( D ) Quantitative analysis of poly - GA aggregates in the spinal cord using immunohistochemistry . Images from complete spinal cord sections at 1 mm intervals . One - way ANOVA , Tukey ' s post hoc test , F ( 5 , 12 ) = 1 . 625 , p = 0 . 2270 . * * * p < 0 . 001 , ns not significant . TG - Ova - ( GA ) 10 vs . TG - PBS p < 0 . 0001 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 2460 . Scale bar indicates 100 μm .

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(D) Quantitative analysis of poly-GA aggregates in the spinal cord using immunohistochemistry. Images from complete spinal cord sections at 1 mm intervals. One-way ANOVA, Tukey's post hoc test, F(5,12)=1.625, p=0.2270. *** p<0.001, ns not significant. TG-Ova-(GA)10 vs. TG-PBS p<0.0001; TG-(GA)15 vs. TG-PBS p=0.2460. Scale bar indicates 100 μm.


( E ) Immunoassay of poly - GA from the insoluble fraction . One - way ANOVA , Tukey ' s post hoc test , F ( 5 , 24 ) = 3 . 098 , p = 0 . 0269 . * * p < 0 . 001 , ns not significant . TG - Ova - ( GA ) 10 vs . TG - PBS p < 0 . 0001 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 1975 .

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

(E) Immunoassay of poly-GA from the insoluble fraction. One-way ANOVA, Tukey's post hoc test, F(5,24)=3.098, p=0.0269. ** p<0.001, ns not significant. TG-Ova-(GA)10 vs. TG-PBS p<0.0001; TG-(GA)15 vs. TG-PBS p=0.1975.


( C , D ) Analysis of microglia / macrophage activation using Iba1 immunohistochemistry from complete spinal cord sections at 1 mm interval . One - way ANOVA , Tukey ' s post hoc test . * * p < 0 . 01 , * p < 0 . 05 , ns not significant . Scale bar indicates 100 μm . F ( 5 , 12 ) = 0 . 6974 , p = 0 . 6357 ; TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0131 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 7650 . TG - PBS vs . WT - PBS p = 0 . 0018 .

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(C, D) Analysis of microglia/macrophage activation using Iba1 immunohistochemistry from complete spinal cord sections at 1 mm interval. One-way ANOVA, Tukey's post hoc test. ** p<0.01, * p<0.05, ns not significant. Scale bar indicates 100 μm. F(5,12)=0.6974, p=0.6357; TG-Ova-(GA)10 vs. TG-PBS p= 0.0131; TG-(GA)15 vs. TG-PBS p=0.7650. TG-PBS vs. WT-PBS p=0.0018.


( E ) The percentage of cells with partial cytoplasmic mislocalization of TDP - 43 was quantified from six images from spinal cord sections at 1 mm intervals were analyzed . One - way ANOVA , Tukey ' s post hoc test . * p < 0 . 05 , ns not significant . F ( 5 , 12 ) = 0 . 6533 , p = 0 . 6650 ; TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0096 ; TG - ( GA ) 15 vs . TG - PBS p = 0 . 4215 .

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(E) The percentage of cells with partial cytoplasmic mislocalization of TDP-43 was quantified from six images from spinal cord sections at 1 mm intervals were analyzed. One-way ANOVA, Tukey's post hoc test. * p<0.05, ns not significant. F(5,12)=0.6533, p=0.6650; TG-Ova-(GA)10 vs. TG-PBS p= 0.0096; TG-(GA)15 vs. TG-PBS p=0.4215.


( F ) Immunoassay of NFL level in cerebrospinal fluid . One - way ANOVA , Tukey ' s post hoc test , F ( 3 , 11 ) = 1 . 911 , p = 0 . 1862 . * * p < 0 . 01 . TG - Ova - ( GA ) 10 vs . TG - PBS p = 0 . 0081 .

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(F) Immunoassay of NFL level in cerebrospinal fluid. One-way ANOVA, Tukey's post hoc test, F(3,11)=1.911, p=0.1862. ** p<0.01. TG-Ova-(GA)10 vs. TG-PBS p=0.0081.


( B ) The ratio of unique histone iCLIP - seq read population to unique histone RNA - seq read population was calculated and shown , with the unique histone RNA - seq read population set as " 1 " . Error bars represent standard deviations from three replicates of ALYREF iCLIP data . Statistical analysis was performed using Student ' s t - test . * * * P < 0 . 001 .

