( E ) Immunoblot of BAT stimulated with 8 - Br - cAMP for 5 minutes in presence of Wrtm , H89 or ESI - 09 for indicated . experiments were performed in triplicates , a representative replicate is presented .

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

(E) Immunoblot analysis of BAT cells stimulated with 8-Br-cAMP for 5 minutes in the presence of Wrtm, H89 or ESI-09 for the indicated proteins. All experiments were performed in triplicates, a representative replicate is presented.


NE cold activate mTORC2 in vivo . ( A ) Immunoblot of BAT from control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for indicated ( n = 3 / group ) .

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

NE and cold activate mTORC2 in vivo. (A) Immunoblot analysis of BAT from control mice treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).


( B ) Immunoblot of BAT from AdRiKO control mice treated with either norepinephrine ( NE ) or vehicle for 30 minutes for indicated ( n = 3 / group ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 treated with either norepinephrine (NE) or vehicle for 30 minutes for the indicated proteins (n=3/group).


( C ) Immunoblot of BAT from AdRiKO control mice housed at either 22 ° C or 4 ° C for 2h for indicated ( 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

(C) Immunoblot analysis of BAT from AdRiKO and control mice housed at either 22°C or 4°C for 2h for the indicated proteins (n=6/group, each lane represents a mix of 3 mice).


( D ) of AdRiKO control mice housed at 22 ° C ( n = 11 ( control ) , n = 9 ( AdRiKO ) ) .

O O O O 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) Body temperature of AdRiKO and control mice housed at 22°C (n=11 (control), n=9 (AdRiKO)).


( E ) of AdRiKO control mice housed at 30 ° C for 2 ( n = 8 / group ) .

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

(E) Body temperature of AdRiKO and control mice housed at 30°C for 2 weeks (n=8/group).


( F ) loss upon cold exposure of AdRiKO control mice ( n = 20 ( control ) , n = 17 ( AdRiKO ) ) . Data represent mean ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 ) . 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

(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 of sWAT sections from AdRiKO control mice ( n = 5 / group ) .

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 ( NEFAs ) in plasma of AdRiKO control mice ( n = 6 / group ) .

O O O B-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

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


( C ) Glycerol in plasma of AdRiKO 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

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


( D ) Representative H & amp ; amp ; E of BAT sections from AdRiKO 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

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


( E ) Triglycerides ( TGs ) in BAT of AdRiKO 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

(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 control mice ( n = 6 / group ) . Data represent mean ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . 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

(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 β - oxidation . ( A ) mRNA of indicated genes in BAT of AdRiKO 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

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

(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 of indicated genes in BAT of AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h ( 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

(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 of BAT from AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h for indicated ( 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 content of BAT from AdRiKO 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

(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 control mice housed at 22 ° C or at 4 ° C for 4h ( n = 3 / group ) .

(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 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

(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 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

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 control mice upon cold exposure ( n = 8 / group ) . Data represent mean ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . 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

(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 glycolysis ( A ) 2 - Deoxyglucose - 6 - phosphate ( 2DG6P ) accumulation in BAT of AdRiKO 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 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

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 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

(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 of BAT from AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h for indicated ( 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

(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 of isolated plasma membranes from BAT of AdRiKO control mice housed at 22 ° C or at 4 ° C for 8h for indicated ( 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

(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 of mitochondrial fractions from BAT of AdRiKO control mice housed at 22 ° C or at 4 ° C for 4h for indicated ( 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

(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 ) hexokinase activity in BAT of AdRiKO 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - 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 - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . 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 O O O O O O O O O O O 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 thermogenic defect in AdRiKO mice . ( A ) HKII mRNA expression level in BAT of AdRiKO control mice infected with either AAV9 - HKII or AAV9 - empty ( n = 8 / group ) .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of BAT from AdRiKO 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 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 ) of AdRiKO 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 ) upon cold exposure of AdRiKO control mice infected with either AAV8 - Akt2S474D or AAV8 - empty . left panel represents after each hour of cold exposure while right panel represents as a bar graph for 3h cold exposure time point ( n = 11 / group ) . a = significant difference between AdRiKO control mice infected with AAV8 - empty , b = significant difference between AdRiKO control mice infected with AAV8 - Akt2S474D , d = significant difference between AdRiKO mice infected with AAV8 - empty 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 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 ± . Statistically significant differences between AdRiKO control mice were determined with unpaired Student ' s t - are indicated with asterisks ( * = p < 0 . 05 ; * * = p < 0 . 01 , * * * = p < 0 . 001 ) . Statistically significant differences between were determined with unpaired Student ' s t - are indicated with a number sign ( # = p < 0 . 05 ; # # = p < 0 . 01 ; # # # = p < 0 . 001 ) . 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


B , C . Distribution of m6A peaks across host mRNAs ( B ) EBV mRNAs ( C ) was analyzed on MeRIP - seq data .

O O 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

B, C. Distribution of the m6A peaks across host mRNAs (B) and EBV mRNAs (C) was analyzed based on the MeRIP-seq data.


B . BZLF1 m6A were measured using MeRIP - qPCR in CNE2EBV , Raji B95 - 8 with latent EBV infection or lytic reactivation . TPA ( 30 ng / ml ) NaB ( 2 mM ) were used to treat for 24 h to reactivate EBV . was harvested for MeRIP - qPCR assays . fold enrichment was determined by calculating 2 - ΔCt of MeRIP sample relative to input sample . mean value of results in mock - treated was normalized to 1 .

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 B-CONTROLLED_VAR 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

B. The BZLF1 m6A levels were measured using MeRIP-qPCR in CNE2EBV, Raji and B95-8 cells with latent EBV infection or lytic reactivation. TPA (30 ng/ml) and NaB (2 mM) were used to treat the cells for 24 h to reactivate EBV. The cellular RNA was harvested for MeRIP-qPCR assays. The fold enrichment was determined by calculating the 2-ΔCt of the MeRIP sample relative to the input sample. The mean value of the results in mock-treated cells was normalized to 1.


B . EBV infection efficiency in HK1 transfected with indicated siRNAs was analyzed by FACS . were infected with EBV - GFP at 24 h after siRNA transfection . of GFP - positive was quantified by FACS at 24 hpi . mean value of GFP - positive rates in siNC transfected HK1 was normalized to 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The EBV infection efficiency in HK1 cells transfected with the indicated siRNAs was analyzed by FACS. The cells were infected with EBV-GFP virus at 24 h after siRNA transfection. The ratio of GFP-positive cells was quantified by FACS at 24 hpi. The mean value of the GFP-positive rates in the siNC transfected HK1 cells was normalized to 1.


M . relative EBV production in C666 transfected with YTHDF1 - specific siRNAs ( siY1 - 1 siY1 - 2 ) or siNC control was measured by qPCR . from - free culture supernatant was harvested at 24 h after siRNA transfection . number of EBV copies in supernatant of was quantified by qPCR using specific primers targeting BamHI - W fragment region of EBV . mean value of EBV copies in siNC was normalized to 1 .

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

M. The relative EBV production in C666 cells transfected with YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control was measured by qPCR. The DNA from the cell-free culture supernatant was harvested at 24 h after siRNA transfection. The number of EBV copies in the supernatant of the cells was quantified by qPCR using specific primers targeting BamHI-W fragment region of EBV. The mean value of EBV copies in the siNC cells was normalized to 1.


