (Bottom) Quantification of BrdU-positive cells in the epidermis (left) and HFs (right) (n=4 biological replicates). The data are shown as the mean ± SE. *0.01 test, followed by Tukey's test. NS, no significance.


α5 integrin ( ITGA5 ) labeling at P2 ( left image : section , right image : whole - mount ) . Scale bar : 100 μm . Blister edges ( epidermal tongue ) and HFs are indicated by arrowheads and arrows , respectively . Blisters are indicated by stars .

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α5 integrin (ITGA5) labeling at P2 (left image: section, right image: whole-mount). Scale bar: 100 μm. Blister edges (epidermal tongue) and HFs are indicated by arrowheads and arrows, respectively. Blisters are indicated by stars.


Sections of K14CreER : H2B - mCherry mice at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . . The data from individual mice are connected by lines . Student ' s t - test . NS , no significance . Whole - mount imaging of K14CreER : R26R - confetti samples at P4 . Scale bar : 200 μm . Sections of Lrig1CreER : H2B - mCherry mouse skin at P4 . Scale bar : 100 μm . ( Bottom ) Quantification of mCherry - positive cells ( n = 3 ) . The data from individual mice are connected by lines . * 0 . 01 test . Whole - mount imaging of Lrig1CreER : R26R - confetti mouse samples at P4 . Scale bar : 200 μm .

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Sections of K14CreER:H2B-mCherry mice at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3).. The data from individual mice are connected by lines. Student's t-test. NS, no significance. Whole-mount imaging of K14CreER:R26R-confetti samples at P4. Scale bar: 200 μm. Sections of Lrig1CreER:H2B-mCherry mouse skin at P4. Scale bar: 100 μm. (Bottom) Quantification of mCherry-positive cells (n=3). The data from individual mice are connected by lines. *0.01 test. Whole-mount imaging of Lrig1CreER:R26R-confetti mouse samples at P4. Scale bar: 200 μm.


H & E ( P2 , left ) , ITGA6 ( P2 , middle ) and LOR staining ( P4 , right ) of Col17a1 - / - mice ( top ) and littermate controls ( bottom ) . The regenerated epidermis is indicated by arrowheads . Scale bar : 200 μm .

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H&E (P2, left), ITGA6 (P2, middle) and LOR staining (P4, right) of Col17a1-/- mice (top) and littermate controls (bottom) . The regenerated epidermis is indicated by arrowheads. Scale bar: 200 μm.


BrdU labeling of Col17a1 - / - skin at P2 . Scale bar : 100 μm . ( Bottom ) Quantification of BrdU - positive cells in the epidermis surrounding blisters ( n = 3 ( control ) and 4 ( Col17a1 - / - ) biological replicates ) . The data are shown as the mean ± SE . Student ' s t - test . NS , no significance .

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BrdU labeling of Col17a1-/- skin at P2. Scale bar: 100 μm. (Bottom) Quantification of BrdU-positive cells in the epidermis surrounding blisters (n= 3 (control) and 4 (Col17a1-/-) biological replicates). The data are shown as the mean ± SE. Student's t-test. NS, no significance.


Lineage tracing of K14CreER : R26R - mCherry : Col17a1 - / - at P4 . Scale bar : 100 μm .

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Lineage tracing of K14CreER:R26R-mCherry:Col17a1-/- at P4. Scale bar: 100 μm.


K10 / K14 ( low magnification , left ) and K14 ( high magnification , middle and right ) labeling of Col7a1 - / - mouse ( bottom ) and littermate control ( top ) blistered skin at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 55 HFs from three control and 143 HFs from four Col7a1 - / - mice ) . The data are shown as violin plots . Student ' s t - test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 244 ( control , L ) , and 132 ( Col7a1 - / - , L ) cells from four mice , respectively ) and in the surrounding intact epidermis ( basal cells ; n = 200 ( control , NL ) , 299 ( Col7a1 - / - , NL ) , from four mice , respectively ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K10/K14 (low magnification, left) and K14 (high magnification, middle and right) labeling of Col7a1-/- mouse (bottom) and littermate control (top) blistered skin at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=55 HFs from three control and 143 HFs from four Col7a1-/- mice). The data are shown as violin plots. Student's t-test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=244 (control, L), and 132 (Col7a1-/-, L) cells from four mice, respectively) and in the surrounding intact epidermis (basal cells; n=200 (control, NL), 299 (Col7a1-/-, NL), from four mice, respectively). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


K10 / K14 ( left ) and K14 ( high magnification , right ) labeling of WT blistered skin treated with CaCl2 ( middle and bottom ) or PBS ( top ) at P2 . Scale bar : 30 μm . Quantification of BrdU - positive cells per μm HF length ( n = 83 ( PBS ) , 95 ( 1 . 8 mM CaCl2 ) , and 97 ( 9 . 0 mM CaCl2 ) HFs from four mice ) . One - way ANOVA test , followed by Tukey ' s test . NS , no significance . Length of the major axis of keratinocytes in the regenerated epidermis ( n = 433 ( PBS , L ) , 451 ( 1 . 8 mM CaCl2 , L ) , and 425 ( 9 . 0 mM CaCl2 , L ) cells from four mice ) and in the surrounding intact epidermis ( basal cells ; n = 311 ( PBS , NL ) , 279 ( 1 . 8 mM CaCl2 , NL ) , 302 ( 9 . 0 mM CaCl2 , NL ) cells from four mice ) . NL : nonlesional area . L : lesional area . The data are shown as violin plots . * * * * p < 0 . 0001 , one - way ANOVA test , followed by Tukey ' s test . NS , no significance .

