E . STAT2 in MEFs were decreased by transfection with siRNAs for 48 hrs after which were infected with ZIKV ( MOI = 5 ) for another 48 hrs . silencing efficiency was determined by immunoblotting . Representative panels are shown . experiment was repeated three times .

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

E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The silencing efficiency was determined by immunoblotting. Representative panels are shown. The experiment was repeated three times.


A . A549 were transfected with control plasmid pcDNA 3 . 1 or plasmids encoding FLAG - tagged WT or mutant NS5 . At 48 hrs post - transfection were treated with IFN - α ( 100 U / mL ) for 2 hrs then processed for indirect immunofluorescence . were acquired on a spinning disk confocal microscope with a 40X objective . NS5 positive are indicated by dashed circles . Representative panels are shown . experiment was repeated three times . B . of nuclearSTAT2 was performed using Volocity image analyses software . A minimum of 20 were counted for each sample . N . D . = not detected . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - ) ; N =

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 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

A. A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG-tagged WT or mutant NS5 proteins. At 48 hrs post-transfection cells were treated with IFN-α (100 U/mL) for 2 hrs and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. NS5 positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.B. Quantification of nuclearSTAT2 was performed using Volocity image analyses software. A minimum of 20 cells were counted for each sample. N.D.= not detected. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


C . A549 were transfected with plasmids encoding FLAG - tagged ZIKVNS5 or FLAG alone for 24 hrs then treated with epoxomicin ( 400 nM ) for 24 hrs . were harvested process for immunoprecipitation ( IP ) using a mouse anti - FLAG or anti - myc antibody followed by SDS - PAGE Immunoblotting . Representative panels are shown . experiment was repeated three times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells were transfected with plasmids encoding FLAG-tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin (400 nM) for 24 hrs. Cells were harvested and process for immunoprecipitation (IP) using a mouse anti-FLAG or anti-myc antibody followed by SDS-PAGE and Immunoblotting. Representative panels are shown. The experiment was repeated three times.


D . A549 were transfected with plasmids encoding FLAG - tagged WT , mutant NS5 or FLAG alone for 48 hrs . were harvested process for immunoprecipitation ( IP ) using rabbit anti - STAT2 followed by SDS - PAGE immunoblotting . Arrows indicate co - immunoprecipitated WT NS5 NS5 mutants that are in a with STAT2 . Representative panels are shown . experiment was repeated two times .

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

D. A549 cells were transfected with plasmids encoding FLAG-tagged WT, mutant NS5 proteins or FLAG alone for 48 hrs. Cells were harvested and process for immunoprecipitation (IP) using rabbit anti-STAT2 followed by SDS-PAGE and immunoblotting. Arrows indicate co-immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2. Representative panels are shown. The experiment was repeated two times.


( A ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 12 ) or , as controls , Bcl - xfl / fl ( n = 6 ) RosaCreERT2 + / Ki ( n = 16 ) mice ( 9 - 12 , ) were treated with tamoxifen ( 200 mg / kg / administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of floxed Bcl - x alleles . Mice were monitored for up to 200 days post - treatment with tamoxifen . Data are presented as % survival post - treatment with tamoxifen statistical significance was assessed using Mantel - Cox ( Log - rank ) ; * * * * p < 0 . 0001 .

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 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 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

(A) Bcl-xfl/fl;RosaCreERT2+/Ki (n=12) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=16) mice (age 9-12 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.


( B ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 24 ) or , as controls , Bcl - xfl / fl ( n = 9 ) RosaCreERT2 + / Ki ( n = 6 ) mice ( , aged 8 - 14 , numbers also indicated in figure legend ) were lethally γ - irradiated ( 2 x 5 . 5 Gy , 3 h apart ) reconstituted with bone marrow from UBC - GFP mice ( referred to as GFP - Chimeras ) . After 8 , reconstituted mice were treated with tamoxifen ( 200 mg / kg administered in 3 daily doses oral gavage ) monitored for up to 200 days ( termination of experiment ) . Data are presented as % survival post - treatment with tamoxifen statistical significance was assessed using Mantel - Cox ( Log - rank ) ; * * * * p < 0 . 0001 .

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 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 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

(B) Bcl-xfl/fl;RosaCreERT2+/Ki (n=24) or, as controls, Bcl-xfl/fl (n=9) and RosaCreERT2+/Ki (n=6) mice (males and females, aged 8-14 weeks, numbers also indicated in the figure legend) were lethally γ-irradiated (2 x 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.


( C - F ) Total of ( C ) platelets , ( D ) red blood ( RBC ) , ( E ) hemoglobin ( HGB ) content ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) RosaCreERT2 + / Ki ( n = 12 ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± . Each data point represents an mouse . Statistical significance was assessed using Student ' s t - ; * * * * p < 0 . 0001 . ( G ) Spleen weights were measured in sick mice ( at sacrifice ) or , for healthy control mice , at termination of experiment . Data are presented as mean ± . Each data point represents an mouse numbers are indicated . Statistical significance was assessed using Student ' s t - ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001. (G) Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using the Student's t-test; ****p<0.0001.


( C - F ) Total of ( C ) platelets , ( D ) red blood ( RBC ) , ( E ) hemoglobin ( HGB ) content ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) RosaCreERT2 + / Ki ( n = 12 ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± . Each data point represents an mouse . Statistical significance was assessed using Student ' s t - ; * * * * p < 0 . 0001 .

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

(C-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001.


( H ) Representative image of enlarged spleens from two Bcl - xfl / fl ; RosaCreERT2Ki / + mice - matched control Bcl - xfl / fl RosaCreERT2 + / Ki mice ( 34 days after treatment ) .

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

(H) Representative image of enlarged spleens from two Bcl-xfl/fl;RosaCreERT2Ki/+ mice and age-matched control Bcl-xfl/fl and RosaCreERT2+/Ki mice (34 days after treatment).


( I ) Histological of H & E - stained sections of spleens of tamoxifen - treated mice of indicated genotypes .

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

(I) Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) RosaCreERT2 + / Ki ( n = 12 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , or as controls , Bcl - xfl / fl ; GFP - Chimeras ( n = 6 ) RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( 8 - 14 , ) were treated with tamoxifen ( 200 mg / kg / administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of floxed Bcl - x alleles . ( A ) Total blood ( WBC ) were analyzed by ADVIA in sick mice or at termination of experiment ( healthy control mice ) . Data are presented as mean ± . Each data point represents an mouse n numbers are indicated above . Statistical significance was assessed using Student ' s t - . No statistically significant differences were observed .

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 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 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 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 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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n=6) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ±SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using the Student's t-test. No statistically significant differences were observed.


( B ) neutrophil / lymphocyte ( NLR ) is presented as mean ± for sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) or healthy control RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) at termination of experiment . Data are presented as mean ± . Each data point represents an mouse . Statistical significance was assessed using Student ' s t - ; * p < 0 . 05 .

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

(B) The neutrophil/lymphocyte ratio (NLR) is presented as mean ±SEM for sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21) or healthy control RosaCreERT2+/Ki;GFP-Chimeras (n=6) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; *p<0.05.


( C ) Histological of H & E - stained sections of sternum of sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras or healthy wild - type RosaCreERT2 + / Ki ; GFP - Chimera control mice at indicated time points post - treatment with tamoxifen ( dpt = days post - treatment ) .

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

(C) Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt=days post-treatment).