B . relative mRNA of BZLF1 BRLF1 in latently infected or reactivated ( 24 h post NaB treatment ) CNE2EBV transfected with YTHDF1 - specific siRNAs ( siY1 - 1 siY1 - 2 ) or siNC control . After 24 h of siRNA transfection , CNE2EBV were treated with NaB ( 2 mM ) or mock for 24 h , followed by qRT - PCR . gene expression were normalized to housekeeping gene ACTB .

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 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

B. The relative mRNA levels of BZLF1 and BRLF1 in the latently infected or reactivated (24 h post NaB treatment) CNE2EBV cells transfected with the YTHDF1-specific siRNAs (siY1-1 and siY1-2) or the siNC control. After 24 h of siRNA transfection, CNE2EBV cells were treated with NaB (2 mM) or mock for 24 h, followed by qRT-PCR analysis. All the gene expression levels were normalized to the housekeeping gene ACTB.


F . level of YTHDF1 , BZLF1 ACTB in CNE2EBV following EBV reactivation ( 24 h post - NaB treatment ) , EBV infected HK1 or NPEC2 - Bmi1 at 24 hpi . whole - was extracted at same time as .

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

F. The protein level of YTHDF1, BZLF1 and ACTB in CNE2EBV cells following EBV reactivation (24 h post-NaB treatment), EBV infected HK1 or NPEC2-Bmi1 cells at 24 hpi. The whole-cell protein was extracted at same time as the RNA.


D . relative enrichment of BZLF1 BRLF1 mRNA in CNE2EBV with transfected with siYTHDF2 , siYTHDF3 or siNC siRNA following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV were treated with TPA ( 30 ng / ml ) NaB ( 2 mM ) for 24 h . RIP assays were performed using YTHDF1 - specific antibody or IgG control in these . Fold enrichment was determined by calculating 2 - ΔCt of RIP sample relative to input sample .

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D. The relative enrichment of the BZLF1 and BRLF1 mRNA in CNE2EBV cells with transfected with siYTHDF2, siYTHDF3 or siNC siRNA following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using YTHDF1-specific antibody or IgG control in these cells. Fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


E , F . relative m6A YTHDF1 enrichment of BZLF1 ( E ) or BRLF1 ( F ) mRNAs measured in CNE2EBV transfected with METTL14 - specific siRNAs ( siM14 ) or siNC control following EBV reactivation . After 24 h of siRNA transfection , CNE2EBV were treated with TPA ( 30 ng / ml ) NaB ( 2 mM ) for 24 h , followed by MeRIP RIP assays . fold enrichment was determined by calculating 2 - ΔCt of RIP sample relative to input sample .

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E, F. The relative m6A and YTHDF1 enrichment of the BZLF1 (E) or BRLF1 (F) mRNAs measured in CNE2EBV cells transfected with the METTL14-specific siRNAs (siM14) or siNC control following EBV reactivation. After 24 h of siRNA transfection, CNE2EBV cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h, followed by MeRIP and RIP assays. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


D . interaction between YTHDF1 DCP2 was detected by co - IP in 293T .

D. The interaction between YTHDF1 and DCP2 was detected by co-IP in 293T cells.


F . interactions between endogenous YTHDF1 ZAP , DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV transfected with YTHDF2 - , YTHDF3 - specific siRNAs or siNC control . YTHDF1 , YTHDF2 , YTHDF3 , ZAP , DDX17 , DCP2 were detected in immunoprecipitated complexes using specific antibodies , respectively .

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F. The interactions between endogenous YTHDF1 and ZAP, DDX17 or DCP2 were detected by endogenous IP assays in CNE2EBV cells transfected with YTHDF2-, YTHDF3-specific siRNAs or the siNC control. YTHDF1, YTHDF2, YTHDF3, ZAP, DDX17, and DCP2 were detected in the immunoprecipitated complexes using specific antibodies, respectively.


F . relative mRNA of BZLF1 BRLF1 in C666 transfected with YTHDF1 - specific siRNAs XRN1 - specific siRNAs . C666 were co - transfected with YTHDF1 - specific siRNAs XRN1 - specific siRNAs for 24 h . mRNA were determined using qPCR .

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F. The relative mRNA levels of BZLF1 and BRLF1 in C666 cells transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs. The C666 cells were co-transfected with YTHDF1-specific siRNAs and XRN1-specific siRNAs for 24 h. The mRNA levels were determined using qPCR.


G . relative enrichment of BZLF1 BRLF1 mRNAs by ZAP , DDX17 , or DCP2 in CNE2EBV following EBV reactivation . CNE2EBV were transfected with plasmids encoding myc - ZAP , myc - DDX17 , myc - DCP2 , or vector control . At 24 h post transfection , were treated with TPA ( 30 ng / ml ) NaB ( 2 mM ) for 24 h . RIP assays were performed using Myc - beads . fold enrichment was determined by calculating 2 - ΔCt of RIP sample relative to input sample .

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G. The relative enrichment of BZLF1 and BRLF1 mRNAs by ZAP, DDX17, or DCP2 in CNE2EBV cells following EBV reactivation. CNE2EBV cells were transfected with plasmids encoding myc-ZAP, myc-DDX17, myc-DCP2, or vector control. At 24 h post transfection, the cells were treated with TPA (30 ng/ml) and NaB (2 mM) for 24 h. The RIP assays were performed using Myc-beads. The fold enrichment was determined by calculating the 2-ΔCt of the RIP sample relative to the input sample.


H . Determination of EBV infection efficiency in HK1 transfected with XRN1 - specific siRNA or siNC control . After 24 h of siRNA transfection , HK1 were infected with EBV analyzed by FACS at 24 hpi .

O O O O O O O O O O O O O O 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. Determination of the EBV infection efficiency in HK1 cells transfected with the XRN1-specific siRNA or the siNC control. After 24 h of siRNA transfection, HK1 cells were infected with EBV and analyzed by FACS at 24 hpi.


B . Volcano plot of differently - regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4h of NGF deprivation , with - level cutoffs set at log2 fold change > 1 . 0 - log10 adjusted P value > 1 . 3 ( P < 0 . 05 ) , marked by dashed lines . Downregulated sites represented as blue points , up - regulated sites as orange points ( n = 2 independent experiments ) .

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B. Volcano plot of differently-regulated phosphosites of MAPK cascade components from 3DIV cultured embryonic DRG neurons upon 4h of NGF deprivation, with protein-level cutoffs set at log2 fold change >1.0 and -log10 adjusted P value >1.3 (P< 0.05), marked by dashed lines. Downregulated sites represented as blue points, and up-regulated sites as orange points (n = 2 independent experiments).


C . Representative Western blots of different MAPKs expressed in DRG neurons ( DIV3 ) cultured in presence or absence of NGF for 3h subjected to a / fractionation . α - tubulin STMN - 1 are used as controls of Na + / K + - ATPase STMN - 2 as controls of .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative Western blots of the different MAPKs expressed in DRG neurons (DIV3) cultured in the presence or absence of NGF for 3h and subjected to a membrane/cytosolic fractionation. α-tubulin and STMN-1 are used as controls of cytosolic proteins and Na+/K+-ATPase and STMN-2 as controls of membrane proteins.