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K10/K14 (left) and K14 (high magnification, right) labeling of WT blistered skin treated with CaCl2 (middle and bottom) or PBS (top) at P2. Scale bar: 30 μm. Quantification of BrdU-positive cells per μm HF length (n=83 (PBS), 95 (1.8 mM CaCl2), and 97 (9.0 mM CaCl2) HFs from four mice). One-way ANOVA test, followed by Tukey's test. NS, no significance. Length of the major axis of keratinocytes in the regenerated epidermis (n=433 (PBS, L), 451 (1.8 mM CaCl2, L), and 425 (9.0 mM CaCl2, L) cells from four mice) and in the surrounding intact epidermis (basal cells; n=311 (PBS, NL), 279 (1.8 mM CaCl2, NL), 302 (9.0 mM CaCl2, NL) cells from four mice). NL: nonlesional area. L: lesional area. The data are shown as violin plots. ****p<0.0001, one-way ANOVA test, followed by Tukey's test. NS, no significance.


( D ) The indicated amounts of recombinant wild - type and mutant PPM1H or PPM1J ( with a His - Sumo N - terminal tag , expressed in E . coli ) were incubated in vitro with 2 . 5 µg pThr72 phosphorylated Rab8a ( left ) or pThr73 phosphorylated Rab10 ( right ) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7 . 5 buffer . Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos - tag gel electrophoresis that separates phosphorylated ( slow migrating ) and dephosphorylated Rabs . The gel was stained with Instant Blue Coomassie . D288A is a substrate - trapping ( inactive ) variant of PPM1H and was used as a control .

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(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.


( D ) Kinetics of phosphate hydrolysis . 25 nM recombinant wild - type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a ( GTPγS or GDP ) as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of released phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 phosphorylated Rab8a [ GTP bound conformation ] ( blue ) and pThr72 phosphorylated Rab8a [ GDP bound conformation ] ( red ) . The experiments were repeated twice , and both data points are shown in curves . Line fittings for the left and middle panel were performed using the mean of the two values . Kinetic constants ( Kcat , Vmax , Km ) were obtained using GraphPad software , and their uncertainties ( ± ) correspond to the SE of mean .

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(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.


( E ) Kinetic analysis of PPM1H as in ( D ) using 50 nM PPM1H and phosphopeptide substrates , as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 Rab8a phospho - peptide ( blue ) and Rab10 pThr73 phospho - peptide ( red ) . Each experiment was performed twice ( individual data points shown ) .

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(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).


( F ) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis . 50 nM recombinant wild - type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein ( blue ) or Rab8a pThr72 phosphorylated peptide ( red ) for indicated times and analysed as described in Materials and Methods . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates .

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(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.


( C ) His6 - SUMO - tagged full - length variants of PPM1H were used for catalytic assays ( left panel ) . 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie . Right , mass photometry histogram for 40nM His6 - SUMO - PPM1H WT ( blue ) and His6 - SUMO - PPM1H 2Glu ( brown ) , where WT is 130kDa ( ± 15 . 8 kDa , with 1004 single molecules counted ) and 2Glu is 83kDa ( ± 13 . 9 kDa , with 4117 single molecules counted ) . The calculated molecular weight of the fusion protein is approximately 68 . 5 kDa .

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(C) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.


( D ) In vitro malachite green assay time course of recombinant His6 - SUMO - tagged PPM1H wild - type ( blue ) , 2Glu ( red ) , or D288A ( grey ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical replicates .

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(D) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


( C ) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins ( 25 nM for the protein substrate , 50 nM for the peptide substrate ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS ) . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates . ( D ) In vitro malachite green assay against 32 μM pThr72 - Rab8a peptide ( left ) and pThr73 - Rab10 peptide mimic ( right ) . The experiments were repeated 3 times . The error bars represent standard error ( SE ) of mean of the technical replicates .

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(C) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. (D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates.


( E , F ) ( E ) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi - 2 for 90 min and then lysed . 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg / ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system . Left , each lane represents cell extract obtained from a different dish of cells ( two biological replicates per condition without MLi - 2 treatment , one biological replicate per condition with MLi - 2 treatment ) . Right , the ratio of phospho - Rab8a / total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild - type expressing cells . ( F ) As in ( E ) assessing phospho - Rab10 levels . For quantitation the two replicates from the presented blots were used . The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates .

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(E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.


( B ) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical replicates .

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(B) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


( C ) 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie .

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(C) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.


( E ) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS ) using the malachite green assay time course . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates . Quality of purified proteins ( right panel ) is shown using the protocol in ( C ) .

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(E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C).


( F ) HEK293 cells overexpressing indicated constructs were treated and analysed as described in ( D ) , with pRab10 analysed in the upper panel , and pRab8a in the lower panel .

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(F) HEK293 cells overexpressing indicated constructs were treated and analysed as described in (D), with pRab10 analysed in the upper panel, and pRab8a in the lower panel.


( B ) Incremental deletions of the N - terminus from residues 1 - 37 to 1 - 44 , one residue at a time . Upon deletion of R43 ( 44 - end ) , a reduced level of soluble PPM1H expression has no significant catalytic activity .