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

(B) The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and shown, with the unique histone RNA-seq read population set as "1". Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P< 0.001.


( C ) ALYREF RIP - qPCRs to examine ALYREF binding on histone mRNAs . Relative RIP efficiencies are shown . A tRNA was used as a negative control for ALYREF binding . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant.


( D ) The distribution of histone reads in 5 ' UTR , CDS and 3 ' UTR in rRNA - depleted nuclear RNA - seq and ALYREF - iCLIP - seq .

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(D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq.


( E ) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA ' s contribution to the plot .

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

(E) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


( F ) Screenshots of two histone mRNAs showing clustered ALYREF binding sites . The 3 ' UTR regions are highlighted .

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(F) Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3' UTR regions are highlighted.


( G ) Distribution profile of clustered ALYREF binding sites on histone mRNA 3 ' UTRs relative to the cleavage site ( CS ) . The CS is set as " 0 " in the x axis , and the relative position to the CS is marked . The y axis shows the total occurrence of binding sites in three repeats at each nucleotide .

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(G) Distribution profile of clustered ALYREF binding sites on histone mRNA 3' UTRs relative to the cleavage site (CS). The CS is set as "0" in the x axis, and the relative position to the CS is marked. The y axis shows the total occurrence of binding sites in three repeats at each nucleotide.


IPs from RNase A - treated S - phase HeLa cell lysate using the SLBP antibody or IgG ( A ) Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

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

IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A) Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots


IPs from RNase A - treated S - phase HeLa cell lysate using the ALYREF antibody or IgG ( B ) . Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

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

IPs from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody or IgG (B). Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots


( C ) Pull - downs of in vitro translated 35S labelled SLBP and luciferase ( Cntl ) using MBP or MBP - ALYREF in the presence of RNase A . The proteins pulled down were visualized by coomassie staining ( left ) or phosphoimager ( right ) . 3 % of input was loaded .

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(C) Pull-downs of in vitro translated 35S labelled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by coomassie staining (left) or phosphoimager (right). 3% of input was loaded.


( D ) GST - SLBP , GST - UAP56 and GST were used for pull - down of purified MBP - ALYREF or MBP in the presence of RNase A . Proteins pulled down were separated by SDS - PAGE , followed by coomassie staining . 37 . 5 % of input proteins were loaded .

(D) GST-SLBP, GST-UAP56 and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE, followed by coomassie staining. 37.5% of input proteins were loaded.


( E ) ( Top ) Domain schematic representation of SLBP . ( Bottom ) Flag IPs from RNase A - treated HeLa cell lysate individually expressing indicated Flag tagged proteins , followed by western blotting using Flag and ALYREF antibodies . 3 % of input was loaded . The * indicates a band probably resulted from degradation of Flag - SLBP . The white line delineates the boundary where irrelevant lanes have been removed from the same

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

(E) (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing indicated Flag tagged proteins, followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( E ) ( Top ) Domain schematic representation of SLBP . followed by western blotting using Flag and ALYREF antibodies . 3 % of input was loaded . The * indicates a band probably resulted from degradation of Flag - SLBP . The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA - SLBP stable expression cells transfected with plasmids expressing ALYREF fragments .

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(E) (Top) Domain schematic representation of SLBP. followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments.


( G ) Western blotting to examine the KD efficiency of SLBP . GAPDH was used as a loading control . The white line delineates the boundary where irrelevant lanes have been removed from the same

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(G) Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


Cntl - or SLBP - siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody . The immunoprecipitates were subjected to western analysis ( H ) Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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

Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to western analysis (H) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.


Cntl - or SLBP - siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody . The immunoprecipitates were subjected to RT - qPCRs ( I ) . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

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Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to RT-qPCRs (I). Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.


( J ) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA ' s contribution to the plot .

(J) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


( B ) List of histone genes with defective 3 ' - end processing detected by polyA + and polyA - RNA - seq . PolyA + ALYREF / Cntl shows polyA + RNA - seq RPM ratio of each histone gene in ALYREF KD versus Cntl .

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(B) List of histone genes with defective 3'-end processing detected by polyA+ and polyA- RNA-seq. PolyA+ ALYREF/Cntl shows polyA+ RNA-seq RPM ratio of each histone gene in ALYREF KD versus Cntl.


( C ) Screenshots of two exemplified histone mRNAs with 3 ' - end processing defects are shown . Transcription direction is indicated . Black arrowheads and the corresponding lines mark the position of CS .