( D ) Histological of H & E - stained sections of livers of sick mice or - matched healthy control wild - type mice or healthy RosaCreERT2 + / Ki ; GFP - Chimeras at termination of experiment ( dpt = days post - treatment ) .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the livers of sick mice or age-matched healthy control wild-type mice or healthy RosaCreERT2+/Ki;GFP-Chimeras at the termination of the experiment (dpt=days post-treatment).


( E ) ALT ( left panel ) , AST ( middle panel ) bilirubin ( right panel ) in serum were determined in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 18 ) or in healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 4 ) at termination of experiment . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using Student ' s t - ; * * p < 0 . 01 . nd = not detected .

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

(E) ALT (left panel), AST (middle panel) and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n=4) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **p<0.01. nd=not detected.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( 8 - 14 , ) were treated with tamoxifen ( 200 mg / kg / administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of floxed Bcl - x alleles . ( A ) Kidney weights were measured in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( at sacrifice ) or , for healthy control mice , at termination of experiment . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using Student ' s t - ; * p < 0 . 05 , * * * p < 0 . 001 .

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 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 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Kidney weights were measured in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; *p<0.05, ***p<0.001.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( 8 - 14 , ) were treated with tamoxifen ( 200 mg / kg / administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of floxed Bcl - x alleles .

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 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 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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles.


( B ) Picture showing both kidneys from - matched healthy control wild - type mice ( left ) sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) .

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

(B) Picture showing both kidneys from age-matched healthy control wild-type mice (left) and sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right).


( C ) Histological of H & E - stained sections of kidneys of - matched healthy control mice or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras as indicated , showing from left to right , shrunken scarred kidney , segmental chronic tubulo - interstitial disease , accumulation of debris amorphous material within collecting of papilla . higher magnification shows tubular epithelial degeneration , apoptosis segmental secondary glomerular sclerosis . Pictures are representative of at least 5 mice for each genotype treatment .

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

(C) Histological analysis of H&E-stained sections of the kidneys of age-matched healthy control mice or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as indicated, showing from left to right, shrunken scarred kidney, segmental chronic tubulo-interstitial disease, accumulation of cellular debris and amorphous material within the collecting ducts of the papilla. The higher magnification shows tubular epithelial degeneration, apoptosis and segmental secondary glomerular sclerosis. Pictures are representative of at least 5 mice for each genotype and treatment.


( D ) Multiphoton of fixed sections of kidneys of - matched healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( left ) or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) . Pictures are representative of at least 3 mice for each genotype treatment . green = second harmony , blue = short wavelength autofluorescence .

O O 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

(D) Multiphoton analysis of fixed sections of the kidneys of age-matched healthy RosaCreERT2+/Ki;GFP-Chimeras (left) or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). Pictures are representative of at least 3 mice for each genotype and treatment. green=second harmony, blue=short wavelength autofluorescence.


( A ) TUNEL of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at indicated time points post - tamoxifen treatment ( 200 mg / kg / administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) . ( B ) TUNEL + were quantified using a personalized script for Image J software . Kidney sections were divided into ~ 40 microscopic total numbers of blue brown ( TUNEL + ) nuclei were determined . percentages of TUNEL + nuclei were calculated for each picture . average value for each kidney section was then determined plotted as a single data point . Error bars represent from at least 3 independent samples for each genotype treatment . Statistical significance was assessed using one - way ANOVA with Tukey ' s multiple comparisons ( comparing control RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 2 , 24 h post - γ - irradiation n = 9 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 6 , 24 h post - γ - irradiation n = 5 ) control RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 7 , End point n = 8 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 8 , End point n = 6 ) ( * * * p < 0 . 001 ) .

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

(A) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells. (B) TUNEL+ cells were quantified using a personalized script for the Image J software. Kidney sections were divided into ~40 microscopic images and the total numbers of blue and brown (TUNEL+) nuclei were determined. The percentages of TUNEL+ nuclei were calculated for each picture. The average value for each kidney section was then determined and plotted as a single data point. Error bars represent SEM from at least 3 independent samples for each genotype and treatment. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test (comparing control RosaCreERT2+/Ki (24 h post-tamoxifen n=2, 24 h post-γ-irradiation n=9) with Bcl-xfl/fl;RosaCreERT2+/Ki (24 h post-tamoxifen n=6, 24 h post-γ-irradiation n=5) and control RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=7, End point n=8) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=8, End point n=6) (***p<0.001).


( A ) TUNEL of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at indicated time points post - tamoxifen treatment ( 200 mg / kg / administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) .

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 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

(A) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells.


( A , B ) Data are presented as % survival statistical significance was assessed using Mantel - Cox ( Log - rank ) comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . survival curves for Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras

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

(A, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras


( A , B ) Data are presented as % survival statistical significance was assessed using Mantel - Cox ( Log - rank ) comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . survival curves for Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP -

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


( C ) Histological of H & E - stained sections of kidneys of sick mice of indicated genotypes at indicated time points ( dpt = days post - tamoxifen treatment ) . Pictures are representative of at least 3 mice for each genotype treatment .

O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment.


( D - F ) ( D ) Total red blood ( RBC ) , ( E ) hematocrit ( HCT ) ( F ) hemoglobin ( HGB ) content were determined by ADVIA in blood of sick mice of indicated genotypes or at termination of experiment for healthy controls . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using Student ' s t - comparing tamoxifen - treated RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 20 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 12 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 15 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 10 ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 17 ) ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Data for Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras RosaCreERT2 + / Ki ; GFP - Chimeras

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

(D-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras


( D - F ) ( D ) Total red blood ( RBC ) , ( E ) hematocrit ( HCT ) ( F ) hemoglobin ( HGB ) content were determined by ADVIA in blood of sick mice of indicated genotypes or at termination of experiment for healthy controls . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using Student ' s t - comparing tamoxifen - treated RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 20 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 12 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 15 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 10 ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 17 ) ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Data for Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras RosaCreERT2 + / Ki ; GFP -

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


( A ) GFP - Chimeras were treated with BCL - XL inhibitor A1331852 ( n = 8 , 5 doses by oral gavage , 25 mg / kg each dose ) . Control groups ( n = 8 each group ) include untreated GFP - Chimeras un - irradiated C57BL / 6 - Ly5 . 1 ( wild - type ) mice treated with BCL - XL inhibitor A1331852 or left untreated . Mice were monitored for up to 200 days post - treatment ( dpt ) . Data are presented as % survival .

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 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

(A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimeras and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Mice were monitored for up to 200 days post-treatment (dpt). Data are presented as % survival.


( B ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( , aged 10 ) were treated with Cyclophosphamide ( n = 7 , 150 mg / kg , 1 dose i . v . ) or 5 - Fluorouracil ( 5 - FU , n = 7 , 100 mg / kg , 1 dose i . v . ) after 5 days additionally treated with BCL - XL inhibitor A1331852 ( 5 doses by oral gavage , 100 mg / kg each dose ) . Control groups ( n = 7 each group ) include C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( , aged 10 ) treated with A1331852 alone , Cyclophosphamide alone , 5 - FU alone or left untreated . Mice were monitored for up to 150 days post - treatment ( dpt ) . Data are presented as % survival .

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

(B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Mice were monitored for up to 150 days post-treatment (dpt). Data are presented as % survival.