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(B) Incremental deletions of the N-terminus from residues 1-37 to 1-44, one residue at a time. Upon deletion of R43 (44-end), a reduced level of soluble PPM1H expression has no significant catalytic activity.


( A ) In vitro Methyltransferase activity . Recombinant proteins Nsp10 and Nsp16 wt or Nsp16mut were combined with cap0 RNA and Adenosyl - L - methionine ( [ methyl - 3H ] SAM ) for 90 min at 37 ° C . Methylation of cap0 RNA was quantified using a scintillation counter ( Beckmann ) . ( - ) control did not contain any recombinant protein . Results are plotted as mean of triplicate reactions with error bars representing SD . One out of three independent biological repeats is shown . Data information : Statistical analysis was done using a one - way ANOVA followed by Tukey ' s multiple comparisons test .

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(A) In vitro Methyltransferase activity. Recombinant proteins Nsp10 and Nsp16 wt or Nsp16mut were combined with cap0 RNA and Adenosyl-L-methionine ([methyl-3H] SAM) for 90 min at 37°C. Methylation of cap0 RNA was quantified using a scintillation counter (Beckmann).(-) control did not contain any recombinant protein. Results are plotted as mean of triplicate reactions with error bars representing SD. One out of three independent biological repeats is shown. Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.


( B ) MTase activity of Nsp16 upon viral infection . Cellular mRNA was purified 24 h postinfection with SARS - CoV - 2 wt or Nsp16mut ( triplicate infections , biological replicates ) . Purified mRNA was incubated with 100U of the recombinant MTase VACV VP39 ( NEB ) and [ methyl - 3H ] SAM for 90 min at 37 ° C . Methylation of mRNA from SARS - CoV - 2 infected cells was quantified using a scintillation counter ( DPM3H ; Beckmann ) . ( - ) control did not contain cellular RNA . Results are plotted as mean of triplicate infections with error bars representing SD . One out of three independent experiments is shown ( n = 3 ) . Data information : Statistical analysis was done using a one - way ANOVA followed by Tukey ' s multiple comparisons test .

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(B) MTase activity of Nsp16 upon viral infection. Cellular mRNA was purified 24 h postinfection with SARS-CoV-2 wt or Nsp16mut (triplicate infections, biological replicates). Purified mRNA was incubated with 100U of the recombinant MTase VACV VP39 (NEB) and [methyl-3H] SAM for 90 min at 37 °C. Methylation of mRNA from SARS-CoV-2 infected cells was quantified using a scintillation counter (DPM3H; Beckmann). (-) control did not contain cellular RNA. Results are plotted as mean of triplicate infections with error bars representing SD. One out of three independent experiments is shown (n=3). Data information: Statistical analysis was done using a one-way ANOVA followed by Tukey's multiple comparisons test.


( A ) Calu - 3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut at 0 . 015 RNA copies / cell . Viral load in the supernatant was quantified at the indicated time points by RT - qPCR targeting viral polymerase RdRp . Results are plotted as mean of triplicate infections with error bars representing the SD ( biological replicates ) . One out of three independent experiments is shown ( n = 3 ) . Three days postinfection , protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment . Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90 . Data information : Statistical analysis was done using two - way ANOVA followed by Bonferroni ' s multiple comparisons test . ns = not significant ( p > 0 . 05 ) .

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(A) Calu-3 cells were incubated with the indicated amounts of type I IFN 24 h prior to infection with normalized amounts of SARS-CoV-2 wt (WT) or Nsp16mut at 0.015 RNA copies/cell. Viral load in the supernatant was quantified at the indicated time points by RT-qPCR targeting viral polymerase RdRp. Results are plotted as mean of triplicate infections with error bars representing the SD (biological replicates). One out of three independent experiments is shown (n=3). Three days postinfection, protein expression of IFIT1 was analyzed by immunoblot to control for type I IFN treatment. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90. Data information: Statistical analysis was done using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( C ) Calu - 3 cells lacking MDA5 ( MDA5 KO ) , RIG - I ( RIG - I KO ) , or control KO cells ( Ctrl ) were seeded into 96well plates and infected with increasing amounts of SARS - CoV - 2 wt ( WT ) or Nsp16mut in triplicates ( biological replicates ) . Type I IFN release was quantified after 48 h by incubating supernatants with HEK - Blue IFN - α / β reporter cells . SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD . ( D ) Calu - 3 cells ( Ctrl ) were seeded into 96 well plates and infected with 10 RNA copies / cell of SARS - CoV - 2 wt ( WT ) or Nsp16mut in the presence of increasing amounts of Remdesivir ( RDV ) . Type I IFN release was quantified after 48 h by IFN bioassay on HEK - Blue IFN - α / β and is depicted as mean of quadruplicates with error bars representing SD . In parallel , infectivity of SARS - CoV - 2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT - qPCR targeting viral polymerase RdRp transcripts . Data information : Statistical analysis was performed using two - way ANOVA followed by Bonferroni ' s multiple comparisons test . ns = not significant ( p > 0 . 05 ) .