D . of of localized in fraction after 3h of NGF deprivation ( n = 6 - 14 biological replicates ; series of unpaired t followed by a Holm - Sidak correction ) . Data information : Graphs represent mean ± in ( D ) * p < 0 . 05 * * p < 0 . 01 .

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D. Quantification of the ratio of protein localized in the membrane fraction after 3h of NGF deprivation (n = 6-14 biological replicates; series of unpaired t tests followed by a Holm-Sidak correction). Data information: Graphs represent mean ± SEM in (D) *p < 0.05 and **p < 0.01.


F . Representative Western blots of total palmitoylated DLK ( upper panel ) of DLK palmitoylation ( lower panel ) from cultured DRG neurons subjected to 3h of NGF deprivation ( n = 5 independent experiments ; Mann - Whitney U ) . Data information : Graphs represent mean ± in ( F ) . * p < 0 . 05 * * p < 0 . 01 .

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F. Representative Western blots of total and palmitoylated DLK (upper panel) and quantification of DLK palmitoylation levels (lower panel) from cultured DRG neurons subjected to 3h of NGF deprivation (n = 5 independent experiments; Mann-Whitney U test). Data information: Graphs represent mean ± SEM in (F). *p < 0.05 and **p < 0.01.


A . Representative stills from time - lapse recordings of DIV6 DRG neurons overexpressing DLK - GFP . Arrowheads point to moving DLK - GFP positive structures . B . Kymographs made from complete time - lapse recording shown in A . left shows original kymograph , right shows an illustration of manually traced structure displacements for better visualization . Data information : Scale bar represents 10 μm in ( B ) 5 μm in ( A )

A. Representative stills from time-lapse recordings of DIV6 DRG neurons overexpressing DLK-GFP. Arrowheads point to individual moving DLK-GFP positive structures. B. Kymographs made from the complete time-lapse recording shown in A. The left shows the original kymograph, and the right shows an illustration of the manually traced structure displacements for better visualization. Data information: Scale bar represents 10 μm in (B) 5 μm in (A)


C . Representative of DIV3 DRG neurons nucleofected with a non - targeting siRNA ( NT siRNA ) or a siRNA against DLK ( DLK siRNA ) , stained for endogenous DLK β3 - tubulin ( TUBB3 ) . On right , of DLK mean in neurons expressing control NT or DLK siRNAs ( n = 25 neurons / condition ; Unpaired t ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 10 μm in ( C ) 2 μm in zooms in ( C ) .

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C. Representative images of DIV3 DRG neurons nucleofected with a non-targeting siRNA (NT siRNA) or a siRNA against DLK (DLK siRNA), and stained for endogenous DLK and β3-tubulin (TUBB3). On the right, quantification of the DLK mean intensity in neurons expressing control NT or DLK siRNAs (n = 25 neurons/condition; Unpaired t test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) 2 μm in zooms in (C).


D - F . Representative kymographs ( D ) , of GFP - positive particle number ( E ) of anterograde , retrograde , reverse or non - moving GFP - positive particles ( F ) from DLK - GFP time - lapse recordings in DIV6 DRG neurons , in control situation ( + NGF ) or after 3h of NGF deprivation ( - NGF ) ( n = 45 - 46 neurons / condition ; Mann - Whitney U in E Chi - square in F ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents , 5 μm in ( D )

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D-F. Representative kymographs (D), quantification of GFP-positive particle number (E) and percentage of anterograde, retrograde, reverse or non-moving GFP-positive particles (F) from DLK-GFP time-lapse recordings in DIV6 DRG neurons, in control situation (+ NGF) or after 3h of NGF deprivation (-NGF) (n = 45-46 neurons/ condition; Mann-Whitney U test in E and Chi-square test in F). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents , 5 μm in (D)


G . Representative of DIV3 cultured embryonic DRG neurons maintained in presence ( + NGF ) or absence ( - NGF ) of NGF for 3h stained for endogenous DLK . On right , of number of DLK - positive in control situation or after 3h of NGF deprivation ( n = 18 - 19 neurons / condition ; Mann - Whitney U ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 5 μm in ( G )

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G. Representative images of DIV3 cultured embryonic DRG neurons maintained in the presence (+NGF) or absence (-NGF) of NGF for 3h and stained for endogenous DLK. On the right, quantification of the number of DLK- positive puncta in control situation or after 3h of NGF deprivation (n = 18-19 neurons/condition; Mann-Whitney U test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 5 μm in (G)


H . Representative kymograph stills from a time lapse recording of 6DIV neurons overexpressing a GFP - tagged N - terminal region of DLK ( NtermDLK - GFP ) . Arrowheads point to moving NtermDLK - GFP positive structures . Data information : Scale bar represents 5 μm in ( H )

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H. Representative kymograph and stills from a time lapse recording of 6DIV neurons overexpressing a GFP-tagged N-terminal region of DLK (NtermDLK-GFP). Arrowheads point to individual moving NtermDLK-GFP positive structures. Data information: Scale bar represents 5 μm in (H)


I - J . Representative Western blots of p - cJun GAPDH ( I ) , of relative c - Jun phosphorylation ( J ) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK - GFP , subjected to NGF withdrawal for 3h ( n = 7 - 8 biological replicates ; series of unpaired t / Mann - Whitney U followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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I-J. Representative Western blots of p-cJun and GAPDH (I), and quantification of relative c-Jun phosphorylation levels (J) from DIV6 cultured embryonic DRG neurons overexpressing GFP or NtermDLK-GFP, and subjected to NGF withdrawal for 3h (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


L . Representative of HeLa expressing different HA - tagged wild - type ( WT ) palmitoyl - mutant ( CS ) DLK constructs ( DLK - WT - ER , DLK - CS - ER , DLK - WT - CAAX , DLK - CS - CAAX , DLK - WT - MTS , DLK - CS - MTS ) , stained with markers of endoplasmic reticulum ( KDEL ) , plasma ( CellMask ) mitochondria ( cytochrome C ) . Data information : Scale bar represents 5 μm in ( L )

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L. Representative images of HeLa cells expressing the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS), and stained with markers of the endoplasmic reticulum (KDEL), plasma membrane (CellMask) and mitochondria (cytochrome C). Data information: Scale bar represents 5 μm in (L)


M - N . Representative Western blots of DLK , p - cJun GAPDH ( M ) of relative c - Jun phosphorylation ( N ) from HeLa transfected with HA alone ( HA ) different HA - tagged wild - type ( WT ) palmitoyl - mutant ( CS ) DLK constructs ( DLK - WT - ER , DLK - CS - ER , DLK - WT - CAAX , DLK - CS - CAAX , DLK - WT - MTS , DLK - CS - MTS ) ( n = 7 - 8 biological replicates ; series of unpaired t / Mann - Whitney U with DLK - WT or DLK - CS followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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M-N. Representative Western blots of DLK, p-cJun and GAPDH (M) and quantification of relative c-Jun phosphorylation levels (N) from HeLa cells transfected with HA alone (HA) and the different HA-tagged wild-type (WT) and palmitoyl-mutant (CS) DLK constructs (DLK-WT-ER, DLK-CS-ER, DLK-WT-CAAX, DLK-CS-CAAX, DLK-WT-MTS, DLK-CS-MTS) (n = 7-8 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT or DLK-CS followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