( C ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( , aged 10 ) were treated with BCL - XL inhibitor A1331852 ( n = 7 , 5 doses by oral gavage , 25 or 100 mg / kg each dose ) . Total platelet were determined by ADVIA at indicated time points . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using one - way ANOVA with Tukey ' s multiple comparisons comparing untreated drug - treated mice within each group ( * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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 O O O O O O O O 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) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with the BCL-XL inhibitor A1331852 (n=7, 5 doses by oral gavage, 25 or 100 mg/kg body weight each dose). Total platelet counts were determined by ADVIA at the indicated time points. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (**p<0.01, ***p<0.001, ****p>0.0001).


Total red blood ( RBC ) , platelet , hemoglobin ( HGB ) content hematocrit ( HCT ) were determined by ADVIA as indicated in blood of drug - treated GFP - Chimeras or control mice ( n = 8 each group ) at termination of experiment Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using one - way ANOVA with Tukey ' s multiple comparisons comparing untreated drug - treated mice within each group ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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

Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of drug-treated GFP-Chimeras or control mice (n=8 each group) at the termination of the experiment Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).


Total red blood ( RBC ) , platelet , hemoglobin ( HGB ) content hematocrit ( HCT ) were determined by ADVIA as indicated in blood of in drug - treated C57BL / 6 - Ly5 . 1 ( wild - type ) mice or control mice at indicated time points ( n = 7 each group ) . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using one - way ANOVA with Tukey ' s multiple comparisons comparing untreated drug - treated mice within each group ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p > 0 . 0001 ) .

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

Total red blood cell count (RBC), platelet count, hemoglobin (HGB) content and hematocrit (HCT) were determined by ADVIA as indicated in the blood of in drug-treated C57BL/6-Ly5.1 (wild-type) mice or control mice at the indicated time points (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test comparing untreated and drug-treated mice within each group (*p<0.05, **p<0.01, ***p<0.001, ****p>0.0001).


( A ) GFP - Chimeras were treated with BCL - XL inhibitor A1331852 ( n = 8 , 5 doses by oral gavage , 25 mg / kg each dose ) . Control groups ( n = 8 each group ) include untreated GFP - Chimera un - irradiated C57BL / 6 - Ly5 . 1 ( wild - type ) mice treated with BCL - XL inhibitor A1331852 or left untreated . Kidney ( left panel ) spleen weights ( right panel ) were measured in drug - treated GFP - Chimeras or control mice at termination of experiment ( n = 8 each group ) . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using one - way ANOVA with Tukey ' s multiple comparisons . No statistically significant differences were observed .

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 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 O O O O O O O O O

(A) GFP-Chimeras were treated with the BCL-XL inhibitor A1331852 (n=8, 5 doses by oral gavage, 25 mg/kg body weight each dose). Control groups (n=8 each group) include untreated GFP-Chimera and un-irradiated C57BL/6-Ly5.1 (wild-type) mice treated with the BCL-XL inhibitor A1331852 or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated GFP-Chimeras or control mice at the termination of the experiment (n=8 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.


( B ) C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( , aged 10 ) were treated with Cyclophosphamide ( n = 7 , 150 mg / kg , 1 dose i . v . ) or 5 - Fluorouracil ( 5 - FU , n = 7 , 100 mg / kg , 1 dose i . v . ) after 5 days additionally treated with BCL - XL inhibitor A1331852 ( 5 doses by oral gavage , 100 mg / kg each dose ) . Control groups ( n = 7 each group ) include C57BL / 6 - Ly5 . 1 ( wild - type ) mice ( , aged 10 ) treated with BCL - XL inhibitor A1331852 alone , Cyclophosphamide alone , 5 - FU alone or left untreated . Kidney ( left panel ) spleen weights ( right panel ) were measured in drug - treated mice or control mice at termination of experiment ( n = 7 each group ) . Data are presented as mean ± . Each data point represents one mouse . Statistical significance was assessed using one - way ANOVA with Tukey ' s multiple comparisons . No statistically significant differences were observed .

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

(B) C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) were treated with Cyclophosphamide (n=7, 150 mg/kg body weight, 1 dose i.v.) or 5-Fluorouracil (5-FU, n=7, 100 mg/kg body weight, 1 dose i.v.) and after 5 days additionally treated with the BCL-XL inhibitor A1331852 (5 doses by oral gavage, 100 mg/kg body weight each dose). Control groups (n=7 each group) include C57BL/6-Ly5.1 (wild-type) mice (females, aged 10 weeks) treated with the BCL-XL inhibitor A1331852 alone, Cyclophosphamide alone, 5-FU alone or left untreated. Kidney (left panel) and spleen weights (right panel) were measured in drug-treated mice or control mice at the termination of the experiment (n=7 each group). Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test. No statistically significant differences were observed.


( C ) Histological of H & E - stained sections of kidneys of drug - treated GFP - Chimeras or control mice at termination of experiment . Pictures are representative of at least 3 mice for each treatment 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

(C) Histological analysis of H&E-stained sections of the kidneys of drug-treated GFP-Chimeras or control mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.


( D ) Histological of H & E - stained sections of kidneys of drug - treated mice or control untreated mice at termination of experiment . Pictures are representative of at least 3 mice for each treatment 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

(D) Histological analysis of H&E-stained sections of the kidneys of drug-treated mice or control untreated mice at the termination of the experiment. Pictures are representative of at least 3 mice for each treatment group.


location of causal candidate LN1 is indicated as is position of linked TAS rs2303565 associated with ALS . structures of transcripts indicate exons ( blue ) introns ( gray with > ) . color key indicates states as defined by ChromHMM of epigenomic data from NIH Roadmap Epigenomics Consortium . states are shown for brain tissues , neural progenitor ( NPCs ) , a variety of other tissues . Next to color key , NCBI dbVar ID for TE , SNP ID for TAS , genic location of TAS , TE name , r2 value are shown . * r2 value verified by genotyping PCR . ChIP - seq peaks of transcription factors from ENCODE are shown below ChromHMM states .

The location of causal candidate LN1 is indicated as is the position of the linked TAS rs2303565 associated with ALS. The structures of transcripts indicate the exons (blue) and introns (gray with >). The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, neural progenitor cells (NPCs), and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states.


location of causal candidate LN2 is indicated as is position of linked TAS rs11759769 associated with migraine disorder . structures of transcripts indicate exons ( blue ) introns ( gray with > ) . L1MA5 indicates position of an L1 element fixed in genome . color key indicates states as defined by ChromHMM of epigenomic data from NIH Roadmap Epigenomics Consortium . states are shown for brain tissues , NPCs , a variety of other tissues . Next to color key , NCBI dbVar ID for TE , SNP ID for TAS , genic location of TAS , TE name , r2 value are shown . * r2 value verified by genotyping PCR . ChIP - seq peaks of transcription factors from ENCODE are shown below ChromHMM states peaks for two transcription factors known for heterochromatin formation located around site of LN2 insertion are labeled .

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

The location of causal candidate LN2 is indicated as is the position of the linked TAS rs11759769 associated with migraine disorder. The structures of transcripts indicate the exons (blue) and introns (gray with >). L1MA5 indicates the position of an L1 element fixed in the genome. The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, NPCs, and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states and peaks for two transcription factors known for heterochromatin formation located around the site of LN2 insertion are labeled.


location of causal candidate LN16 is indicated as is position of linked TAS rs12185268 associated with PD . structures of transcripts indicate exons ( blue ) introns ( gray with > ) . color key indicates states as defined by ChromHMM of epigenomic data from NIH Roadmap Epigenomics Consortium . states are shown for brain tissues , NPCs , a variety of other tissues . Next to color key , NCBI dbVar ID for TE , SNP ID for TAS , genic location of TAS , TE name , r2 value are shown . * r2 value verified by genotyping PCR . ChIP - seq peaks of transcription factors from ENCODE are shown below ChromHMM states . position of LN16 TE is enlarged in an inset .