(C) Screenshots of two exemplified histone mRNAs with 3'-end processing defects are shown. Transcription direction is indicated. Black arrowheads and the corresponding lines mark the position of CS.


( D ) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3 ' READS + .

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(D) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3'READS+.


( E , F ) RT - qPCRs to detect histone mRNA levels in the polyA + ( E ) or the polyA - fraction ( F ) in cells treated with Cntl , ALYREF or ARS2 siRNA . The arrowhead and arrows indicate the cleavage site and primer location , respectively . The bars show the relative abundance of polyA + forms to the actin mRNA ( E ) and that of polyA - forms relative to the 18S rRNA ( F ) . Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(E, F) RT-qPCRs to detect histone mRNA levels in the polyA+ (E) or the polyA- fraction (F) in cells treated with Cntl, ALYREF or ARS2 siRNA. The arrowhead and arrows indicate the cleavage site and primer location, respectively. The bars show the relative abundance of polyA+ forms to the actin mRNA (E) and that of polyA- forms relative to the 18S rRNA (F). Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Western blotting to examine ALYREF protein level ( G ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression cells treated with Cntl or ALYREF siRNA . The white line delineates the boundary where irrelevant lanes have been removed from the same

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Western blotting to examine ALYREF protein level (G) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


RT - qPCRs to detect polyA + histone mRNA levels ( H ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression cells treated with Cntl or ALYREF siRNA . The bars show the relative abundance of histone mRNAs to the GAPDH mRNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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RT-qPCRs to detect polyA+ histone mRNA levels (H) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The bars show the relative abundance of histone mRNAs to the GAPDH mRNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


IPs were carried out from RNase A - treated S - phase HeLa cell lysate using the Lsm11 antibody and IgG ( A ) followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

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

IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the Lsm11 antibody and IgG (A) followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


IPs were carried out from RNase A - treated S - phase HeLa cell lysate using the ALYREF antibody and IgG ( B ) , followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

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

IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody and IgG (B), followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( C ) Pull - down of purified His - Cntl ( PGK1 ) or His - Lsm11 using equal amount of GST and GST - ALYREF proteins in the presence of RNase A . Proteins pulled down were separated by SDS - PAGE followed by coomassie staining . * indicates a nonspecific band .

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(C) Pull-down of purified His-Cntl (PGK1) or His-Lsm11 using equal amount of GST and GST-ALYREF proteins in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE followed by coomassie staining. * indicates a nonspecific band.


Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody . The immunoprecipitates were subjected western analysis ( D ) . * indicates the antibody heavy chain .

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Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected western analysis (D). * indicates the antibody heavy chain.


Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody . The immunoprecipitates were subjected to RT - qPCRs ( E ) * indicates the antibody heavy chain . The bars show the relative IP efficiencies of histone pre - mRNAs to U7 snRNA . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected to RT-qPCRs (E) * indicates the antibody heavy chain. The bars show the relative IP efficiencies of histone pre-mRNAs to U7 snRNA. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( A ) ( Top ) Illustration of the HIST2H2AA3 reporter construct . ( Bottom ) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl , ALYREF , THOC2 ( THO ) and UAP56 . DAPI staining served as a nuclear marker . N and C indicate nuclear and cytoplasmic FISH signals , respectively . N / C ratios were determined for 20 cells in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( B ) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript - specific probe in HeLa cells depleted of Cntl , ALYREF , THOC2 ( THO ) and UAP56 . N and C indicate nuclear and cytoplasmic FISH signals , respectively . N / C ratios were calculated Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(B) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript-specific probe in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were calculated Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Western analysis to examine the purities of nuclear and cytoplasmic fractions ( C ) in Cntl and UAP56 KD cells . MTR4 and Tubulin were used as the nuclear and cytoplasmic marker , respectively .

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Western analysis to examine the purities of nuclear and cytoplasmic fractions (C) in Cntl and UAP56 KD cells. MTR4 and Tubulin were used as the nuclear and cytoplasmic marker, respectively.


RT - qPCRs to examine N / C distribution of each histone mRNA ( D ) in Cntl and UAP56 KD cells . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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RT-qPCRs to examine N/C distribution of each histone mRNA (D) in Cntl and UAP56 KD cells. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( F ) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl , ALYREF and SLBP KD cells . Data information : Error bars represent standard deviations from biological repeats n = 2 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(F) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl, ALYREF and SLBP KD cells. Data information: Error bars represent standard deviations from biological repeats n=2 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( G ) NXF1 iCLIP signals along the histone mRNA . The histone mRNA is divided into 10 fractions based on their mRNA size . Each fraction represents 10 % of each histone mRNA . The tenth fractions that contains the SL is boxed by green dashed lines .

