( 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 -

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

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

(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 ( 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 .

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

(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 ( 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 ) .

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

(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 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 ) .

O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

(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 ( 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

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 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 B-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

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 - 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 .

O 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 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

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 , 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR 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 B-MEASURED_VAR O 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 + / + and TBK1 - / - MEFs were serum starved ( 20 hr . ) and 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 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

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 . 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 ) .

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


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 .

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

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 . ) , and 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 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 + / + and TBK1 - / - MEFs were serum - starved ( 20 hr . ) and 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 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 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 and mTORC2 signaling : TBK1 + / + and 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 O B-CONTROLLED_VAR O 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 + / + and TBK1 - / - MEFs were stimulated with insulin as in C .

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

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 and 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 B-CONTROLLED_VAR 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

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 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 ) .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_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 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

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 B-CONTROLLED_VAR 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 ) , and stimulated - / + epidermal growth factor ( EGF ) [ 25 ng / mL ] ( 30 min ) . Whole cell 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 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 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

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 ) . Cells 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 O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 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 .

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

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

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

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 + / + 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 .

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

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 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 I-CONTROLLED_VAR 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 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 .

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

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 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

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 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 .

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 B-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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 ) .

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 B-CONTROLLED_VAR 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 + / + 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 .

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

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

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

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 O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR 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 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 ) .

O O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

O O B-CONTROLLED_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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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-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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

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

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

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 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 .

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

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-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 B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

O O O O O O B-MEASURED_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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 .

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

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 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 ) .

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

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 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 .

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 B-CONTROLLED_VAR 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 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 ) .

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 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

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 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 ,

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

O O O O O O B-MEASURED_VAR O O O O O O O B-CONTROLLED_VAR 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 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 ) 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 ,

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

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

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


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

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

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

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

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 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 ) .

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

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).


F , G Quantitative analysis ( F ) and representative blots ( G ) showing increased mitochondrial association with Rheb and LC3 - II in mature cortical neurons upon ∆ ψm depolarization . Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post - nuclear supernatant ( P ) , mitochondria - enriched membrane fraction ( M ) , and cytosolic supernatant ( S ) . Equal amounts of protein ( 20 μg ) were sequentially immunoblotted with antibodies against mitophagic / autophagic proteins Rheb , LC3 , and Rab7 , mitochondrial protein VDAC , and cytosolic protein GAPDH on the same membranes after stripping between each antibody application . The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH , compared to post - nuclear supernatant . Protein levels in mitochondrial fractions of CCCP - treated neurons were normalized to those in neurons incubated with DMSO . Data were quantified from four independent repeats .

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F,G Quantitative analysis (F) and representative blots (G) showing increased mitochondrial association with Rheb and LC3-II in mature cortical neurons upon ∆ψm depolarization. Cortical neurons at 14 DIV were incubated with DMSO or 10 μM CCCP for 24 hr and then subjected to fractionation into post-nuclear supernatant (P), mitochondria-enriched membrane fraction (M), and cytosolic supernatant (S). Equal amounts of protein (20 μg) were sequentially immunoblotted with antibodies against mitophagic/autophagic proteins Rheb, LC3, and Rab7, mitochondrial protein VDAC, and cytosolic protein GAPDH on the same membranes after stripping between each antibody application. The purity of mitochondrial fractions was confirmed by enrichment of VDAC and less abundance of GAPDH, compared to post-nuclear supernatant. Protein levels in mitochondrial fractions of CCCP-treated neurons were normalized to those in neurons incubated with DMSO. Data were quantified from four independent repeats.


Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP - treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA ( H ) .

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Quantitative analysis showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Mean fluorescence intensity ratios evoked by the two excitation wavelengths at individual mitochondria in the CCCP-treated axons expressing scrambled shRNA or Rheb shRNA were quantified and normalized to those of control neurons transfected with scrambled shRNA (H).


representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ ψm depolarization . The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm , respectively . Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light . The ratio has been false colored with the indicated heat map , with high intensity indicative of ROGFP fluorescence in a more oxidative

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representative images showing that Rheb RNAi augments axonal retention of oxidized mitochondria upon ∆ψm depolarization. The fluorescence of MitoROGFP was emitted at 510 nm and excited at 405 nm or 488 nm, respectively. Ratiometric images were generated from fluorescence excited by 405 nm light relative to that excited by 488 nm light. The ratio has been false colored with the indicated heat map, with high intensity indicative of ROGFP fluorescence in a more oxidative level