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

The location of causal candidate LN16 is indicated as is the position of the linked TAS rs12185268 associated with PD. The structures of transcripts indicate the exons (blue) and introns (gray with >). The color key indicates the chromatin states as defined by ChromHMM analysis of epigenomic data from the NIH Roadmap Epigenomics Consortium. Chromatin states are shown for brain tissues, NPCs, and a variety of other tissues. Next to the color key, NCBI dbVar ID for TE, SNP ID for TAS, genic location of TAS, TE name, and r2 value are shown. * r2 value verified by genotyping PCR. ChIP-seq peaks of transcription factors from ENCODE are shown below ChromHMM chromatin states. The position of the LN16 TE is enlarged in an inset.


Approximately 500 bp of genomic , with without Alu inserts was assayed for transcription activity when inserted upstream of a minimal promoter in a luciferase vector transfected into NCRM1 . orientation ( triangle ) of Alu its associated genomic ( yellow box ) is diagramed at top . Forward orientation ( Fwd ) indicates plus strand of reference genome . minimal promoter is symbolized by a black rectangle with an arrow indicating direction of transcription . causal TE candidates tested , their linked TASs , their ChromHMM status are indicated at right . Luciferase values are normalized to transfection efficiency ( renilla ) for each of four independent transfections subsequently to luciferase expression from minimal promoter alone . Mean values ( ± SD ) were determined , statistical significance was analyzed by Student ' s two - sample unequal variance t - with a one - distribution . Each locus was examined in four independent experiments a representative dataset is shown here . ☨ : p < 0 . 05 in two experiments p > 0 . 05 in two experiments . ns : P > 0 . 05 .

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

Approximately 500 bp of genomic sequence, with and without Alu inserts was assayed for transcription activity when inserted upstream of a minimal promoter in a luciferase vector transfected into NCRM1 cells. The orientation (white triangle) of the Alu and its associated genomic sequence (yellow box) is diagramed at top. Forward orientation (Fwd) indicates plus strand sequence of the reference genome. The minimal promoter is symbolized by a black rectangle with an arrow indicating the direction of transcription. The causal TE candidates tested, their linked TASs, and their ChromHMM status are indicated at right. Luciferase values are normalized to transfection efficiency (renilla) for each of four independent transfections and subsequently to luciferase expression from the minimal promoter alone. Mean values (± SD) were determined, and statistical significance was analyzed by Student's two-sample unequal variance t-Test with a one-tailed distribution. Each locus was examined in four independent experiments and a representative dataset is shown here. ☨: p < 0.05 in two experiments and p > 0.05 in two experiments. ns: P > 0.05.


B . TBK1 / IKKε phosphorylate mTOR S2159 directly in vitro : In vitro kinase ( IVK ) assays with recombinant ( re ) active TBK1 or IKKε [ 50 ng ] ( Invitrogen ) recombinant GST - mTOR substrate [ 200 ng ] for 30 min at 30oC . Reactions were pre - incubated on ice 30 min with amlexanox [ 500 , 250 or 50 μM ] , BX - 795 [ 10 μM ] or MRT - 67307 [ 10 μM ] immunoblotted ( IB ) as indicated .

O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_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 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. TBK1/IKKε phosphorylate mTOR S2159 directly in vitro: In vitro kinase (IVK) assays with recombinant (re) active TBK1 or IKKε [50 ng] (Invitrogen) and recombinant GST-mTOR substrate [200 ng] for 30 min at 30oC. Reactions were pre-incubated on ice 30 min with amlexanox [500, 250 or 50 μM], BX-795 [10 μM] or MRT-67307 [10 μM] and immunoblotted (IB) as indicated.


C . TBK1 / IKKε phosphorylate full - mTOR on S2159 : Myc - mTOR wild type ( WT ) S2159A were immunoprecipitated ( IP ) from transfected HEK293 incubated with re - TBK1 or re - IKKε . IVK assays were performed as above immunoblotted ( IB ) as indicated .

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

C. TBK1/IKKε phosphorylate full-length mTOR on S2159: Myc-mTOR wild type (WT) and S2159A were immunoprecipitated (IP) from transfected HEK293 cells and incubated with re-TBK1 or re-IKKε. IVK assays were performed as above and immunoblotted (IB) as indicated.


J . LPS increases mTOR P - S2159 in vivo : Mice ( C57BL6 , 6 wks old ) were injected intraperitoneally with PBS or LPS [ 1 mg / kg - BW ] ( 2 hr . ) . mTOR was immunoprecipitated from spleen tissue , IPs WCL were immunoblotted as indicated . graph depicts of mTOR P - S2159 relative to total mTOR in spleen tissue - / + LPS . n = 8 from 3 independent experiments + / - SD . * p = . 004 relative to PBS treated control mice by paired t - ( two - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. LPS increases mTOR P-S2159 in vivo: Mice (C57BL6, 6 wks old) were injected intraperitoneally with PBS or LPS [1 mg/kg-BW] (2 hr.). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P-S2159 relative to total mTOR in spleen tissue -/+ LPS. n=8 from 3 independent experiments +/- SD. *p=.004 relative to PBS treated control mice by paired t-test (two-tailed).


E . TBK1 IKKε overexpression increases mTOR P - S2159 within mTORC1 : HEK293 - TLR3 were co - transfected with Flag - TBK1 or Flag - IKKε , Myc - mTOR , HA - raptor . HA - raptor immunoprecipitates whole lysates ( WCL ) were immunoblotted ( IB ) as indicated .

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

E. TBK1 and IKKε overexpression increases mTOR P-S2159 within mTORC1: HEK293-TLR3 cells were co-transfected with Flag-TBK1 or Flag-IKKε, Myc-mTOR, and HA-raptor. HA-raptor immunoprecipitates and whole cell lysates (WCL) were immunoblotted (IB) as indicated.


F . Flag - TBK1 Flag - IKKε co - immunoprecipitate with endogenous mTORC1 : HEK293T were transfected with Flag - TBK1 or Flag - IKKε wild type ( + ) or kinase dead ( KD ) . Endogenous raptor immunoprecipitates WCL were immunoblotted ( IB ) as indicated .

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

F. Flag-TBK1 and Flag-IKKε co-immunoprecipitate with endogenous mTORC1: HEK293T cells were transfected with Flag-TBK1 or Flag-IKKε wild type (+) or kinase dead (KD). Endogenous raptor immunoprecipitates and WCL were immunoblotted (IB) as indicated.


G . mTOR is phosphorylated on S2159 in wild type but not TBK1 null MEFs : TBK1 + / + TBK1 - / - MEFs were serum starved ( 20 hr . ) stimulated - / + EGF [ 25 ng / mL ] . WCL was immunoblotted ( IB ) as indicated .

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

G. mTOR is phosphorylated on S2159 in wild type but not TBK1 null MEFs: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.) and stimulated -/+ EGF [25 ng/mL]. WCL was immunoblotted (IB) as indicated.