(G) NXF1 iCLIP signals along the histone mRNA. The histone mRNA is divided into 10 fractions based on their mRNA size. Each fraction represents 10% of each histone mRNA. The tenth fractions that contains the SL is boxed by green dashed lines.


( A ) ( Top ) Illustration of the reporter constructs . The black and red lines indicate H2AA3 - SL and H2AA3 - SLD PCR products , respectively . The dashed line indicates the FISH probe . ( Bottom ) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3 - SL and H2AA3 - SLD PCR products . C and N indicate cytoplasmic and nuclear FISH signals , respectively . C / N ratios were determined for 20 cells in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(A) (Top) Illustration of the reporter constructs. The black and red lines indicate H2AA3-SL and H2AA3-SLD PCR products, respectively. The dashed line indicates the FISH probe. (Bottom) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3-SL and H2AA3-SLD PCR products. C and N indicate cytoplasmic and nuclear FISH signals, respectively. C/N ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( B ) ( Top ) Illustration of the reporter constructs . The dashed line indicates the FISH probe . FISH to detect mRNA distribution at 24 hr after transfection of the H1C - SL - Rb ( cH1C ) and H1C - SLD - Rb ( pH1C ) constructs . C / N ratios were quantified Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(B) (Top) Illustration of the reporter constructs. The dashed line indicates the FISH probe. FISH to detect mRNA distribution at 24 hr after transfection of the H1C-SL-Rb (cH1C) and H1C-SLD-Rb (pH1C) constructs. C/N ratios were quantified Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( Top ) Illustration of the reporter constructs . PCR products of cG - SL and cG - SLD were used instead . cG , β - globin cDNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(Top) Illustration of the reporter constructs. PCR products of cG-SL and cG-SLD were used instead. cG, β-globin cDNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . The immunoprecipitates were subjected to western analysis to detect ALYREF ( D )

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H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis to detect ALYREF (D)


H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . RT - qPCRs to detect RNAs ( E ) . The bars in the graph shows the relative ALYREF IP efficiency , with that for the H1C - SL - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. RT-qPCRs to detect RNAs (E). The bars in the graph shows the relative ALYREF IP efficiency, with that for the H1C-SL-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( F ) H1C - SLD - Rb construct with wild - type ( H1C - SLDWT - Rb ) or mutant ( H1C - SLDmut - Rb ) U7 - binding sequence , together with VSV M , was co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . The immunoprecipitates were subjected to western analysis ( left panel ) and RT - qPCRs ( right panel ) . The bars in the right graph show the relative ALYREF IP efficiency , with that for the H1C - SLDWT - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(F) H1C-SLD-Rb construct with wild-type (H1C-SLDWT-Rb) or mutant (H1C-SLDmut-Rb) U7-binding sequence, together with VSV M, was co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis (left panel) and RT-qPCRs (right panel). The bars in the right graph show the relative ALYREF IP efficiency, with that for the H1C-SLDWT-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( A ) Western blotting to examine the expression levels of H2B and H4 in Cntl , ALYREF and ARS2 KD cells . GAPDH was used as a loading control . The white line delineates the boundary where irrelevant lanes have been removed from the same

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(A) Western blotting to examine the expression levels of H2B and H4 in Cntl, ALYREF and ARS2 KD cells. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( B ) Growth curve of Cntl and ALYREF KD cells . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * * P < 0 . 01 .

(B) Growth curve of Cntl and ALYREF KD cells. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01.


( C ) Cell cycle analysis of Cntl , ALYREF and ARS2 KD cells . Quantifications of cells in G0 / G1 ( 1N ) , G2 ( 2N ) and S - phase ( intermediate ) of the cell cycle are shown in the upper right corner of each histogram .

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(C) Cell cycle analysis of Cntl, ALYREF and ARS2 KD cells. Quantifications of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle are shown in the upper right corner of each histogram.