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(C) Calu-3 cells lacking MDA5 (MDA5 KO), RIG-I (RIG-I KO), or control KO cells (Ctrl) were seeded into 96well plates and infected with increasing amounts of SARS-CoV-2 wt (WT) or Nsp16mut in triplicates (biological replicates). Type I IFN release was quantified after 48 h by incubating supernatants with HEK-Blue IFN-α/β reporter cells. SEAP activity in the supernatant of the reporter cells is shown as mean of quadruplicates with error bars representing SD. (D) Calu-3 cells (Ctrl) were seeded into 96 well plates and infected with 10 RNA copies/cell of SARS-CoV-2 wt (WT) or Nsp16mut in the presence of increasing amounts of Remdesivir (RDV). Type I IFN release was quantified after 48 h by IFN bioassay on HEK-Blue IFN-α/β and is depicted as mean of quadruplicates with error bars representing SD. In parallel, infectivity of SARS-CoV-2 wt and Nsp16 in the presence of increasing amounts of RDV was determined at the indicated time points by RT-qPCR targeting viral polymerase RdRp transcripts. Data information: Statistical analysis was performed using two-way ANOVA followed by Bonferroni's multiple comparisons test. ns = not significant (p>0.05).


( B ) Three days after SARS - CoV - 2 Nsp16mut infection of A549 cells described in ( A ) , expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N protein , and the housekeeping gene HSP90 .

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(B) Three days after SARS-CoV-2 Nsp16mut infection of A549 cells described in (A), expression of IFIT1 and viral nucleocapsid protein N was analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, and the housekeeping gene HSP90.


( D ) IFIT1 expression levels in Calu - 3 Ctrl , IFIT1 KO # 1 and # 2 lines treated with type I IFN ( 24 h ; 100 U / ml ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90 .

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(D) IFIT1 expression levels in Calu-3 Ctrl, IFIT1 KO #1 and #2 lines treated with type I IFN (24 h; 100 U/ml) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1 or the housekeeping gene HSP90.


( F ) Three days postinfection , A549 cells described in ( E ) were analyzed by immunoblot . Membranes were probed with antibodies targeting IFIT1 , SARS - CoV - 2 N protein , or the housekeeping gene HSP90 .

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(F) Three days postinfection, A549 cells described in (E) were analyzed by immunoblot. Membranes were probed with antibodies targeting IFIT1, SARS-CoV-2 N protein, or the housekeeping gene HSP90.


( H ) IFIT1 expression was analyzed in mock treated , SARS - CoV - 2 wt ( WT ) or Nsp16mut infected cells at 48 hpi by immunoblot . Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90 .

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(H) IFIT1 expression was analyzed in mock treated, SARS-CoV-2 wt (WT) or Nsp16mut infected cells at 48 hpi by immunoblot. Membranes were probed with antibodies targeting IFIT1 and the housekeeping gene HSP90.


( D ) Top : Schematic of GFP expression reporter system , with the CBASS promoter , capH , and capP genes from E . coli MS115 - 1 and the CBASS core genes replaced with GFP . Bottom : Western blot showing GFP expression in cells with the wild - type GFP reporter or constructs lacking either capP or capH genes . RNAP : RNA polymerase loading control .

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(D) Top: Schematic of GFP expression reporter system, with the CBASS promoter, capH, and capP genes from E. coli MS115-1 and the CBASS core genes replaced with GFP. Bottom: Western blot showing GFP expression in cells with the wild-type GFP reporter or constructs lacking either capP or capH genes. RNAP: RNA polymerase loading control.


( E ) Western blots of the CBASS expression reporter system with FLAG - NucC showing FLAG - NucC expression after infection with phage λ cI - ( multiplicity of infection : 10 ) . MPI : minutes post infection . α - RNAP : anti - RNA polymerase loading control . Low RNAP expression at later time points is due to cell death .

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(E) Western blots of the CBASS expression reporter system with FLAG-NucC showing FLAG-NucC expression after infection with phage λ cI- (multiplicity of infection: 10). MPI: minutes post infection. α-RNAP: anti-RNA polymerase loading control. Low RNAP expression at later time points is due to cell death.


( B ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ) to three 40 - bp DNAs : 1 - 40 ( black circles ) , 31 - 70 ( Site 1 ; green squares ) , and 81 - 120 ( Site 2 ; orange triangles ) . Fit Kd and Hill coefficient for each DNA is shown . Error bars indicate standard deviation from three technical replicates .

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(B) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to three 40-bp DNAs: 1-40 (black circles), 31-70 (Site 1; green squares), and 81-120 (Site 2; orange triangles). Fit Kd and Hill coefficient for each DNA is shown. Error bars indicate standard deviation from three technical replicates.


( D ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ; wild - type or indicated point mutants ) to the Site 2 DNA ( bases 81 - 120 in panel A ) . Fit Kd and Hill coefficient for each DNA is shown .

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(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; wild-type or indicated point mutants) to the Site 2 DNA (bases 81-120 in panel A). Fit Kd and Hill coefficient for each DNA is shown.


( E ) GFP expression reporter assay showing loss of suppression upon mutation of CapH . RNAP : RNA polymerase loading control .

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(E) GFP expression reporter assay showing loss of suppression upon mutation of CapH. RNAP: RNA polymerase loading control.


( D ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ; full - length ( black circles ) or NTD ( green squares ) ) to the Site 2 DNA . WT Kd = 0 . 30 + / - 0 . 08 μM , Hill coefficient = 2 . 0 + / - 0 . 7 ; no binding detected for CapHNTD ) . Error bars indicate standard deviation from three technical replicates . ( E ) Fluorescence polarization assay showing binding of E . coli MS115 - 1 CapH ( His6 - MBP tagged ) to the Site 1 ( bases 31 - 70 ) and Site 2 ( bases 81 - 120 ) DNAs . Wild - type CapH binding Site 1 is shown in orange triangles , and binding Site 2 is shown in blue diamonds . CapH ( I99M ) binding Site 1 is shown in black circles , and binding Site 2 is shown in green squares . Fit Kd and Hill coefficient for each combination is shown .