H . TBK1 - IKKε - activating agonists poly ( I : C ) LPS increase mTOR P - S2159 in a BX - 795 sensitive manner in cultured macrophages : RAW264 . 7 macrophages were pre - treated with BX - 795 [ 10 μM ] ( 2 hr . ) stimulated - / + poly ( I : C ) [ 30 μg / ml ] or LPS [ 100 ng / ml ] ( 60 min ) .

O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The TBK1- and IKKε-activating agonists poly(I:C) and LPS increase mTOR P-S2159 in a BX-795 sensitive manner in cultured macrophages: RAW264.7 macrophages were pre-treated with BX-795 [10 μM] (2 hr.) and stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).


I . Poly ( I : C ) LPS increase mTOR P - S2159 in bone marrow - derived macrophages ( BMDMs ) : BMDMs were stimulated - / + poly ( I : C ) [ 30 μg / ml ] or LPS [ 100 ng / ml ] ( 60 min ) .

O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O 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. Poly(I:C) and LPS increase mTOR P-S2159 in primary bone marrow-derived macrophages (BMDMs): BMDMs were stimulated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).


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


A . EGF increases mTORC1 signaling in a TBK1 - dependent manner : TBK1 + / + TBK1 - / - MEFs were serum starved ( 20 hr . ) , pre - treated with Ku - 0063794 [ 1 μM ] ( 30 min ) , stimulated - / + EGF [ 25 ng / mL ] ( 0 - 60 min ) . Whole lysate ( WCL ) were immunoblotted ( IB ) as indicated .

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

A. EGF increases mTORC1 signaling in a TBK1-dependent manner: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+ EGF [25 ng/mL] (0-60 min). Whole cell lysate (WCL) were immunoblotted (IB) as indicated.


B . Ectopic expression of TBK1 rescues mTORC1 signaling in TBK1 - / - MEFs : TBK1 - / - MEFs were transiently transfected with vector control ( V ) , wild type ( WT ) , or kinase dead ( KD ) Flag - TBK1 , serum - starved ( 20 hr . ) , analyzed as above .

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

B. Ectopic expression of TBK1 rescues mTORC1 signaling in TBK1-/- MEFs: TBK1-/- MEFs were transiently transfected with vector control (V), wild type (WT), or kinase dead (KD) Flag-TBK1, serum-starved (20 hr.), and analyzed as above.


C . EGF - receptor signaling remains intact in TBK1 - / - MEFs : TBK1 + / + TBK1 - / - MEFs were serum - starved ( 20 hr . ) stimulated - / + EGF [ 25 ng / mL ] ( 0 , 5 , or 15 min ) .

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

C. EGF-receptor signaling remains intact in TBK1-/- MEFs: TBK1+/+ and TBK1-/- MEFs were serum-starved (20 hr.) and stimulated -/+EGF [25 ng/mL] (0, 5, or 15 min).


D . TBK1 is required for EGF - stimulated mTORC1 mTORC2 signaling : TBK1 + / + TBK1 - / - MEFs were stimulated with EGF as in A .

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

D. TBK1 is required for EGF-stimulated mTORC1 and mTORC2 signaling: TBK1+/+ and TBK1-/- MEFs were stimulated with EGF as in A.


E . TBK1 is not required for insulin - stimulated mTORC1 signaling : TBK1 + / + TBK1 - / - MEFs were stimulated with insulin as in C .

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

E. TBK1 is not required for insulin-stimulated mTORC1 signaling: TBK1+/+ and TBK1-/- MEFs were stimulated with insulin as in C.


F . Side - by - side comparison of EGF - vs . insulin - stimulated mTORC1 mTORC2 signaling in TBK1 + / + vs . TBK1 - / - MEFs : MEFs were treated as in C .

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

F. Side-by-side comparison of EGF- vs. insulin-stimulated mTORC1 and mTORC2 signaling in TBK1+/+ vs. TBK1-/- MEFs: MEFs were treated as in C.


G . Pharmacologic TBK1 inhibition reduces EGF - induced mTORC1 signaling : HEK293 were serum starved ( 20 hr . ) , pre - treated with Ku - 0063794 [ 1 μM ] ( 30 min ) or amlexanox [ 50 μM ] ( 2 hr . ) , stimulated - / + EGF [ 25 ng / mL ] ( 30 min ) or insulin ( INS ) [ 100 nM ] ( 30 min ) .

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 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

G. Pharmacologic TBK1 inhibition reduces EGF-induced mTORC1 signaling: HEK293 cells were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min) or amlexanox [50 μM] (2 hr.), and stimulated -/+EGF [25 ng/mL] (30 min) or insulin (INS) [100 nM] (30 min).


H . EGF but not insulin increases mTOR P - S2159 : TBK1 + / + vs . TBK1 - / - MEFs : MEFs were treated as in C .

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

H. EGF but not insulin increases mTOR P-S2159: TBK1+/+ vs. TBK1-/- MEFs: MEFs were treated as in C.


A . mTORC1 signaling remains intact in MEFs lacking mTORC2 function : Rictor - / - MEFs stably expressing vector control ( V ) or rescued stably with HA - rictor were serum starved ( 20 hr . ) , pre - treated with Ku - 0063794 [ 1 μM ] ( 30 min ) , stimulated - / + epidermal growth factor ( EGF ) [ 25 ng / mL ] ( 30 min ) . Whole lysate ( WCL ) were immunoblotted as indicated .

O O B-MEASURED_VAR 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 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 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

A. mTORC1 signaling remains intact in MEFs lacking mTORC2 function: Rictor-/- MEFs stably expressing vector control (V) or rescued stably with HA-rictor were serum starved (20 hr.), pre-treated with Ku-0063794 [1 μM] (30 min), and stimulated -/+epidermal growth factor (EGF) [25 ng/mL] (30 min). Whole cell lysate (WCL) were immunoblotted as indicated.


B . TBK1 is required for mTORC1 signaling in MEFs with reduced Akt S473 phosphorylation : TBK1 expression was reduced using lentivirally - delivered TBK1 shRNA in Rictor - / - MEFs stably expressing vector control ( V ) . were EGF stimulated as above , except Torin1 [ 100 nM ] was used to inhibit mTOR .

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

B. TBK1 is required for mTORC1 signaling in MEFs with reduced Akt S473 phosphorylation: TBK1 expression was reduced using lentivirally-delivered TBK1 shRNA in Rictor-/- MEFs stably expressing vector control (V). Cells were EGF stimulated as above, except Torin1 [100 nM] was used to inhibit mTOR.


A . mTOR S2159 phosphorylation is required for EGF stimulated mTORC1 signaling : HEK293 were co - transfected with vector control , wild type , or rapamycin - resistant ( RR ) AU1 - mTOR alleles ( RR or RR / S2159A ) together with HA - S6K1 . were serum starved ( 20 hr . ) , treated - / + rapamycin ( 30 min ) to ablate endogenous mTORC1 function , stimulated - / + EGF [ 25 ng / mL ] ( 30 min ) . HA - S6K1 was immunoprecipitated , immunoprecipitates ( IP ) whole lysate ( WCL ) were immunoblotted ( IB ) as indicated .

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

A. mTOR S2159 phosphorylation is required for EGF stimulated mTORC1 signaling: HEK293 cells were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were serum starved (20 hr.), treated -/+ rapamycin (30 min) to ablate endogenous mTORC1 function, and stimulated -/+EGF [25 ng/mL] (30 min). HA-S6K1 was immunoprecipitated, and immunoprecipitates (IP) and whole cell lysate (WCL) were immunoblotted (IB) as indicated.