Representative Western blots of GFP , p - cJun GAPDH ( B ) from HeLa non transfected ( NT ) or transfected with GFP alone ( GFP ) , GFP - tagged DLK wild - type ( DLK - WT - GFP ) , kinase dead mutant ( DLK - KD - GFP ) , leucine zipper domain mutant ( DLK - LZ - GFP ) a palmitoyl - site mutant ( DLK - CS - GFP )

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Representative Western blots of GFP, p-cJun and GAPDH (B) from HeLa cells non transfected (NT) or transfected with GFP alone (GFP), GFP-tagged DLK wild-type (DLK-WT-GFP), kinase dead mutant (DLK-KD-GFP), leucine zipper domain mutant (DLK-LZ-GFP) and a palmitoyl-site mutant (DLK-CS-GFP)


of relative c - Jun phosphorylation ( C ) from HeLa non transfected ( NT ) or transfected with GFP alone ( GFP ) , GFP - tagged DLK wild - type ( DLK - WT - GFP ) , kinase dead mutant ( DLK - KD - GFP ) , leucine zipper domain mutant ( DLK - LZ - GFP ) a palmitoyl - site mutant ( DLK - CS - GFP ) ( n = 4 - 7 biological replicates ; series of unpaired t / Mann - Whitney U with DLK - WT - GFP followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 .

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quantification of relative c-Jun phosphorylation levels (C) from HeLa cells non transfected (NT) or transfected with GFP alone (GFP), GFP-tagged DLK wild-type (DLK-WT-GFP), kinase dead mutant (DLK-KD-GFP), leucine zipper domain mutant (DLK-LZ-GFP) and a palmitoyl-site mutant (DLK-CS-GFP) (n = 4-7 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001.


D . Representative zooms of HeLa expressing GFP alone or different GFP - tagged DLK mutants . Data information : Scale bar represents 100 μm in ( D ) zooms in ( D ) .

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D. Representative images and zooms of HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants. Data information: Scale bar represents 100 μm in (D) zooms in (D).


E - F . Representative Western blots of GFP , α - tubulin Na + / K + - ATPase from fractions ( E ) of of DLK localized in fraction ( F ) from HeLa expressing GFP alone or different GFP - tagged DLK mutants ( n = 4 - 5 biological replicates ; series of unpaired t / Mann - Whitney U with DLK - WT - GFP followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 .

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E-F. Representative Western blots of GFP, α-tubulin and Na+/K+-ATPase from cytosolic and membrane fractions (E) and quantification of the percentage of DLK localized in the membrane and cytosolic fraction (F) from HeLa cells expressing GFP alone or the different GFP-tagged DLK mutants (n = 4-5 biological replicates; series of unpaired t tests /Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001.


G - I . Representative stills ( G ) , representative kymographs ( H ) of number of GFP - positive particles ( I ) from time - lapse recordings of different GFP - tagged DLK mutants in DIV6 cultured embryonic DRG neurons ( n = 31 - 65 neurons / condition ; series of Mann - Whitney U with DLK - WT - GFP followed by a Holm - Sidak correction ) . Data information : Arrowheads point to DLK - GFP positive structures in G graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 . Scale bar represents 10 μm in ( G ) , 5 μm in ( H )

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G-I. Representative stills (G), representative kymographs (H) and quantification of the number of GFP-positive particles (I) from time-lapse recordings of the different GFP-tagged DLK mutants in DIV6 cultured embryonic DRG neurons (n = 31-65 neurons/condition; series of Mann-Whitney U tests with DLK-WT-GFP followed by a Holm-Sidak correction). Data information: Arrowheads point to individual DLK-GFP positive structures in G All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bar represents 10 μm in (G) , 5 μm in (H)


J - L . Representative stills ( J ) , kymographs ( K ) of number of DLK - GFP positive particles ( L ) from time - lapse recordings of DIV6 DRG neurons overexpressing GFP - tagged DLK treated with control ( DMSO ) or DLK inhibitor GNE - 3511 ( DLKi ) for 3h ( n = 22 - 46 neurons / condition ; Unpaired t ) . Data information : Arrowheads point to DLK - GFP positive structures in J . graphs represent mean ± . * p < 0 . 05 * * * p < 0 . 001 . Scale bar represents 10 μm in ( J ) , 5 μm in ( K )

O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-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

J-L. Representative stills (J), kymographs (K) and quantification of the number of DLK-GFP positive particles (L) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with control (DMSO) or DLK inhibitor GNE-3511 (DLKi) for 3h (n = 22-46 neurons/condition; Unpaired t test). Data information: Arrowheads point to individual DLK-GFP positive structures in J. All graphs represent mean ± SEM. *p < 0.05 and ***p < 0.001. Scale bar represents 10 μm in (J), 5 μm in (K)


A . Representative confocal deconvolved thresholded of DIV3 cultured embryonic DRG neurons co - stained for endogenous DLK ( green ) marker for synaptic vesicles Rab3 ( magenta ) . Data information : Scale bar represents , 5 μm in ( A ) 2 μm in zooms in ( A ) .

A. Representative confocal deconvolved and thresholded images of DIV3 cultured embryonic DRG neurons co-stained for endogenous DLK (green) and the marker for synaptic vesicles Rab3 (magenta). Data information: Scale bar represents , 5 μm in (A) and 2 μm in zooms in (A).


B . Colocalization between DLK ( black values ) or same image after 90 ° rotation ( grey values ) , markers for different intracellular compartments ( Rab3 , Rab6 , Rab7 , Rab11 mitochondria ) in DIV3 cultured embryonic DRG neurons ( n = 13 - 17 / condition ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Colocalization analysis between DLK (black values) or same image after 90° rotation (grey values), and markers for different intracellular compartments (Rab3, Rab6, Rab7, Rab11 and mitochondria) in DIV3 cultured embryonic DRG neurons (n = 13-17 images/condition). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


C . Representative of HeLa expressing HA alone different HA - tagged DLK constructs : a wild - type form of DLK ( DLK - WT - HA ) palmitoyl - mutant DLK ( DLK - CS - HA ) . Data information : Scale bar represents 20 μm in ( 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

C. Representative images of HeLa cells expressing HA alone and the different HA-tagged DLK constructs: a wild-type form of DLK (DLK-WT-HA) and the palmitoyl-mutant DLK (DLK-CS-HA). Data information: Scale bar represents 20 μm in (C)


D . Western blot from total lysate ( Input ) immunoprecipitates ( IP ) of HA - tag immunoprecipitations in HeLa overexpressing HA alone different HA - tagged DLK constructs , DLK - WT - HA DLK - CS - HA in absence of any detergent .

O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot analysis from total lysate (Input) and immunoprecipitates (IP) of HA-tag protein immunoprecipitations in HeLa cells overexpressing HA alone and the different HA-tagged DLK constructs, DLK-WT-HA and DLK-CS-HA in the absence of any detergent.


E - F . Lipidomic of immunoprecipitates obtained from HA - tagged immunoprecipitations from extracts of HeLa overexpressing HA alone , DLK - WT - HA DLK - CS - HA . of lipid classes ( E ) sphingomyelin fatty acid species ( F ) measured in immunoprecipitates ( n = 3 - 4 independent experiments ; series of unpaired t / Mann - Whitney U followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . CE , cholesteryl ester ; CER , ceramide ; DAG , diacylglycerol ; DCER , dihydroceramides ; HCER , hexosylceramides , LCER , lactosylceramides ; LPC , lysophosphatidylcholine ; LPE , lysophosphatidylethanolamine ; PC , phosphatidylcholine ; PE , phosphatidylethanolamine ; SM , sphingomyelin ; TAG , triacylglycerol .