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(D) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged; full-length (black circles) or NTD(green squares)) to the Site 2 DNA. WT Kd = 0.30 +/- 0.08 μM, Hill coefficient = 2.0 +/- 0.7; no binding detected for CapHNTD). Error bars indicate standard deviation from three technical replicates. (E) Fluorescence polarization assay showing binding of E. coli MS115-1 CapH (His6-MBP tagged) to the Site 1 (bases 31-70) and Site 2 (bases 81-120) DNAs. Wild-type CapH binding Site 1 is shown in orange triangles, and binding Site 2 is shown in blue diamonds. CapH(I99M) binding Site 1 is shown in black circles, and binding Site 2 is shown in green squares. Fit Kd and Hill coefficient for each combination is shown.


( A ) Anti - GFP western blot showing coexpression in E . coli of an MBP - CapH - GFP fusion construct with wild - type or catalytic - dead ( E98Q ) CapP . Full - length MBP - CapH - GFP is indicated with a yellow arrowhead , and the C - terminal product of CapP cleavage is indicated with a white arrowhead . RNAP : RNA polymerase loading control .

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(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type or catalytic-dead (E98Q) CapP. Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. RNAP: RNA polymerase loading control.


( B ) In vitro cleavage of purified MBP - CapH - GFP ( yellow arrowhead ) into N - terminal and C - terminal products ( white arrowheads ) by CapP is stimulated by DNA . For both ssDNA and dsDNA , the highest concentration is 350 ng / μL , followed by three 5 - fold dilutions . Red asterisk indicates the band that was analyzed by Edman degradation .

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(B) In vitro cleavage of purified MBP-CapH-GFP (yellow arrowhead) into N-terminal and C-terminal products (white arrowheads) by CapP is stimulated by DNA. For both ssDNA and dsDNA, the highest concentration is 350 ng/μL, followed by three 5-fold dilutions. Red asterisk indicates the band that was analyzed by Edman degradation.


( E ) In vitro cleavage of MBP - CapH - GFP ( wild - type or R83A mutant ) by CapP , in the presence of 10 μM ssDNA .

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(E) In vitro cleavage of MBP-CapH-GFP (wild-type or R83A mutant) by CapP, in the presence of 10 μM ssDNA.


( F ) GFP reporter assay showing effect of a CapP E98Q catalytic - dead mutant , CapH R83A mutant , or removal of CapH residues 83 - 107 ( capH 1 - 82 ) on GFP expression . RNAP : RNA polymerase loading control .

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(F) GFP reporter assay showing effect of a CapP E98Q catalytic-dead mutant, CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on GFP expression. RNAP: RNA polymerase loading control.


( G ) CBASS expression reporter system with FLAG - NucC , showing effect of a CapH R83A mutant , or removal of CapH residues 83 - 107 ( capH 1 - 82 ) on FLAG - NucC expression . α - RNAP : anti - RNA polymerase loading control .

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(G) CBASS expression reporter system with FLAG-NucC, showing effect of a CapH R83A mutant, or removal of CapH residues 83-107 (capH 1-82) on FLAG-NucC expression. α-RNAP: anti-RNA polymerase loading control.


( A ) Anti - GFP western blot showing coexpression in E . coli of an MBP - CapH - GFP fusion construct with wild - type CapP after exposure to zeocin ( 100 μg / mL ) . Full - length MBP - CapH - GFP is indicated with a yellow arrowhead , and the C - terminal product of CapP cleavage is indicated with a white arrowhead . α - RNAP : anti - RNA polymerase loading control .

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(A) Anti-GFP western blot showing coexpression in E. coli of an MBP-CapH-GFP fusion construct with wild-type CapP after exposure to zeocin (100 μg/mL). Full-length MBP-CapH-GFP is indicated with a yellow arrowhead, and the C-terminal product of CapP cleavage is indicated with a white arrowhead. α-RNAP: anti-RNA polymerase loading control.


( B ) Western blot of the CBASS expression reporter system with FLAG - NucC , showing FLAG - NucC expression after exposure to zeocin ( 100 μg / mL ) . α - RNAP : anti - RNA polymerase loading control . ( C ) Western blot of the CBASS expression reporter system with FLAG - NucC , showing FLAG - NucC expression after exposure to mitomycin C ( 1 μg / mL ) . α - RNAP : anti - RNA polymerase loading control .

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(B) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to zeocin (100 μg/mL). α-RNAP: anti-RNA polymerase loading control. (C) Western blot of the CBASS expression reporter system with FLAG-NucC, showing FLAG-NucC expression after exposure to mitomycin C (1 μg/mL). α-RNAP: anti-RNA polymerase loading control.


A ) Representative zooms of WT MEFs transfected with GFP - tagged Miro1 and Miro2 ( GFPMiro1 and GFPMiro2 ) . Control is GFP fused to the first 70 amino acids of Tom70 . Tom20 and catalase stain mitochondria and peroxisomes , respectively . Merge is of Miro ( green ) and catalase ( magenta ) with colocalisation shown as white . White arrowheads highlight an area where an isolated peroxisome is situated . B ) Quantification of the extent of GFP signal on peroxisomes . Data information : B ) One - way ANOVA with Newman - Keuls post hoc test was used for all comparisons ( n = 18 cells per condition over 3 independent experiments ) . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 in comparison to Control , respectively , and # # and # # # denote p < 0 . 01 and p < 0 . 001 in comparison to GFPMiro1 , respectively . Data are represented as mean ± SEM . Scale bar is 5 μm .