Aberrant accumulation of mitophagosomes co - labeled by antibodies against LC3 and cytochrome c ( Cyto c ) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains . Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse

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Aberrant accumulation of mitophagosomes co-labeled by antibodies against LC3 and cytochrome c (Cyto c) in the hippocampal mossy fibers of mutant hAPP Tg mouse brains. Note that mitophagosomes indicated by white arrows are not readily observed in WT mouse brains


The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified from the total number of brain slice sections ( n ) indicated on the top of the

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The average number of mitophagosomes per section (320 μm × 320 μm) was quantified from the total number of brain slice sections (n) indicated on the top of the bars


C Clustering of mitophagosomes ( white arrows ) co - labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen / dystrophic presynaptic terminals indicated by synaptophysin ( SYP ) surrounding amyloid plaques in AD mouse brains .

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C Clustering of mitophagosomes (white arrows) co-labeled by antibodies against LC3 and Cyto c or p62 and HSP60 within swollen/dystrophic presynaptic terminals indicated by synaptophysin (SYP) surrounding amyloid plaques in AD mouse brains.


Quantitative analysis showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in

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Quantitative analysis showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses


representative TEM images showing presynaptic retention of mitophagosome - like structures in the hippocampal regions of mutant hAPP mouse brains . White arrow - labeled mitophagosomes are not readily detected in WT mouse brains . Black arrows marked amphisomes or degradative autophagic vacuoles ( AVd ) .

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representative TEM images showing presynaptic retention of mitophagosome-like structures in the hippocampal regions of mutant hAPP mouse brains. White arrow-labeled mitophagosomes are not readily detected in WT mouse brains. Black arrows marked amphisomes or degradative autophagic vacuoles (AVd).


F , G Mitophagic accumulation at Aβ - enriched synapses in AD mouse brains . Note that the levels of mitophagic / autophagic proteins ( red box ) along with Aβ ( green box ) are increased in synaptic fractions isolated from AD mouse brains . Equal amounts ( 10 μg ) of synaptosomal preparations ( Syn ) and post - nuclear supernatants ( PNS ) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP / Aβ , Rheb , LC3 , p62 , and Rab7 , as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH ( F ) . Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls . Data were quantified from five repeats ( G ) .

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F,G Mitophagic accumulation at Aβ-enriched synapses in AD mouse brains. Note that the levels of mitophagic/autophagic proteins (red box) along with Aβ (green box) are increased in synaptic fractions isolated from AD mouse brains. Equal amounts (10 μg) of synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and mutant hAPP mouse brains were sequentially immunoblotted with antibodies against hAPP/Aβ, Rheb, LC3, p62, and Rab7, as well as mitochondrial proteins TOM20 and SOD2 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and less abundance of p115 and GAPDH (F). Relative protein levels in the synaptosomal preparations from mutant hAPP mice were compared to those in WT littermate controls. Data were quantified from five repeats (G).


H , I Representative blots ( H ) and quantitative analysis ( I ) showing increased levels of Rheb along with LC3 - II , p62 , and Rab7 ( red box ) in Mito fractions purified from AD mouse brains . The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115 , relative to PNS fractions . Data were quantified from four repeats .

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H,I Representative blots (H) and quantitative analysis (I) showing increased levels of Rheb along with LC3-II, p62, and Rab7 (red box) in Mito fractions purified from AD mouse brains. The purity of Mito fractions was confirmed by enrichment of mitochondrial proteins VDAC and TOM20 and by the absence of GAPDH and p115, relative to PNS fractions. Data were quantified from four repeats.


Representative images showing increased mitochondrial association with Rheb in AD axons , relative to that of WT littermate controls . Note that CCCP treatment enhances Rheb localization to mitochondria . The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of

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Representative images showing increased mitochondrial association with Rheb in AD axons, relative to that of WT littermate controls. Note that CCCP treatment enhances Rheb localization to mitochondria. The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP


quantitative analysis The percentages of white arrow - marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified ,

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quantitative analysis The percentages of white arrow-marked mitochondria that were targeted by Rheb in WT and mutant hAPP axons in the absence and presence of CCCP were quantified, respectively


C - Representative dual - channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons . mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were