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

E-F. Lipidomic analysis of the immunoprecipitates obtained from the HA-tagged protein immunoprecipitations from extracts of HeLa cells overexpressing HA alone, DLK-WT-HA and DLK-CS-HA. Quantification of lipid classes (E) and sphingomyelin fatty acid species (F) measured in the immunoprecipitates (n = 3-4 independent experiments; series of unpaired t tests /Mann-Whitney U tests followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. CE, cholesteryl ester; CER, ceramide; DAG, diacylglycerol; DCER, dihydroceramides; HCER, hexosylceramides, LCER, lactosylceramides; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SM, sphingomyelin; TAG, triacylglycerol.


G - I . Representative Western blots of DLK , p - cJun GAPDH ( G ) of relative c - Jun phosphorylation ( H ) DLK ( I ) from 3DIV cultured embryonic DRG neurons treated with D609 for 3h in presence or absence of NGF ( n = 8 biological replicates ; series of unpaired t with control NGF withdrawal condition followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

G-I. Representative Western blots of DLK, p-cJun and GAPDH (G) and quantification of relative c-Jun phosphorylation (H) and DLK levels (I) from 3DIV cultured embryonic DRG neurons treated with D609 for 3h in the presence or absence of NGF (n = 8 biological replicates; series of unpaired t tests with control NGF withdrawal condition followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


Representative stills ( J ) from time - lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP - tagged DLK treated with D609 for 3h ( n = 29 - 46 neurons Arrowheads in J point to DLK - GFP positive structures . Data information : Scale bar represents 10 μm in ( J )

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

Representative stills (J) from time-lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP-tagged DLK and treated with D609 for 3h (n = 29-46 neurons Arrowheads in J point to individual DLK-GFP positive structures. Data information: Scale bar represents 10 μm in (J)


kymographs of number of DLK - GFP positive particles ( K ) from time - lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP - tagged DLK treated with D609 for 3h ( n = 29 - 46 neurons ; Mann - Whitney U ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents , 5 μm in ( K )

O O O O O O 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

kymographs and quantification of the number of DLK-GFP positive particles (K) from time-lapse recordings from 6DIV cultured embryonic DRG neurons overexpressing GFP-tagged DLK and treated with D609 for 3h (n = 29-46 neurons; Mann-Whitney U test). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents , 5 μm in (K)


L . Representative Western blots of DLK , α - tubulin Na + / K + - ATPase from , insoluble fractions of 3DIV cultured embryonic DRG neurons treated with D609 for 3h .

O O O O O O 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

L. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase from cytosolic, membrane and insoluble fractions of 3DIV cultured embryonic DRG neurons treated with D609 for 3h.


A . Western blot of endogenous DLK , p - cJun , Rab6 GAPDH , of relative c - Jun phosphorylation in 3DIV cultured embryonic DRG neurons treated with dynasore ( DYN ) or brefeldin A ( BFA ) for 3h in presence or absence of NGF ( n = 6 - 12 biological replicates ; series of unpaired t / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

A. Western blot analysis of endogenous DLK, p-cJun, Rab6 and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) or brefeldin A (BFA) for 3h in the presence or absence of NGF (n = 6-12 biological replicates; series of unpaired t tests /Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


B . Representative Western blots of p - MKK4 , MKK4 GAPDH , quantifications of relative MKK4 phosphorylation in 3DIV cultured embryonic DRG neurons treated with dynasore ( DYN ) for 3h in presence or absence of NGF ( n = 6 biological replicates , series of unpaired t with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

O O 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 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

B. Representative Western blots of p-MKK4, MKK4 and GAPDH, and quantifications of the relative MKK4 phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) for 3h in the presence or absence of NGF (n = 6 biological replicates, series of unpaired t tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


Representative stills ( C ) , kymographs ( D ) of orientation ( E ) of DLK - GFP positive particles from time - lapse recordings of DIV6 DRG neurons overexpressing GFP - tagged DLK treated with dynasore ( DYN ) or brefeldin A ( BFA ) for 3h ( n = 34 - 101 neurons , Chi - square in E Arrowheads in C point to DLK - GFP positive structures . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 10 μm in ( C ) , 5 μm in ( D )

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

Representative stills (C), kymographs (D) and quantification of the orientation (E) of DLK-GFP positive particles from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with dynasore (DYN) or brefeldin A (BFA) for 3h (n = 34-101 neurons, Chi-square test in E Arrowheads in C point to individual DLK-GFP positive structures. Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (C) and , 5 μm in (D)


of number of DLK - GFP positive particles ( F ) from time - lapse recordings of DIV6 DRG neurons overexpressing GFP - tagged DLK treated with dynasore ( DYN ) or brefeldin A ( BFA ) for 3h ( n = 34 - 101 neurons , series of Mann - Whitney with control followed by a Holm - Sidak correction in F ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

quantification of number of DLK-GFP positive particles (F) from time-lapse recordings of DIV6 DRG neurons overexpressing GFP-tagged DLK and treated with dynasore (DYN) or brefeldin A (BFA) for 3h (n = 34-101 neurons, series of Mann-Whitney tests with control followed by a Holm-Sidak correction in F). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


G - H . Representative Western blots of DLK , α - tubulin Na + / K + - ATPase ( G ) quantifications ( H ) from fractions of 3DIV cultured embryonic DRG neurons with dynasore ( DYN ) for 3h in absence of NGF ( n = 5 - 11 biological replicates , series of unpaired t / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

G-H. Representative Western blots of DLK, α-tubulin and Na+/K+-ATPase (G) and quantifications (H) from cytosolic and membrane fractions of 3DIV cultured embryonic DRG neurons with dynasore (DYN) for 3h in the absence of NGF (n = 5-11 biological replicates, series of unpaired t tests /Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


I - K . Representative stills ( I ) , kymographs ( J ) of number of DLK - GFP positive particles ( K ) from cultured embryonic DRG neurons treated with chlorpromazine ( CHP ) , dansylcadaverine ( DCV ) or methyl - β - cyclodextrin ( MCD ) for 3h ( n = 12 neurons , series of Mann - Whitney U with control situation followed by a Holm - Sidak correction ) . Arrowheads in I point to DLK - GFP positive structures . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 10 μm in ( I ) , 5 μm in ( J ) .

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

I-K. Representative stills (I), kymographs (J) and quantification of the number of DLK-GFP positive particles (K) from cultured embryonic DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 3h (n = 12 neurons, series of Mann-Whitney U tests with control situation followed by a Holm-Sidak correction). Arrowheads in I point to individual DLK-GFP positive structures. Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 10 μm in (I), 5 μm in (J).