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A) Representative zooms of WT MEFs transfected with GFP-tagged Miro1 and Miro2 (GFPMiro1 and GFPMiro2). Control is GFP fused to the first 70 amino acids of Tom70. Tom20 and catalase stain mitochondria and peroxisomes, respectively. Merge is of Miro (green) and catalase (magenta) with colocalisation shown as white. White arrowheads highlight an area where an isolated peroxisome is situated. B) Quantification of the extent of GFP signal on peroxisomes. Data information: B) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.


C ) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full - length Miro1 , but not Miro1 lacking the transmembrane domain .

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C) Pulldown of overexpressed Pex19 in Cos7 cells with a Pex19 antibody shows interaction with full-length Miro1, but not Miro1 lacking the transmembrane domain.


E ) Representative images of Miro1 truncation constructs expressed in DKO MEFs . Mitochondria and peroxisomes are stained with Tom20 and catalase , respectively . White arrowheads highlight an area where an isolated peroxisome is situated .

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E) Representative images of Miro1 truncation constructs expressed in DKO MEFs. Mitochondria and peroxisomes are stained with Tom20 and catalase, respectively. White arrowheads highlight an area where an isolated peroxisome is situated.F


. F ) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs . Data information : F ) One - way ANOVA with Newman - Keuls post hoc test was used for all comparisons ( n = 18 cells per condition over 3 independent experiments ) . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 in comparison to Control , respectively , and # # and # # # denote p < 0 . 01 and p < 0 . 001 in comparison to GFPMiro1 , respectively . Data are represented as mean ± SEM . Scale bar is 5 μm .

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.F) Quantification of the extent of peroxisomal localisation of the Miro1 truncation constructs. Data information: F) One-way ANOVA with Newman-Keuls post hoc test was used for all comparisons (n=18 cells per condition over 3 independent experiments). *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001 in comparison to Control, respectively, and ## and ### denote p < 0.01 and p < 0.001 in comparison to GFPMiro1, respectively. Data are represented as mean ± SEM. Scale bar is 5 μm.


G ) Co - immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells . GFP - tagged Miro1 truncation constructs were pulled down with GFP - Trap agarose beads and Pex19 was probed with myc antibody .

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G) Co-immunoprecipation of Miro1 truncation constructs and mycPex19 following transfection into Cos7 cells. GFP-tagged Miro1 truncation constructs were pulled down with GFP-Trap agarose beads and Pex19 was probed with myc antibody.


A ) Six consecutive frames of live trafficking of peroxisomes ( pxDsRed signal at 1 . 5 seconds per frame ) . Yellow arrows show the trajectory of a fast - moving peroxisome in both WT and DKO MEFs . Scale bar is 5 μm .

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A) Six consecutive frames of live trafficking of peroxisomes (pxDsRed signal at 1.5 seconds per frame). Yellow arrows show the trajectory of a fast-moving peroxisome in both WT and DKO MEFs. Scale bar is 5 μm.


B ) Blind scoring of the number of long - range peroxisomal trafficking events in WT , Miro1KO and Miro2KO and DKO MEFs ( n = 42 cells over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For B ) a Kruskall - Wallis with a Dunn ' s correction posthoc test was used to test for significance . For B ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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B) Blind scoring of the number of long-range peroxisomal trafficking events in WT, Miro1KO and Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For B) a Kruskall-Wallis with a Dunn's correction posthoc test was used to test for significance. For B) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


C ) and D ) Quantification of individual track length and velocity , respectively ( n = 24 cells over three independent experiments ) . Data information : Analysis for C ) , D ) is a one - way ANOVA with Newman - Keuls post - hoc test . * * denotes p < 0 . 01 . For C ) , D ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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C) and D) Quantification of individual track length and velocity, respectively (n=24 cells over three independent experiments). Data information: Analysis for C), D) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For C), D) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


E ) Representative blots of Miro1 , Miro2 and actin from whole cell lysates of wild - type and Miro1 - floxed ERT Cre - recombinase MEFs treated with and without 4 - OH tamoxifen for 48 hours . Lysates were taken one day after the end of treatment .

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E) Representative blots of Miro1, Miro2 and actin from whole cell lysates of wild-type and Miro1-floxed ERT Cre-recombinase MEFs treated with and without 4-OH tamoxifen for 48 hours. Lysates were taken one day after the end of treatment.


F ) Quantification of long - range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two - minute movie ( n = 18 cells per condition over three independent experiments ) . Data information : Statistical significance in F ) was calculated by two - way ANOVA . For F ) no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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F) Quantification of long-range peroxisomal trafficking events by blind scoring of pxDsRed signal from a two-minute movie (n=18 cells per condition over three independent experiments). Data information: Statistical significance in F) was calculated by two-way ANOVA. For F) no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


G ) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells .

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G) Representative blot of Miro1 and Miro2 following knockdown of either Miro1 or Pex14 in HeLa cells.


H ) Quantification of long - range peroxisomal trafficking events ( visualised with pxGFP ) in HeLa cells transfected with scrambled , Miro1 and Pex14 siRNA , by blind scoring of a five - minute movie ( n = 18 cells over three independent experiments ) . Data information : For H ) is a one - way ANOVA with Newman - Keuls post - hoc test . * * denotes p < 0 . 01 . For H ) , no statistical difference between conditions was observed , unless stated . Data are represented as mean ± SEM .