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C- Representative dual-channel kymographs showing defective retrograde transport and mitophagic accumulation within AD axons. mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified


quantitative analysis , the numbers of Mito , AVs , and Mito - AVs , and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified ,

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quantitative analysis , the numbers of Mito, AVs, and Mito-AVs, and the percentage of mitochondria within AVs in WT or mutant hAPP axons with and without CCCP were quantified, respectively


quantitative analysis Relative motility of AVs and Mito - AVs / mitophagosomes in WT or mutant hAPP axons with and without CCCP were

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quantitative analysis Relative motility of AVs and Mito-AVs/mitophagosomes in WT or mutant hAPP axons with and without CCCP were quantified


F , G Quantitative analysis ( F ) and representative blots ( G ) showing that ∆ ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains . Note increased localization of Rheb along with LC3 - II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment ( red box ) . Data were quantified from three independent repeats . P : post - nuclear supernatants ; M : mitochondria - enriched membrane fractions ; S : cytosolic supernatants .

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F,G Quantitative analysis (F) and representative blots (G) showing that ∆ψm depolarization augments mitophagy activation in cortical neurons cultured from AD mouse brains. Note increased localization of Rheb along with LC3-II and Rab7 to mitochondria in mutant hAPP Tg neurons after CCCP treatment (red box). Data were quantified from three independent repeats. P: post-nuclear supernatants; M: mitochondria-enriched membrane fractions; S: cytosolic supernatants.


Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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Quantitative analysis Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons , which is exacerbated after dissipating ∆ ψm . Mean fluorescence intensity ratios evoked by the two excitation wavelengths ( 405 nm and 488 nm ) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT

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representative images showing abnormal accumulation of oxidatively damaged mitochondria in AD axons, which is exacerbated after dissipating ∆ψm. Mean fluorescence intensity ratios evoked by the two excitation wavelengths (405 nm and 488 nm) at individual mitochondria in mutant hAPP axons with and without CCCP treatment were quantified and normalized to those of WT controls


A , B Representative images ( A ) and quantitative analysis ( B ) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin - cKO mouse brains . Note that white arrow - labeled mitophagosomes are not readily observed in WT mouse brains . The average number of mitophagosomes per section ( 320 μm × 320 μm ) was quantified .

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A,B Representative images (A) and quantitative analysis (B) showing aberrant accumulation of mitophagosomes labeled by LC3 and Cyto c antibodies in the hippocampal mossy fibers of snapin-cKO mouse brains. Note that white arrow-labeled mitophagosomes are not readily observed in WT mouse brains. The average number of mitophagosomes per section (320 μm × 320 μm) was quantified.


Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin - mutant mouse brains . The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Equal amounts ( 10 μg ) of Syn and post - nuclear supernatants (

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Representative blots showing aberrant mitophagic retention at synaptic terminals in snapin-mutant mouse brains. The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Equal amounts (10 μg) of Syn and post-nuclear supernatants (PNS


quantitative analysis The levels of Rheb along with LC3 - II , p62 , and Rab7 are significantly elevated in the synaptosomal fractions ( Syn ) purified from snapin - deficient mouse brains , relative to those of WT littermate controls WT and snapin - mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application . The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH . Data were quantified from five independent repeats .

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quantitative analysis The levels of Rheb along with LC3-II, p62, and Rab7 are significantly elevated in the synaptosomal fractions (Syn) purified from snapin-deficient mouse brains, relative to those of WT littermate controls WT and snapin-mutant mouse brains were sequentially immunoblotted with antibodies against TOM20 on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by relative enrichment of SYP and PSD95 and less abundance of GAPDH. Data were quantified from five independent repeats.


E , F Representative blots ( E ) and quantitative analysis ( F ) showing mitophagic accumulation in the brains of snapin conditional KO ( cKO ) mice by crossing with Thy1 - cre Tg mice . Note increased levels of Rheb along with LC3 - II , p62 , and Rab7 in mitochondrial ( Mito ) fractions isolated from snapin - deficient mouse brains , relative to those of WT littermate controls ( red box ) . Data were quantified from five independent repeats . PNS : post - nuclear supernatants .

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E,F Representative blots (E) and quantitative analysis (F) showing mitophagic accumulation in the brains of snapin conditional KO (cKO) mice by crossing with Thy1-cre Tg mice. Note increased levels of Rheb along with LC3-II, p62, and Rab7 in mitochondrial (Mito) fractions isolated from snapin-deficient mouse brains, relative to those of WT littermate controls (red box). Data were quantified from five independent repeats. PNS: post-nuclear supernatants.