L . Representative Western blots of p - cJun GAPDH , of relative c - Jun phosphorylation in 3DIV cultured embryonic DRG neurons treated with chlorpromazine ( CHP ) , dansylcadaverine ( DCV ) or methyl - β - cyclodextrin ( MCD ) for 3h in absence of NGF ( n = 6 - 24 biological replicates , series of Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

L. Representative Western blots of p-cJun and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 3h in the absence of NGF (n = 6-24 biological replicates, series of Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


M . Representative Western blots of p - cJun GAPDH , of relative c - Jun phosphorylation in 3DIV cultured embryonic DRG neurons treated with vincristine ( VNC ) dynasore ( DYN ) for 3h ( n = 7 biological replicates , series of Mann - Whitney U with vincristine condition followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

M. Representative Western blots of p-cJun and GAPDH, and quantification of relative c-Jun phosphorylation levels in 3DIV cultured embryonic DRG neurons treated with vincristine (VNC) and dynasore (DYN) for 3h (n = 7 biological replicates, series of Mann-Whitney U tests with vincristine condition followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


A . Western blot of caspase - 3 ( cCasp3 ) GAPDH , quantifications of cCasp3 in 3DIV cultured embryonic DRG neurons treated with dynasore ( DYN ) for 8h in absence of NGF ( n = 6 - 13 biological replicates , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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 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

A. Western blot analysis of cleaved caspase-3 (cCasp3) and GAPDH, and quantifications of cCasp3 levels in 3DIV cultured embryonic DRG neurons treated with dynasore (DYN) for 8h in the absence of NGF (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


B . Quantifications of cCasp3 from 3DIV DRG neurons treated with chlorpromazine ( CHP ) , dansylcadaverine ( DCV ) or methyl - β - cyclodextrin ( MCD ) for 8h in absence of NGF ( n = 6 - 13 biological replicates , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 .

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

B. Quantifications of cCasp3 levels from 3DIV DRG neurons treated with chlorpromazine (CHP), dansylcadaverine (DCV) or methyl-β-cyclodextrin (MCD) for 8h in absence of NGF (n = 6-13 biological replicates, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001.


C . Representative of DRG neurons expressing GFP , Dnm - 1 - WT - HA or Dnm1 - DN - HA form , cultured in absence of NGF for 8h , stained with antibodies against cCasp3 ( magenta ) HA tag ( green ) . On right , of of cCasp3 positive over GFP or HA positive ( n = 10 - 15 from 3 independent cultures , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 20 μm in ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images of DRG neurons expressing GFP, Dnm-1-WT-HA or Dnm1-DN-HA form, cultured in the absence of NGF for 8h, and stained with antibodies against cCasp3 (magenta) and HA tag (green). On the right, quantification of the ratio of cCasp3 positive cells over GFP or HA positive cells (n = 10-15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 20 μm in (C)


D . Representative of axons from E12 . 5 DRG explants cultured for 2 days , subsequently deprived of NGF treated with different concentrations of dynasore ( DYN ) for 24 h , visualized by immunostaining for βIII - tubulin . of axonal degeneration index as of fragmented axon over total axon ( n = 13 - 15 explants , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 20 μm in ( D ) 10 μm in zooms in ( D ) .

O 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 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

D. Representative images of axons from E12.5 DRG explants cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore (DYN) for 24 h, and visualized by immunostaining for βIII-tubulin. Quantification of the axonal degeneration index as the ratio of fragmented axon area over total axon area (n = 13-15 explants, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 20 μm in (D) and 10 μm in zooms in (D).


E . Representative of axons from cultured embryonic DRG neurons cultured for 2 days , subsequently deprived of NGF treated with different concentrations of dynasore ( DYN ) for 24 h , visualized by imaging SiR - tubulin probe . On right , of fragmented axons using degeneration index ( n = 15 from 3 independent cultures , series of unpaired t - / Mann - Whitney U with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 100 μm in ( E )

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 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

E. Representative images of axons from cultured embryonic DRG neurons cultured for 2 days, subsequently deprived of NGF and treated with different concentrations of dynasore (DYN) for 24 h, and visualized by imaging SiR-tubulin probe. On the right, quantification of fragmented axons using the degeneration index (n = 15 images from 3 independent cultures, series of unpaired t-test/Mann-Whitney U tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 100 μm in (E)


F . Representative of axons from cultured embryonic DRG neurons expressing GFP , Dnm - 1 - WT - HA or Dnm1 - DN - HA form deprived of NGF for 24h , visualized with an antibody against neurofilament heavy chain ( NFH ) . On right , of fragmented axons using degeneration index in ( n = 8 - 13 from 3 independent cultures , series of unpaired t - with control NGF withdrawal followed by a Holm - Sidak correction ) . Data information : graphs represent mean ± . * p < 0 . 05 * * p < 0 . 01 * * * p < 0 . 001 . Scale bar represents 40 μm in ( F )

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F. Representative images of axons from cultured embryonic DRG neurons expressing GFP, Dnm-1-WT-HA or Dnm1-DN-HA form and deprived of NGF for 24h, and visualized with an antibody against neurofilament heavy chain (NFH). On the right, quantification of fragmented axons using the degeneration index in (n = 8-13 images from 3 independent cultures, series of unpaired t-test tests with control NGF withdrawal followed by a Holm-Sidak correction). Data information: All graphs represent mean ± SEM. *p < 0.05 **p < 0.01 and ***p < 0.001. Scale bar represents 40 μm in (F)


A , HBMEC were challenged with type I ( RH ) or type II ( ME49 ) strains of T . gondii for indicated time points . lysates were used to probe for total Akt phospho - Ser473 Akt by immunoblot .

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A, Primary HBMEC were challenged with type I (RH) or type II (ME49) strains of T. gondii for the indicated time points. Cell lysates were used to probe for total Akt and phospho-Ser473 Akt by immunoblot.


B , C , Mouse endothelial ( mHEVc ; B ) human retinal pigment epithelial ( RPE , C ) were challenged with RH T . gondii followed by assessment of Akt phosphorylation .

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B, C, Mouse endothelial cells (mHEVc; B) and human retinal pigment epithelial cells (RPE, C) were challenged with RH T. gondii followed by assessment of Akt phosphorylation.


D , HBMEC were challenged with either live or killed T . gondii . lysates were subject to immunoblotting as indicated .

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D, HBMEC were challenged with either live or killed T. gondii. Cell lysates were subject to immunoblotting as indicated.


E , HBMEC were transiently transfected with a plasmid encoding PH - Akt - GFP . treated with or without LY294002 ( 20 µM ) for 1 h prior to challenge with T . gondii - RFP . were examined by immunofluorescence at 5 min post - challenge to examine accumulation of PH - Akt - GFP around .

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E, HBMEC were transiently transfected with a plasmid encoding PH-Akt-GFP. Cells treated with or without LY294002 (20 µM) for 1 h prior to challenge with T. gondii-RFP. Cells were examined by immunofluorescence at 5 min post-challenge to examine accumulation of PH-Akt-GFP around the parasites.


F , HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T . gondii . Densitometry data represent means ± of 4 experiments . A vertical was inserted between densitometry data from control LY294002 - treated to indicate that band densities from infected treated with or without LY294002 are compared to bands from their respective uninfected , which were given an arbitrary number of 1 .

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F, HBMEC were incubated with LY294002 or vehicle for 1 h prior to challenge with T. gondii. Densitometry data represent means ± SEM of 4 experiments. A vertical line was inserted between densitometry data from control and LY294002-treated cells to indicate that band densities from infected cells treated with or without LY294002 are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1.


G , HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA . expressing either control siRNA or PI3K p110α siRNA were challenged with T . gondii 48 h after transfection . lysates were subject to immunoblotting as indicated . Densitometry data are shown as above represent means ± of 3 experiments . Results shown are representative of 3 - 4 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, HBMEC were transiently transfected with control siRNA or PI3K p110α siRNA. Cells expressing either control siRNA or PI3K p110α siRNA were challenged with T. gondii 48 h after transfection. Cell lysates were subject to immunoblotting as indicated. Densitometry data are shown as above and represent means ± SEM of 3 experiments. Results shown are representative of 3-4 independent experiments.


A , HBMEC , mHEVc human RPE were incubated with or without Akt inhibitor IV ( 1 . 25 µM ) for 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy 2 h 24 h post - challenge .

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A, HBMEC, mHEVc and human RPE cells were incubated with or without Akt inhibitor IV (1.25 µM) for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


B , HBMEC were transfected with control siRNA or Akt siRNA . were then challenged with T . gondii 48 h after transfection . Monolayers were examined microscopically 24 h post - challenge .

B, HBMEC were transfected with control siRNA or Akt siRNA. Cells were then challenged with T. gondii 48 h after transfection. Monolayers were examined microscopically 24 h post-challenge.


C , RAW 264 . 7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy 2 h 24 h post - challenge .

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C, RAW 264.7 were incubated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy 2 h and 24 h post-challenge.


D , mHEVc - LC3 - EGFP were incubated with or without Akt inhibitor IVfollowed by challenge with T . gondii - RFP . Monolayers were examined by microscopy 5 h post - challenge . Arrowheads indicate accumulation of LC3 around .

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D, mHEVc-LC3-EGFP cells were incubated with or without Akt inhibitor IVfollowed by challenge with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge. Arrowheads indicate accumulation of LC3 around the parasite.


E , HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii ( T ) then processed for electron microscopy at 5 h post - challenge . at bottom represent magnification of areas within boxes . Arrow indicates PVM ; arrowhead indicates double structure around vacuole .

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E, HBMEC were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii (T) and then processed for electron microscopy at 5 h post-challenge. Images at the bottom represent magnification of the areas within the boxes. Arrow indicates the PVM; arrowhead indicates the double membrane structure around the vacuole.


F , Control or Akt inhibitor IV - treated HBMEC were challenged with T . gondii - YFP . Expression of LAMP - 1 was examined by microscopy 8 h post - challenge . Arrowheads indicate accumulation of LAMP - 1 around

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F, Control or Akt inhibitor IV-treated HBMEC were challenged with T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. Arrowheads indicate accumulation of LAMP-1 around the parasite


G , H , mHEVc were transfected with Beclin 1 siRNA ( G ) , Atg7 siRNA ( H ) or control siRNA . After 48 h , were treated with or without Akt inhibitor IV for 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy at 24 h .

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G, H, mHEVc cells were transfected with Beclin 1 siRNA (G), Atg7 siRNA (H) or control siRNA. After 48 h, cells were treated with or without Akt inhibitor IV for 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 24 h.


I , mHEVc were treated with or without Akt inhibitor IV infected with T . gondii . 1 h post infection were treated with or without leupeptin plus pepstatin ( Lys inhibitors ) . Monolayers were examined microscopically 24 h post - challenge .

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I, mHEVc were treated with or without Akt inhibitor IV and infected with T. gondii. 1 h post infection cells were treated with or without leupeptin plus pepstatin (Lys inhibitors). Monolayers were examined microscopically 24 h post-challenge.


J , Mouse microglia were incubated with or without Akt inhibitor IV . 3 - methyl adenine ( 3MA ; 10 mM ) or vehicle were added 2 h post - challenge with T . gondii . Monolayers were examined microscopically 24 h post - challenge . Results are shown as mean ± are representative of 3 independent experiments .

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J, Mouse microglia were incubated with or without Akt inhibitor IV. 3-methyl adenine (3MA; 10 mM) or vehicle were added 2 h post-challenge with T. gondii. Monolayers were examined microscopically 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


A , HBMEC were treated with or without pertussis toxin ( PTx ; 100 ng / mL ) for 4 h prior to challenge with T . gondii . lysates were obtained at 5 , 15 or 30 min post - incubation with T . gondii used to examine total Akt phospho - Akt serine 473 by immunoblot . Densitometry data represent means ± of 3 experiments . A vertical was inserted between densitometry data from control PTx - treated to indicate that band densities from infected treated with or without PTx were compared to bands from their respective uninfected , which were given an arbitrary number of 1 . HBMEC were also treated with or without LPA ( 10 µM ) in presence or absence of PTx . lysates were obtained at 5 min subjected to

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

A, HBMEC were treated with or without pertussis toxin (PTx; 100 ng/mL) for 4 h prior to challenge with T. gondii. Cell lysates were obtained at 5, 15 or 30 min post-incubation with T. gondii and used to examine total Akt and phospho-Akt serine 473 by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and PTx-treated cells to indicate that band densities from infected cells treated with or without PTx were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. HBMEC were also treated with or without LPA (10 µM) in the presence or absence of PTx. Cell lysates were obtained at 5 min and subjected to immunoblotting


B , HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h . lysates were used to examine total Akt phospho - serine 473 Akt by immunoblot . Results shown are representative of 3 independent experiments .

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B, HBMEC were transfected with MyD88 siRNA or control siRNA followed by challenge with T gondii after 48 h. Cell lysates were used to examine total Akt and phospho-serine 473 Akt by immunoblot. Results shown are representative of 3 independent experiments.


A , HBMEC were challenged with RH T . gondii lysates were obtained to probe for total EGFR , phospho - tyrosine 1068 EGFR phospho - tyrosine 1148 EGFR by immunoblot .

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A, HBMEC cells were challenged with RH T. gondii and cell lysates were obtained to probe for total EGFR, phospho-tyrosine 1068 EGFR and phospho- tyrosine 1148 EGFR by immunoblot.


B , HBMEC were challenged with live vs killed tachyzoites . Total EGFR phospho - tyrosine 1068 EGFR expression was examined by immunoblot .

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B, HBMEC were challenged with live vs killed tachyzoites. Total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot.


C , D , Human RPE ( C ) mouse microglia ( D ) were challenged with RH T . gondii total EGFR phospho - tyrosine 1068 EGFR expression was examined by immunoblot . Results shown are representative of 3 independent experiments .

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C, D, Human RPE cells (C) and mouse microglia (D) were challenged with RH T. gondii and total EGFR and phospho-tyrosine 1068 EGFR expression was examined by immunoblot. Results shown are representative of 3 independent experiments.


A , mHEVc were transfected with control siRNA or EGFR siRNA . Expression of EGFR actin were assessed by immunoblot 48 h post - transfection . mHEVc expressing either control siRNA or EGFR siRNA were challenged with T . gondii . lysates were used to examine expression total Akt or phospho - Ser473 Akt by immunoblot .

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A, mHEVc cells were transfected with control siRNA or EGFR siRNA. Expression of EGFR and actin were assessed by immunoblot 48 h post-transfection. mHEVc cells expressing either control siRNA or EGFR siRNA were challenged with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot.