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H) Quantification of long-range peroxisomal trafficking events (visualised with pxGFP) in HeLa cells transfected with scrambled, Miro1 and Pex14 siRNA, by blind scoring of a five-minute movie (n=18 cells over three independent experiments). Data information: For H) is a one-way ANOVA with Newman-Keuls post-hoc test. ** denotes p < 0.01. For H), no statistical difference between conditions was observed, unless stated. Data are represented as mean ± SEM.


A ) Representative images of WT , Miro1KO , Miro2KO and DKO MEFs seeded onto Y - shaped fibronectin micropatterns stained for mitochondria ( Tom20 in red ) and peroxisomes ( Catalase in white ) . Scale bar represents 10 μm . Data information : n = 60 cells over three independent experiments .

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A) Representative images of WT, Miro1KO, Miro2KO and DKO MEFs seeded onto Y-shaped fibronectin micropatterns stained for mitochondria (Tom20 in red) and peroxisomes (Catalase in white). Scale bar represents 10 μm. Data information: n = 60 cells over three independent experiments.


B ) Normalised cumulative distribution of mitochondria in WT , Miro1KO , Miro2KO and DKO MEFs . C ) Distance at which 95 % of mitochondria are distributed ( Mito95 ) for all four genotypes of MEF . Data information : n = 60 cells over three independent experiments . For C ) one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± SEM .

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B) Normalised cumulative distribution of mitochondria in WT, Miro1KO, Miro2KO and DKO MEFs. C) Distance at which 95% of mitochondria are distributed (Mito95) for all four genotypes of MEF. Data information: n = 60 cells over three independent experiments. For C) one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


D ) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT , Miro1KO , Miro2KO and DKO MEFs . E ) Bar graph comparing the average distance at which 95 % of the peroxisomal signal is distributed ( Perox95 ) . Data information : n = 60 cells over three independent experiments . For E ) a one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance . * * * represents p < 0 . 001 . Data are represented as mean ± SEM .

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D) Normalised cumulative distribution curves comparing the peroxisomal distribution from the centre of the cell to the periphery of WT, Miro1KO, Miro2KO and DKO MEFs. E) Bar graph comparing the average distance at which 95% of the peroxisomal signal is distributed (Perox95). Data information: n = 60 cells over three independent experiments. For E) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance. *** represents p < 0.001. Data are represented as mean ± SEM.


A ) Snapshots and tracks of pxDsRed signal in WT , Miro1KO , Miro2KO and DKO MEFs following live imaging at 1 . 5 seconds per frame for two minutes . B ) Quantification of median net displacement of individual peroxisomes ( pxDsRed signal ) for WT , Miro1KO , Miro2KO and DKO MEFs ( n = 42 cells over six independent experiments . Two different MEFs lines were used for each genotype ) . Data information : For multiple comparisons in B ) a one - way ANOVA with Newman - Keuls post - hoc test was used to test for statistical significance .

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A) Snapshots and tracks of pxDsRed signal in WT, Miro1KO, Miro2KO and DKO MEFs following live imaging at 1.5 seconds per frame for two minutes. B) Quantification of median net displacement of individual peroxisomes (pxDsRed signal) for WT, Miro1KO, Miro2KO and DKO MEFs (n=42 cells over six independent experiments. Two different MEFs lines were used for each genotype). Data information: For multiple comparisons in B) a one-way ANOVA with Newman-Keuls post-hoc test was used to test for statistical significance.


C ) Still images every 30 seconds of ER and peroxisomes ( ER - DsRed pseudo - coloured to green and pxGFP pseudo - coloured to magenta , respectively ) in WT and DKO MEFs taken from two - minute movies by live - cell spinning - disk microscopy . Arrows track individual peroxisomes associated with an ER tubule . Scale bar is 5 μm .

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C) Still images every 30 seconds of ER and peroxisomes (ER-DsRed pseudo-coloured to green and pxGFP pseudo-coloured to magenta, respectively) in WT and DKO MEFs taken from two-minute movies by live-cell spinning-disk microscopy. Arrows track individual peroxisomes associated with an ER tubule. Scale bar is 5 μm.


D ) Quantification of the relative ER displacement over a two - minute movie using ER - DsRed signal in WT and DKO MEFs ( n = 20 cells per condition over three independent experiments ) . Data information : Student ' s t - test was used to calculate statistical significance . * and * * * represent p < 0 . 05 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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D) Quantification of the relative ER displacement over a two-minute movie using ER-DsRed signal in WT and DKO MEFs (n=20 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


E ) Median net displacement of peroxisomes in WT and DKO MEFs . n = 20 cells per condition over three independent experiments .

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E) Median net displacement of peroxisomes in WT and DKO MEFs. n=20 cells per condition over three independent experiments.


F ) Median net displacement of peroxisomes in DKO MEFs with and without myc - tagged variant 4 of Miro1 ( mycv4 ) ( n = 12 cells per condition over three independent experiments ) . Data information : Student ' s t - test was used to calculate statistical significance . * and * * * represent p < 0 . 05 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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F) Median net displacement of peroxisomes in DKO MEFs with and without myc-tagged variant 4 of Miro1 (mycv4) (n=12 cells per condition over three independent experiments). Data information: Student's t-test was used to calculate statistical significance. * and *** represent p<0.05 and p<0.001, respectively. Data are represented as mean ± SEM.