B . TBK1 is required for EGF stimulated mTOR auto - phosphorylation : TBK1 + / + TBK1 - / - MEFS were serum starved , EGF stimulated , analyzed as in A .

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

B. TBK1 is required for EGF stimulated mTOR auto-phosphorylation: TBK1+/+ and TBK1-/- MEFS were serum starved, EGF stimulated, and analyzed as in A.


C . EGF time course of mTOR auto - phosphorylation : TBK1 + / + TBK1 - / - MEFS were serum starved , EGF stimulated for 0 - 60 min , analyzed as in A .

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

C. EGF time course analysis of mTOR auto-phosphorylation: TBK1+/+ and TBK1-/- MEFS were serum starved, EGF stimulated for 0-60 min, and analyzed as in A.


D . TBK1 is required for EGF stimulated mTOR auto - phosphorylation within mTORC1 : TBK1 + / + TBK1 - / - MEFS were serum starved , pre - treated with Ku - 0063794 [ 1 μM ] , EGF stimulated as in A . Raptor was immunoprecipitated , IPs WCL were analyzed .

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

D. TBK1 is required for EGF stimulated mTOR auto-phosphorylation within mTORC1: TBK1+/+ and TBK1-/- MEFS were serum starved, pre-treated with Ku-0063794 [1 μM], and EGF stimulated as in A. Raptor was immunoprecipitated, and IPs and WCL were analyzed.


E . mTOR S2159 phosphorylation is required for EGF stimulated mTOR auto - phosphorylation : HEK293 were transfected with Myc - mTOR wild type ( WT ) , S2159A , kinase dead ( KD ) . were then serum starved , pre - treated with Ku - 0063794 ( 30 min ) , stimulated - / + EGF as in A .

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

E. mTOR S2159 phosphorylation is required for EGF stimulated mTOR auto-phosphorylation: HEK293 cells were transfected with Myc-mTOR wild type (WT), S2159A, and kinase dead (KD). Cells were then serum starved, pre-treated with Ku-0063794 (30 min), and stimulated -/+ EGF as in A.


A . Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 TLR4 in cultured macrophages : RAW264 . 7 cultured in full serum were pre - treated with amlexanox ( [ 50 μM or 100 μM ] ) ( 2 hr . ) , rapamycin [ 20 ng / mL ] ( 30 min ) , or Ku - 0063794 [ 1 μM ] ( 30 min ) stimulated - / + poly ( I : C ) [ 30 μg / ml ] or LPS [ 100 ng / ml ] ( 60 min ) . Whole lysate ( WCL ) was immunoblotted as indicated .

O O O B-MEASURED_VAR 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

A. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in cultured macrophages: RAW264.7 cells cultured in full serum were pre-treated with amlexanox ([50 μM or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Whole cell lysate (WCL) was immunoblotted as indicated.


B . Knockdown of TBK1 with shRNA reduces TLR3 - TLR4 - stimulated mTORC1 signaling : RAW264 . 7 macrophages were co - infected with shRNA - containing lentiviruses targeting TBK1 IKKε shRNA or infected with scrambled control ( Scr ) , selected in puromycin , treated as in A .

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

B. Knockdown of TBK1 with shRNA reduces TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-infected with shRNA-containing lentiviruses targeting TBK1 and IKKε shRNA or infected with scrambled control (Scr), selected in puromycin, and treated as in A.


C . Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 TLR4 in macrophages : Bone marrow derived macrophages - monocytes ( BMDMs ) were pre - treated with amlexanox [ 100 μM ] ( 2 hr . ) or Ku - 0063794 [ 1 μM ] ( 30 min ) stimulated - / + poly ( I : C ) or LPS as in A .

O O O B-CONTROLLED_VAR 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

C. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 and TLR4 in primary macrophages: Bone marrow derived macrophages-monocytes (BMDMs) were pre-treated with amlexanox [100 μM] (2 hr.) or Ku-0063794 [1 μM] (30 min) and stimulated -/+ poly(I:C) or LPS as in A.


D . Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 in HEK293 - TLR3 . were serum starved ( 20 hr . ) , pre - treated with amlexanox or Ku - 0063794 as in C above , stimulated - / + poly ( I : C ) [ 50 ug / mL ] ( 60 min ) .

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

D. Pharmacologic TBK1 inhibition reduces mTORC1 signaling upon activation of TLR3 in HEK293-TLR3 cells. Cells were serum starved (20 hr.), pre-treated with amlexanox or Ku-0063794 as in C above, and stimulated -/+ poly(I:C) [50 ug/mL] (60 min).


E . TLR4 / LPS - stimulated mTORC1 signaling requires TBK1 : TBK1 + / + TBK1 - / - MEFs were serum starved ( 20 hr . ) , pre - treated with amlexanox [ 50 μM ] ( 2 hr . ) or Torin1 [ 100 nM ] ( 30 min ) , stimulated - / + LPS as in A .

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

E. TLR4/LPS-stimulated mTORC1 signaling requires TBK1: TBK1+/+ and TBK1-/- MEFs were serum starved (20 hr.), pre-treated with amlexanox [50 μM] (2 hr.) or Torin1 [100 nM] (30 min), and stimulated -/+ LPS as in A.


A . TBK1 activity is required for TLR3 - TLR4 - stimulated mTORC1 catalytic activity : RAW264 . 7 macrophages were pre - treated with amlexanox ( [ 50 μM ] or 100 μM ] ) ( 2 hr . ) , rapamycin [ 20 ng / mL ] ( 30 min ) , or Ku - 0063794 [ 1 μM ] ( 30 min ) treated - / + poly ( I : C ) [ 30 μg / ml ] or LPS [ 100 ng / ml ] ( 60 min ) . Raptor was immunoprecipitated ( IP ) , IPs whole lysate ( WCL ) were immunoblotted ( IB ) as indicated .

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

A. TBK1 activity is required for TLR3- and TLR4-stimulated mTORC1 catalytic activity: RAW264.7 macrophages were pre-treated with amlexanox ([50 μM] or 100 μM]) (2 hr.), rapamycin [20 ng/mL] (30 min), or Ku-0063794 [1 μM] (30 min) and treated -/+ poly (I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min). Raptor was immunoprecipitated (IP), and IPs and whole cell lysate (WCL) were immunoblotted (IB) as indicated.


B . C . mTOR S2159 phosphorylation is required for TLR3 - TLR4 - stimulated mTORC1 signaling : RAW264 . 7 macrophages were co - transfected with vector control , wild type , or rapamycin - resistant ( RR ) AU1 - mTOR alleles ( RR or RR / S2159A ) together with HA - S6K1 . were treated with rapamycin ( + ) to ablate endogenous mTORC1 function stimulated with poly ( I : C ) ( A ) or LPS ( B ) as in A . HA - S6K1 was immunoprecipitated , IPs WCL was immunoblotted as indicated .

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

B. and C. mTOR S2159 phosphorylation is required for TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were co-transfected with vector control, wild type, or rapamycin-resistant (RR) AU1-mTOR alleles (RR or RR/S2159A) together with HA-S6K1. Cells were treated with rapamycin (+) to ablate endogenous mTORC1 function and stimulated with poly(I:C) (A) or LPS (B) as in A. HA-S6K1 was immunoprecipitated, and IPs and WCL was immunoblotted as indicated.