B , Mouse microglia were treated with or without AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . lysates were used to examine expression total Akt or phospho - Ser473 Akt by immunoblot . Densitometry data represent means ± of 3 experiments . A vertical was inserted between densitometry data from control siRNA EGFR siRNA or control AG1478 - treated to indicate that band densities from infected control or infected subjected to EGFR blockade were compared to bands from their respective uninfected , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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B, Mouse microglia were treated with or without AG1478 (1 µM) 1 h prior to challenge with T. gondii. Cell lysates were used to examine the expression total Akt or phospho-Ser473 Akt by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control siRNA and EGFR siRNA or control and AG1478-treated cells to indicate that band densities from infected control cells or infected cells subjected to EGFR blockade were compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


A , HBMEC were incubated with AG1478 ( 1 µM ) 1 h prior to challenge with T . gondii . Monolayers were examined by microscopy at 2 24 h .

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A, HBMEC were incubated with AG1478 (1 µM) 1 h prior to challenge with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


B , Human RPE transfected with either EGFR or control siRNA were challenged with T . gondii followed by examination of monolayers by microscopy at 24 h .

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B, Human RPE cells transfected with either EGFR or control siRNA were challenged with T. gondii followed by examination of monolayers by light microscopy at 24 h.


C , Mouse bone marrow - derived macrophages were incubated with AG1478 challenged with T . gondii . Monolayers were examined by microscopy at 2 24 h .

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C, Mouse bone marrow-derived macrophages were incubated with AG1478 and challenged with T. gondii. Monolayers were examined by light microscopy at 2 and 24 h.


D , Parental CHO CHO - EGFR were challenged with RH T . gondii . Monolayers were examined microscopically 2 h or 24 h post - challenge .

D, Parental CHO and CHO-EGFR cells were challenged with RH T. gondii. Monolayers were examined microscopically 2 h or 24 h post-challenge.


E , mHEVc - LC3 - EGFP transfected with control siRNA or EGFR siRNA were challenged with T . gondii - RFP . Monolayers were examined by microscopy 5 h post - challenge to determine of endothelial with LC3 accumulation around .

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E, mHEVc-LC3-EGFP cells transfected with control siRNA or EGFR siRNA were challenged with T. gondii-RFP. Monolayers were examined by fluorescence microscopy 5 h post-challenge to determine the percentage of endothelial cells with LC3 accumulation around the parasite.


F , HBMEC treated with or without AG1478 were challenged with either T . gondii - YFP . Expression of LAMP - 1 was examined by microscopy 8 h post - challenge . percentages of endothelial with LAMP - 1 accumulation of around were determined .

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F, HBMEC cells treated with or without AG1478 were challenged with either T. gondii-YFP. Expression of LAMP-1 was examined by fluorescent microscopy 8 h post-challenge. The percentages of endothelial cells with LAMP-1 accumulation of around the parasite were determined.


G , H , mHEVc transfected with Beclin1 siRNA ( G ) or Atg7 siRNA ( H ) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T . gondii . Monolayers were examined by microscopy 24 h post - challenge . Results are shown as mean ± are representative of 3 independent experiments .

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G, H, mHEVc cells transfected with Beclin1 siRNA (G) or Atg7 siRNA (H) were transfected with EGFR siRNA or treated with or without AG1478 followed by challenge with T. gondii. Monolayers were examined by light microscopy 24 h post-challenge. Results are shown as the mean ± SEM and are representative of 3 independent experiments.


HBMEC were treated with or without GM6001 ( 10 µM ) for 1 h prior to challenge with T . gondii ( A ) or with Pertussis Toxin ( PTx ; 100 ng / ml ) for 4 h prior to challenge ( B ) . lysates were used to examine total EGFR phospho - tyrosine 1068 EGFR by immunoblot . Densitometry data represent means ± of 3 experiments . A vertical was inserted between densitometry data from control GM6001 - or PTX - treated to indicate that band densities from infected treated with or without these inhibitors are compared to bands from their respective uninfected , which were given an arbitrary number of 1 . Results shown are representative of 3 independent experiments .

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HBMEC were treated with or without GM6001 (10 µM) for 1 h prior to challenge with T. gondii (A) or with Pertussis Toxin (PTx; 100 ng/ml) for 4 h prior to parasite challenge (B). Cell lysates were used to examine total EGFR and phospho-tyrosine 1068 EGFR by immunoblot. Densitometry data represent means ± SEM of 3 experiments. A vertical line was inserted between densitometry data from control and GM6001- or PTX-treated cells to indicate that band densities from infected cells treated with or without these inhibitors are compared to bands from their respective uninfected cells, which were given an arbitrary number of 1. Results shown are representative of 3 independent experiments.


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A , B , C HBMEC were challenged with ΔHx ( WT ) , MIC1 ko , MIC3 ko , MIC1 - 3 ko T . gondii at MOIs that yielded similar percentages of infected ( A ) .

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A, B, C HBMEC were challenged with ΔHx (WT), MIC1 ko, MIC3 ko, MIC1-3 ko T. gondii at MOIs that yielded similar percentages of infected cells (A).


lysates were obtained used to examine total EGFR phospho - tyrosine 1068 EGFR ( B ) or total Akt phospho - Ser 473 Akt ( C ) by immunoblot .

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Cell lysates were obtained and used to examine total EGFR and phospho-tyrosine 1068 EGFR (B) or total Akt and phospho-Ser 473 Akt (C) by immunoblot.


HBMEC were incubated with 10 nM of recombinant MIC4 , MIC3 , M2AP or MIC6 for 15 minutes followed by examination of total EGFR phospho - tyrosine 1068 EGFR ( D ) or total Akt phospho - Ser473 ( E ) by immunoblot . Densitometry data represent means ± of 4 experiments . Band densities from infected or treated with MICs were compared to bands from uninfected or untreated ( Un ) , which were given an arbitrary number of 1 . Results shown are representative of 4 independent experiments .

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

HBMEC were incubated with 10 nM of recombinant MIC4, MIC3, M2AP or MIC6 for 15 minutes followed by examination of total EGFR and phospho-tyrosine 1068 EGFR (D) or total Akt and phospho-Ser473 (E) by immunoblot. Densitometry data represent means ± SEM of 4 experiments. Band densities from infected cells or cells treated with MICs were compared to bands from uninfected or untreated cells (Un), which were given an arbitrary number of 1. Results shown are representative of 4 independent experiments.


A , hmCD40 mHEVc expressing LC3 - EGFP were treated with or without CD154 followed by challenge with T . gondii - RFP in presence or absence of EGF ( 50 ng / ml ) or AG1478 ( 1 µM ) . LC3 accumulation around T . gondii was assessed by immunofluorescence . B , mHEVc - LC3 - EGFP were infected with T . gondii - T . gondii . were treated with vehicle or rapamycin ( 1 µM ) 2 h after challenge with T . gondii in presence or absent of EGF or AG1478 . Recruitment of LC3 around was examined at 5 h after challenge with T . gondii .

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A, hmCD40 mHEVc expressing LC3-EGFP were treated with or without CD154 followed by challenge with T. gondii-RFP in the presence or absence of EGF (50 ng/ml) or AG1478 (1 µM). LC3 accumulation around T. gondii was assessed by immunofluorescence. B, mHEVc-LC3-EGFP cells were infected with T. gondii-T. gondii. Cells were treated with vehicle or rapamycin (1 µM) 2 h after challenge with T. gondii in the presence or absent of EGF or AG1478. Recruitment of LC3 around the parasite was examined at 5 h after challenge with T. gondii.