A ) Representative images of catalase staining ( peroxisomes ) in WT , Miro1KO , Miro2KO and DKO MEFs .

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A) Representative images of catalase staining (peroxisomes) in WT, Miro1KO, Miro2KO and DKO MEFs.


B ) Bar graph comparing average peroxisomal area of WT Miro1KO , Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments ) . Data information : Calculation of statistical significance in B ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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B) Bar graph comparing average peroxisomal area of WT Miro1KO, Miro2KO and DKO MEFs ( n = 60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in B) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


C ) Comparison of total peroxisomal number between WT , Miro1KO , Miro2KO and DKO MEFs . n = 60 cells per condition over three independent experiments . Data information : Calculation of statistical significance in C ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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C) Comparison of total peroxisomal number between WT, Miro1KO, Miro2KO and DKO MEFs. n = 60 cells per condition over three independent experiments. Data information: Calculation of statistical significance in C) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


D ) Representative images of Control ( GFP - tagged 1 - 70 of Tom70 ) , GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase ( peroxisomes ) .

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D) Representative images of Control (GFP-tagged 1-70 of Tom70), GFPMiro1 and GFPMiro2 in WT MEFs stained with catalase (peroxisomes).


E ) Representative images of Pex14 ( peroxisomes ) in DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) .

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E) Representative images of Pex14 (peroxisomes) in DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4).


F ) Bar graph comparing average peroxisomal area of Control , GFPMiro1 and GFPMiro2 overexpressing WT MEFs ( n = 60 cells per condition over three independent experiments ) . Data information : Calculation of statistical significance in F ) was a one - way ANOVA with Newman - Keuls post - hoc . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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F) Bar graph comparing average peroxisomal area of Control, GFPMiro1 and GFPMiro2 overexpressing WT MEFs (n=60 cells per condition over three independent experiments). Data information: Calculation of statistical significance in F) was a one-way ANOVA with Newman-Keuls post-hoc. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


G ) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP ( control ) or GFP - tagged variant 4 of Miro1 ( GFPv4 ) ( n = 30 cells per condition over three independent experiments ) . Data information : Statistical test used in G ) was a two - tailed Student ' s t test . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM . Scale bar is 10 μm . Scale bar in zooms is 5 μm .

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G) Comparison of average area of individual peroxisomes between DKO MEFs expressing either GFP (control) or GFP-tagged variant 4 of Miro1 (GFPv4) (n=30 cells per condition over three independent experiments). Data information: Statistical test used in G) was a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM. Scale bar is 10 μm. Scale bar in zooms is 5 μm.


A ) Representative images of proximity - ligation assay ( PLA ) of Fis1 and Drp1 in WT and DKO MEFs . Scale bar is 10 μm . Red dots indicate interaction of Fis1 and Drp1 . Blue is DAPI . B ) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs . Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls ( n = 3 experiments ) . Data information : For B ) statistical significance was quantified by a two - tailed Student ' s t test . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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A) Representative images of proximity-ligation assay (PLA) of Fis1 and Drp1 in WT and DKO MEFs. Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. B) Quantification of fluorescent dots from PLA of Fis1 and Drp1 in WT and DKO MEFs. Dotted line indicates the sum of the Fis1 and Drp1 single antibody controls (n=3 experiments). Data information: For B) statistical significance was quantified by a two-tailed Student's t test. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


C ) Representative images of a Fis1 - Drp1 PLA in DKO MEFs expressing either GFP , GFPv1 ( GFP - tagged variant 1 of Miro1 ) or GFPv4 ( GFP - tagged variant 4 of Miro1 ) n = 30 cells per condition over three independent experiments ) . Scale bar is 10 μm . Red dots indicate interaction of Fis1 and Drp1 . Blue is DAPI . D ) Quantification of PLA fluorescent dots per cells between GFP , GFPv1 or GFPv4 transfected DKO MEFs ( n = 30 cells over three independent experiments ) . Data information : In D ) a one - way ANOVA with a Newman - Keuls post - hoc test was used to calculate statistical significance . * , * * and * * * denote p < 0 . 05 , p < 0 . 01 and p < 0 . 001 , respectively . Data are represented as mean ± SEM .

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C) Representative images of a Fis1-Drp1 PLA in DKO MEFs expressing either GFP, GFPv1 (GFP-tagged variant 1 of Miro1) or GFPv4 (GFP-tagged variant 4 of Miro1) n=30 cells per condition over three independent experiments). Scale bar is 10 μm. Red dots indicate interaction of Fis1 and Drp1. Blue is DAPI. D) Quantification of PLA fluorescent dots per cells between GFP, GFPv1 or GFPv4 transfected DKO MEFs (n=30 cells over three independent experiments). Data information: In D) a one-way ANOVA with a Newman-Keuls post-hoc test was used to calculate statistical significance. *, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively. Data are represented as mean ± SEM.


E ) Western blot of Drp1 , Fis1 and actin from WT and DKO whole cell lysates . F ) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs ( n = 3 ) . Data information F ) , statistical significance was quantified by a two - tailed Student ' s t test .

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E) Western blot of Drp1, Fis1 and actin from WT and DKO whole cell lysates. F) Quantification of normalised band intensity of Drp1 and Fis1 in WT and DKO MEFs (n=3). Data information F), statistical significance was quantified by a two-tailed Student's t test.