A Intracellular localization of total ( HA ) and polymeric ATZ ( 2C1 ) in WT MEF mock - treated , Confocal Laser Scanning Microscopy ( CLSM ) . B Same as A for MEF exposed to 50 nM BafA1 for 12 h . C Same as A , 4 h after BafA1 wash - out . D Quantification of ATZ - positive , LAMP1 - positive endolysosomes ( EL ) ( n = 13 , 10 , 11 cells , respectively ) . One - way ANOVA and Dunnett ' s multiple comparisons test , ns P > 0 . 05 , * * * * P < 0 . 0001 . Data information : Scale bars : 10 μm .

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A Intracellular localization of total (HA) and polymeric ATZ (2C1) in WT MEF mock-treated, Confocal Laser Scanning Microscopy (CLSM). B Same as A for MEF exposed to 50 nM BafA1 for 12 h. C Same as A, 4 h after BafA1 wash-out. D Quantification of ATZ-positive, LAMP1-positive endolysosomes (EL) (n=13, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.


E ATZ polymers immunoisolated from lysates of WT MEF mock treated ( lane 1 ) , incubated for 12 h with BafA1 ( lane 2 ) and 4 h after BafA1 wash - out ( lane 3 ) . Immunoprecipitation ( IP ) of ATZ polymers with polymer - specific 2C1 antibody , transfer on PVDF membrane , revealed with anti - HA antibody on western blot ( WB ) . F Quantification of E , n = 3 , mean ± SEM . Unpaired two - tailed t - test , ns P > 0 . 05 , * P < 0 . 05 . Data information : Scale bars : 10 μm .

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E ATZ polymers immunoisolated from lysates of WT MEF mock treated (lane 1), incubated for 12 h with BafA1 (lane 2) and 4 h after BafA1 wash-out (lane 3). Immunoprecipitation (IP) of ATZ polymers with polymer-specific 2C1 antibody, transfer on PVDF membrane, revealed with anti-HA antibody on western blot (WB). F Quantification of E, n=3, mean ± SEM. Unpaired two-tailed t-test, ns P>0.05, * P<0.05. Data information: Scale bars: 10 μm.


G - K Same as B in WT MEF , in cells exposed to 20 mM CST and in Cnx - , Crt - and ERp57 - KO MEF . L Quantification of ATZ - positive EL ( n = 10 , 9 , 11 , 10 , 11 cells , respectively ) . One - way ANOVA and Dunnett ' s multiple comparisons test , ns P > 0 . 05 , * * * * P < 0 . 0001 . Data information : Scale bars : 10 μm .

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G-K Same as B in WT MEF, in cells exposed to 20 mM CST and in Cnx-, Crt- and ERp57-KO MEF. L Quantification of ATZ-positive EL (n=10, 9, 11, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001. Data information: Scale bars: 10 μm.


A HEK293 cells transfected with empty vector ( lanes 1 , 6 ) , ATZ - HA ( 2 , 7 ) , FAM134B - V5 ( 3 and 8 ) , FAM134B - V5 and ATZ - HA ( 4 , 9 ) , or FAM134BLIR - V5 and ATZ - HA ( 5 , 10 ) , incubated for 12 h with 50 nM BafA1 and then treated with the cross - linker DSP before lysis as described in Methods . Lanes 1 - 5 , WB of the total cell extract ( TCE ) ; lanes 6 - 10 , WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR . The membranes were probed with antiV5 ( upper panels ) , antiCNX , antiHA and antiLC3 antibodies . B Quantification of LC3 co - precipitating with FAM134B - V5 ( A , lanes 8 and 9 ) . Mean ± SEM , n = 3 , unpaired two - tailed t - test , * P < 0 . 05 .

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A HEK293 cells transfected with empty vector (lanes 1, 6), ATZ-HA (2, 7), FAM134B-V5 (3 and 8), FAM134B-V5 and ATZ-HA (4, 9), or FAM134BLIR-V5 and ATZ-HA (5, 10), incubated for 12 h with 50 nM BafA1 and then treated with the cross-linker DSP before lysis as described in Methods. Lanes 1-5, WB of the total cell extract (TCE); lanes 6-10, WB of antiV5 immunoprecipites to isolate complexes containing ectopically expressed FAM134B or FAM134BLIR. The membranes were probed with antiV5 (upper panels), antiCNX, antiHA and antiLC3 antibodies. B Quantification of LC3 co-precipitating with FAM134B-V5 (A, lanes 8 and 9). Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05.


WT MEF . Scale bar : 10 μm . D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes . Scale bar : 10 μm .