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


D . Pharmacologic inhibition of PI3Kα but not Akt reduces TLR3 - TLR4 - stimulated mTORC1 signaling : RAW264 . 7 macrophages were pre - treated with PI3Kα class I inhibitor BYL - 719 [ 10 μM ] , Akt inhibitor MK - 2206 [ 10 μM ] , amlexanox [ 100 uM ] , or Ku - 0063794 [ 1 μM ] ( 30 min ) stimulated - / + poly ( I : C ) or LPS as in A .

O O O O O B-CONTROLLED_VAR O O 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 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

D. Pharmacologic inhibition of PI3Kα but not Akt reduces TLR3- and TLR4-stimulated mTORC1 signaling: RAW264.7 macrophages were pre-treated with the PI3Kα class I inhibitor BYL-719 [10 μM], the Akt inhibitor MK-2206 [10 μM], amlexanox [100 uM], or Ku-0063794 [1 μM] (30 min) and stimulated -/+poly (I:C) or LPS as in A.


A . Rapamycin suppresses TLR3 - stimulated IFNβ production in cultured macrophages : RAW264 . 7 macrophages were pre - treated with rapamycin [ 20 ng / mL ] ( 30 min ) , Ku - 0063794 [ 1 μM ] ( 30 min ) , or amlexanox [ 50 μM ] ( 2 hr . ) stimulated - / + poly ( I : C ) [ 30 μg / ml ] for 6 hr . secretion of IFNβ was measured by ELISA . Results represent mean + / - SD of quadruplicate samples from one experiment . * p = . 0009 relative to + Poly ( I : C ) by paired t - ( two - ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O 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. Rapamycin suppresses TLR3-stimulated IFNβ protein production in cultured macrophages: RAW264.7 macrophages were pre-treated with rapamycin [20 ng/mL] (30 min), Ku-0063794 [1 μM] (30 min), or amlexanox [50 μM] (2 hr.) and stimulated -/+poly (I:C) [30 μg/ml] for 6 hr. The secretion of IFNβ was measured by ELISA. Results represent the mean +/- SD of quadruplicate samples from one experiment. *p=.0009 relative to +Poly(I:C) by paired t-test (two-tailed).


B . Rapamycin suppresses TLR3 - stimulated IFNβ mRNA production in cultured macrophages : RAW264 . 7 macrophages were treated as above but stimulated - / + poly ( I : C ) [ 30 μg / ml ] for 3 hr . q - RT - PCR measured IFNβ gene expression . Results represent mean + / - SD of triplicate samples from one experiment . * p = . 03 relative to no Poly ( I : C ) by paired t - ( one - ) ; * * p = . 04 relative to + Poly ( I : C ) by paired t - ( one - ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rapamycin suppresses TLR3-stimulated IFNβ mRNA production in cultured macrophages: RAW264.7 macrophages were treated as above but stimulated -/+poly (I:C) [30 μg/ml] for 3 hr. q-RT-PCR measured IFNβ gene expression. Results represent the mean +/- SD of triplicate samples from one experiment. *p=.03 relative to no Poly(I:C) by paired t-test (one-tailed); **p=.04 relative to +Poly (I:C) by paired t-test (one-tailed).


C . Rapamycin suppresses TLR3 - stimulated IFNβ production in macrophages : BMDMs were pre - treated with rapamycin or amlexanox analyzed as in A . Results represent mean + / - SD of triplicate samples from one experiment . * p = . 002 relative to + Poly ( I : C ) by paired t - ( two - ) .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rapamycin suppresses TLR3-stimulated IFNβ protein production in primary macrophages: BMDMs were pre-treated with rapamycin or amlexanox and analyzed as in A. Results represent the mean +/- SD of triplicate samples from one experiment. *p=.002 relative to +Poly (I:C) by paired t-test (two-tailed).


D . Rapamycin suppresses TLR3 - stimulated IRF3 translocation in RAW264 . 7 macrophages : were pre - treated with rapamycin stimulated - / + poly ( I : C ) for 3 hr . as in A , fixed , processed for confocal immunofluorescence microscopy using an anti - IRF3 - Alexa 488 antibody Dapi . graph represents mean + / - SD of at least 400 total from three independent experiments . * p = . 002 relative to no Poly ( I : C ) by paired t - ( two - ) ; * * p . 003 relative to + Poly ( I : C ) by paired t - ( two - ) . Scale bar = 10 μM .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rapamycin suppresses TLR3-stimulated IRF3 nuclear translocation in RAW264.7 macrophages: Cells were pre-treated with rapamycin and stimulated -/+ poly (I:C) for 3 hr. as in A, fixed, and processed for confocal immunofluorescence microscopy using an anti-IRF3-Alexa 488 antibody and Dapi staining. The graph represents the mean +/- SD of at least 400 cells total from three independent experiments. *p=.002 relative to no Poly(I:C) by paired t-test (two-tailed); **p.003 relative to +Poly (I:C) by paired t-test (two-tailed). Scale bar = 10 μM.


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A . Reduced TLR3 - induced mTORC1 signaling in mTOR S2159A knock - in macrophages ( mTORA / A ) relative to wild type ( mTOR + / + ) : BMDMs were pre - treated with Torin 1 [ 100 nM ] ( 30 min ) stimulated - / + poly ( I : C ) [ 30 μg / ml ] for 30 min . Whole lysate ( WCL ) was immunoblotted as indicated . graph quantitates three independent experiments each with n = 1 ( n = 3 total ) . level of P - S6K1 - T389 normalized to total in mTOR + / + macrophages stimulated with poly ( I : C ) was set at 100 % . * confidence interval at 95 % ( 52 . 7 - 59 . 1 ) indicates statistical significance between mTOR + / + vs . mTORA / A BMDMs .

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

A. Reduced TLR3-induced mTORC1 signaling in mTOR S2159A knock-in primary macrophages (mTORA/A) relative to wild type (mTOR+/+): BMDMs were pre-treated with Torin 1 [100 nM] (30 min) and stimulated -/+ poly (I:C) [30 μg/ml] for 30 min. Whole cells lysate (WCL) was immunoblotted as indicated. The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with poly (I:C) was set at 100%. *The confidence interval at 95% (52.7-59.1) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs.


B . Reduced TLR4 - induced mTORC1 signaling in mTORA / A macrophages : BMDMs were treated as in A except stimulated - / + LPS [ 100 ng / mL ] ( 30 min ) . graph quantitates three independent experiments each with n = 1 ( n = 3 total ) . level of P - S6K1 - T389 normalized to total in mTOR + / + macrophages stimulated with LPS was set at 100 % . * confidence interval at 95 % ( 58 . 6 - 71 . 2 % ) indicates statistical significance between mTOR + / + vs . mTORA / A BMDMs .

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

B. Reduced TLR4-induced mTORC1 signaling in mTORA/A primary macrophages: BMDMs were treated as in A except stimulated -/+ LPS [100 ng/mL] (30 min). The graph quantitates three independent experiments each with n=1 (n=3 total). The level of P-S6K1-T389 normalized to total protein in mTOR+/+ macrophages stimulated with LPS was set at 100%. *The confidence interval at 95% (58.6-71.2%) indicates statistical significance between mTOR+/+ vs. mTORA/A BMDMs.


C . Reduced TLR3 - stimulated IFNβ production in mTORA / A macrophages : BMDMs were serum starved for 6 hr . stimulated - / + poly ( I : C ) [ 30 μg / ml ] for 6 hr . secretion of IFNβ was measured by ELISA . Results represent mean + / - from three independent experiments , n = 8 samples total . * p = . 0002 relative to mTOR + / + + Poly ( I : C ) by unpaired t - ( equal variance ; two - ) . * * p = . 0001 relative to mTOR + / + + Poly ( I : C ) by paired t - ( two - ) .