G , H Representative TEM images ( G ) and quantitative analysis ( H ) showing abnormal presynaptic retention of mitophagosome - like structures in the hippocampal regions of snapin - deficient mouse brains . Mitophagosomes , indicated by black arrows , are not readily detected in WT mouse brains . Data were quantified from the total number of the imaging fields ( n ) indicated in parentheses ( H ) .

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G,H Representative TEM images (G) and quantitative analysis (H) showing abnormal presynaptic retention of mitophagosome-like structures in the hippocampal regions of snapin-deficient mouse brains. Mitophagosomes, indicated by black arrows, are not readily detected in WT mouse brains. Data were quantified from the total number of the imaging fields (n) indicated in parentheses (H).


I , J Representative images ( I ) and quantitative analysis ( J ) showing that deletion of snapin in mouse brains induces synaptic damage . Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin - mutant mice crossed with CamKII - cre Tg mice at the age of 8 months , relative to that of WT littermate controls .

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I,J Representative images (I) and quantitative analysis (J) showing that deletion of snapin in mouse brains induces synaptic damage. Note that the density of presynaptic terminals in the hippocampal mossy fibers revealed by SYP immunostaining is significantly reduced in snapin-mutant mice crossed with CamKII-cre Tg mice at the age of 8 months, relative to that of WT littermate controls.


Representative dual - channel kymographs ( A ) and quantitative analysis ( B showing that overexpression of Snapin , but not Snapin - L99K , a Snapin mutant deficient in dynein motor binding , reduces mitophagic accumulation within AD axons incubated with CCCP .

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Representative dual-channel kymographs (A) and quantitative analysis (B showing that overexpression of Snapin, but not Snapin-L99K, a Snapin mutant deficient in dynein motor binding, reduces mitophagic accumulation within AD axons incubated with CCCP.


Representative quantitative analysis showing that overexpression of Snapin , but not Snapin - L99K , a

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Representative quantitative analysis showing that overexpression of Snapin, but not Snapin-L99K, a Snapin


D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin - L99K were quantified and normalized to those of mutant hAPP controls . Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment .

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D Mean fluorescence intensity ratios evoked by 405 nm and 488 nm excitation wavelengths at individual mitochondria in mutant hAPP axons expressing Snapin or Snapin-L99K were quantified and normalized to those of mutant hAPP controls. Note that oxidatively stressed mitochondria are reduced within AD axons expressing Snapin after CCCP treatment.


A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV - mCherry or AAV - mCherry - Snapin . mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus ( DG ) of mutant hAPP mouse brains . NeuN , a neuronal nuclear marker , indicates neurons .

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A Representative images showing gene delivery into the hippocampus of AD mice injected with AAV-mCherry or AAV-mCherry-Snapin. mCherry fluorescence indicated by white arrows is present in the soma and processes of transduced neurons in the hippocampal CA3 and dentate gyrus (DG) of mutant hAPP mouse brains. NeuN, a neuronal nuclear marker, indicates neurons.


Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c * : color is converted from blue to red for better

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Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression Cyto c*: color is converted from blue to red for better contrast


Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV - mCherry - Snapin . Note a marked reduction of mitophagosomes ( white arrows ) in mutant hAPP Tg mouse brains with elevated Snapin

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Attenuation of mitophagic accumulation in the hippocampal mossy fibers of AD mice transduced with AAV-mCherry-Snapin. Note a marked reduction of mitophagosomes (white arrows) in mutant hAPP Tg mouse brains with elevated Snapin expression


D , E Quantitative analysis ( D ) and representative images ( E ) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV - mCherry - Snapin . Blue indicates the signal of DAPI staining . The percentage area of SYP - labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV - mCherry ( D ) . Data information : Data were quantified from a total number of 40 - 44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection .

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D,E Quantitative analysis (D) and representative images (E) showing increased density of presynaptic terminals in the hippocampal mossy fibers of AD mice infected with AAV-mCherry-Snapin. Blue indicates the signal of DAPI staining. The percentage area of SYP-labeled presynaptic terminals was quantified and normalized to that of control AD mice injected with AAV-mCherry (D). Data information: Data were quantified from a total number of 40-44 imaging slice sections of three pairs of mutant hAPP Tg mice with AAV injection.