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WT MEF. Scale bar: 10 μm. D Ectopic expression of FAM134BLIR in WT MEF inhibits ATZ delivery to endolysosomes. Scale bar: 10 μm.


E , F Distribution of gold - labeled ATZ - HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR , respectively . EV , ER - derived vesicles , red arrowheads ; EL , endolysosome . G Quantification of ATZ - gold density of E , F ( n = 75 and 79 EL , respectively ) . Unpaired two - tailed t - test , * * * * P < 0 . 0001 .

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E, F Distribution of gold-labeled ATZ-HA by IEM in BafA1 treated WT MEF and WT MEF overexpressing FAM134BLIR, respectively. EV, ER-derived vesicles, red arrowheads; EL, endolysosome. G Quantification of ATZ-gold density of E, F (n=75 and 79 EL, respectively). Unpaired two-tailed t-test, **** P <0.0001.


H Max projection of the same cell as in D after deconvolution . Insets show orthogonal section of select regions . Scale bar : 10 μm .

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H Max projection of the same cell as in D after deconvolution. Insets show orthogonal section of select regions. Scale bar: 10 μm.


I Decay of ATZ polymers ( CHX chase , upper panel ) immunoisolated with the polymer - specific 2C1 antibody ( visualized with antiHA in WB ) in HEK293 cells mock - transfected ( lanes 1 - 4 ) or expressing FAM134BLIR - V5 ( lanes 5 - 8 ) . Middle panel , expression of FAM134BLIR - V5 assessed by WB ; lower panel , loading control . ( J ) Quantification of ( I ) ( Mean ± SEM , n = 3 , unpaired two - tailed t - test , * P < 0 . 05 ) .

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I Decay of ATZ polymers (CHX chase, upper panel) immunoisolated with the polymer-specific 2C1 antibody (visualized with antiHA in WB) in HEK293 cells mock-transfected (lanes 1-4) or expressing FAM134BLIR-V5 (lanes 5-8). Middle panel, expression of FAM134BLIR-V5 assessed by WB; lower panel, loading control. (J) Quantification of (I) (Mean ± SEM, n=3, unpaired two-tailed t-test, * P<0.05).


K Flow cytometry analysis of ATZ - HA polymer levels in MEFs mock treated , exposed to BafA1 , and co - expressing FAM134LIR . MFI : mean fluorescence intensity ( Mean ± SEM , n = 5 , unpaired two - tailed t - test , ns P > 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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K Flow cytometry analysis of ATZ-HA polymer levels in MEFs mock treated, exposed to BafA1, and co-expressing FAM134LIR. MFI: mean fluorescence intensity (Mean ± SEM, n=5, unpaired two-tailed t-test, ns P>0.05, ** P<0.01, *** P <0.001).


A WB analysis showing KO efficiency for CRISPR134B MEF .

A WB analysis showing KO efficiency for CRISPR134B MEF.


WB analysis showing KO efficiency for CRISPR62 MEF .

WB analysis showing KO efficiency for CRISPR62 MEF.


C - E CRISPR WT MEF , CRISPR134B MEF , CRISPR62 MEF , respectively . Scale bars : 10 μm . F Quantification of C - E ( n = 10 , 8 , 7 cells , respectively ) . One - way ANOVA and Dunnett ' s multiple comparisons test , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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C-E CRISPR WT MEF, CRISPR134B MEF, CRISPR62 MEF, respectively. Scale bars: 10 μm. F Quantification of C-E (n=10, 8, 7 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.


A - H Intracellular localization of LAMP1 - positive EL , total ( HA ) and polymeric ( 2C1 ) ATZ in WT MEF exposed to BafA1 for 12 h ( A ) ; in Atg4BKO MEF ( B ) ; in Atg7KO MEF ( C ) ; in Fip200KO MEF ( D ) ; in Ulk1 / 2 double - KO MEF ( E ) ; in Atg13KO MEF ( F ) ; in Atg9KO MEF ( G ) ; in RubiconKO MEF ( H ) . Scale bars : 10 μm . I Quantifications of A - H ( n = 10 , 9 , 9 , 10 , 7 , 15 , 9 , 12 cells ) . One - way ANOVA and Dunnett ' s multiple comparisons test , ns P > 0 . 05 , * * * * P < 0 . 0001 .