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

C. Reduced TLR3-stimulated IFNβ protein production in mTORA/A primary macrophages: BMDMs were serum starved for 6 hr. and stimulated -/+ poly (I:C) [30 μg/ml] for 6 hr. The secretion of IFNβ was measured by ELISA. Results represent the mean +/- SEM from three independent experiments, n=8 samples total. *p=.0002 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.0001 relative to mTOR+/+ + Poly(I:C) by paired t-test (two-tailed).


D . Reduced TLR3 - stimulated IRF3 translocation in mTORA / A macrophages : BMDMs were treated - / + poly ( I : C ) for 3 hr as in A , fixed , processed for confocal immunofluorescence microscopy using an anti - IRF3 - Alexa 488 antibody Dapi stain . graph represents mean + / - of at least 270 total from four independent experiments . * p = . 001 relative to mTOR + / + no Poly ( I : C ) by unpaired t - ( equal variance ; two - ) . * * p = . 02 relative to mTOR + / + + Poly ( I : C ) by unpaired t - ( equal variance ; two - ) . Scale bar = 10 μM .

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

D. Reduced TLR3-stimulated IRF3 nuclear translocation in mTORA/A primary macrophages: BMDMs were treated -/+ poly (I:C) for 3 hr as in A, fixed, and processed for confocal immunofluorescence microscopy using an anti-IRF3-Alexa 488 antibody and Dapi stain. The graph represents the mean +/- SEM of at least 270 cells total from four independent experiments. *p=.001 relative to mTOR+/+ no Poly(I:C) by unpaired t-test (equal variance; two-tailed). **p=.02 relative to mTOR+/+ + Poly(I:C) by unpaired t-test (equal variance; two-tailed). Scale bar = 10 μM.


Representative dual - channel kymographs showing that Rheb is recruited to mitochondria in axons co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial potential ( ∆ ψm ) dissipation . motility of mitochondria ( Mito ) Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to right ( negative slope ) represent anterograde movement ; those to left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

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

Representative dual-channel kymographs showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Time - lapse ( B ) showing colocalization comigration ( arrows ) of Rheb with mitochondria in a retrograde direction along axons during a 148 - s recording period . Cortical neurons were transfected with GFP - Rheb DsRed - Mito at 5 - 7 DIV incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10 - 12 DIV .

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

Time-lapse images (B) showing colocalization and comigration (white arrows) of Rheb with mitochondria in a retrograde direction along axons during a 148-s recording period. Cortical neurons were transfected with GFP-Rheb and DsRed-Mito at 5-7 DIV and incubated with DMSO or 10 μM CCCP for 24 hr prior to imaging at 10-12 DIV.


quantitative showing that Rheb is recruited to mitochondria in axons co - migrates with mitochondria in a predominant retrograde direction upon mitochondrial potential ( ∆ ψm ) dissipation . motility of mitochondria ( Mito ) Rheb - associated mitochondria ( Rheb - Mito ) in axons were quantified , respectively . Vertical lines represent stationary organelles . Slanted lines or curves to right ( negative slope ) represent anterograde movement ; those to left ( positive slope ) indicate retrograde movement . An organelle was considered stationary if it remained immotile ( displacement ≤ 5 μm ) .

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 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

quantitative analysis showing that Rheb is recruited to mitochondria in axons and co-migrates with mitochondria in a predominant retrograde direction upon mitochondrial membrane potential (∆ψm) dissipation. The motility of mitochondria (Mito) and Rheb-associated mitochondria (Rheb-Mito) in axons were quantified, respectively. Vertical lines represent stationary organelles. Slanted lines or curves to the right (negative slope) represent anterograde movement; those to the left (positive slope) indicate retrograde movement. An organelle was considered stationary if it remained immotile (displacement ≤ 5 μm).


Quantitative showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . percentages of mitochondria targeted by Rheb or Rheb - SSVM in presence or absence of CCCP were quantified ,

O O O O O B-MEASURED_VAR 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

Quantitative analysis showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. The percentages of mitochondria targeted by Rheb or Rheb-SSVM in the presence or absence of CCCP were quantified, respectively


representative showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP . Rheb - SSVM , a farnesylation deficient mutant of Rheb , fails to be recruited to mitochondria upon dissipating ∆ ψm . Mitochondria associated with Rheb were marked by arrows .

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

representative images showing robust association of Rheb with depolarized mitochondria within axons treated with CCCP. Rheb-SSVM, a farnesylation deficient mutant of Rheb, fails to be recruited to mitochondria upon dissipating ∆ψm. Mitochondria associated with Rheb were marked by white arrows.


F - H Representative dual - channel kymographs ( F ) quantitative ( G , H ) showing that Rheb - mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation . Note that Rheb , but not Rheb - SSVM , is associated with autophagic vacuoles ( AVs ) within axons upon ∆ ψm dissipation . Rheb - associated AVs move in a predominant retrograde direction along axons . number of Rheb - associated AVs ( Rheb - AVs ) with or without CCCP treatment relative motilities of AVs Rheb - AVs in CCCP - treated axons were quantified ,

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

F-H Representative dual-channel kymographs (F) and quantitative analysis (G, H) showing that Rheb-mediated mitophagy activation depends on Rheb recruitment to mitochondria through Rheb farnesylation. Note that Rheb, but not Rheb-SSVM, is associated with autophagic vacuoles (AVs) within axons upon ∆ψm dissipation. Rheb-associated AVs move in a predominant retrograde direction along axons. The number of Rheb-associated AVs (Rheb-AVs) with or without CCCP treatment and the relative motilities of AVs and Rheb-AVs in CCCP-treated axons were quantified, respectively


Representative dual - channel kymographs showing that Rheb enhances mitophagy in CCCP - treated axons .

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Representative dual-channel kymographs showing that Rheb enhances mitophagy in CCCP-treated axons.


quantitative showing that Rheb enhances mitophagy in CCCP - treated axons . numbers of mitochondria ( Mito ) , AVs , Mito - AVs / mitophagosomes , of Mito within AVs , were

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quantitative analysis showing that Rheb enhances mitophagy in CCCP-treated axons. The numbers of mitochondria (Mito), AVs, and Mito-AVs/mitophagosomes, the percentage of Mito within AVs, were quantified


quantitative of Mito within AVs , motilities of AVs Mito - AVs along axons with without elevated Rheb expression were

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quantitative analysis the percentage of Mito within AVs, and the motilities of AVs and Mito-AVs along axons with and without elevated Rheb expression were quantified


D , E Representative ( D ) quantitative ( E ) showing that mitophagy in axons requires Rheb Nix , but not Parkin . number of mitophagosomes indicated by arrows of Mito within AVs in CCCP - treated axons expressing scrambled shRNA , Rheb shRNA , Nix shRNA , or Parkin shRNA were quantified , respectively ( E ) .

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D,E Representative images (D) and quantitative analysis (E) showing that mitophagy in axons requires Rheb and Nix, but not Parkin. The number of mitophagosomes indicated by white arrows and the percentage of Mito within AVs in CCCP-treated axons expressing scrambled shRNA, Rheb shRNA, Nix shRNA, or Parkin shRNA were quantified, respectively (E).