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A-H Intracellular localization of LAMP1-positive EL, total (HA) and polymeric (2C1) ATZ in WT MEF exposed to BafA1 for 12 h (A); in Atg4BKO MEF (B); in Atg7KO MEF (C); in Fip200KO MEF (D); in Ulk1/2 double-KO MEF (E); in Atg13KO MEF (F); in Atg9KO MEF (G); in RubiconKO MEF (H). Scale bars: 10 μm. I Quantifications of A-H (n=10, 9, 9, 10, 7, 15, 9, 12 cells). One-way ANOVA and Dunnett's multiple comparisons test, ns P>0.05, **** P<0.0001.


A Distribution of gold - labeled ATZ - HA by IEM in WT MEF exposed to BafA1 ( the cell on the right is not transfected , as specificity control for gold labeling ) . ER , endoplasmic reticulum ; EV , ER - derived vesicle ; EL , endolysosomes . Scale bar : 1 μm . B Inset of A . Red asterisks show site of EV : EL membranes fusion

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A Distribution of gold-labeled ATZ-HA by IEM in WT MEF exposed to BafA1 (the cell on the right is not transfected, as specificity control for gold labeling). ER, endoplasmic reticulum; EV, ER-derived vesicle; EL, endolysosomes. Scale bar: 1 μm. B Inset of A. Red asterisks show site of EV:EL membranes fusion


C - E Distribution of gold - labeled ATZ - HA by IEM in WT MEF . ES , ER subdomains . Red asterisk shows site of EV : EL membranes fusion . Scale bar : 1 μm .

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C-E Distribution of gold-labeled ATZ-HA by IEM in WT MEF. ES, ER subdomains. Red asterisk shows site of EV:EL membranes fusion. Scale bar: 1 μm.


F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ - HA ( visualized as white dots ES , ER subdomains . Scale bar : 500 nm .

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F 3D visualization by means of electron tomography of organelles containing gold labeled ATZ-HA (visualized as white dots ES, ER subdomains. Scale bar: 500 nm.


G CLEM showing ATZ ( red ) , LAMP1 - positive EL ( cyan ) and GFP - derived fluorescence ( green ) in correspondence to heavily labeled vesicles , which were identified as autophagosomes for their double membrane ( insets 1 - 3 ) . GFP - LC3 was also stained by immunogold . Scale bar : 1 μm .

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G CLEM showing ATZ (red), LAMP1-positive EL (cyan) and GFP-derived fluorescence (green) in correspondence to heavily labeled vesicles, which were identified as autophagosomes for their double membrane (insets 1-3). GFP-LC3 was also stained by immunogold. Scale bar: 1 μm.


A , B Triple staining of cells with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) and the luminal ER marker sfGFP - KDEL ( green ) .

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A, B Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and the luminal ER marker sfGFP-KDEL (green).


Triple staining of cells with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) and with CNX as ER membranes marker .

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Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CNX as ER membranes marker.


Triple staining of cells with LAMP1 ( cyan ) , HaloTag - ATZ ( red ) and with CLIMP63 as ER membranes marker .

Triple staining of cells with LAMP1 (cyan), HaloTag-ATZ (red) and with CLIMP63 as ER membranes marker.


G Quantification of ER markers and HaloTag - ATZ co - localization in time ( sfGFP - KDEL n = 10 , 10 , 12 , 13 , CNX n = 8 , 10 , 9 , 8 , CLIMP63 n = 8 , 10 , 6 , 10 cells for corresponding time points ) . Mean ± SEM , unpaired two - tailed t - test , * * * * P < 0 . 0001 . Data information : Scale bars 10 μm .

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G Quantification of ER markers and HaloTag-ATZ co-localization in time (sfGFP-KDEL n=10, 10, 12, 13, CNX n=8, 10, 9, 8, CLIMP63 n=8, 10, 6, 10 cells for corresponding time points). Mean ± SEM, unpaired two-tailed t-test, **** P<0.0001. Data information: Scale bars 10 μm.


H Quantification of double - positive endolysosomes at 300 ' ( ATZ : sfGFP - KDEL n = 14 , ATZ : CNX n = 8 , ATZ : CLIMP63 n = 10 cells . One - way ANOVA and Dunnett ' s multiple comparisons test , * * P < 0 . 01 * * * * P < 0 . 0001 ) . Data information : Scale bars 10 μm .

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

H Quantification of double-positive endolysosomes at 300' (ATZ:sfGFP-KDEL n=14, ATZ:CNX n=8, ATZ:CLIMP63 n=10 cells. One-way ANOVA and Dunnett's multiple comparisons test, ** P<0.01 **** P<0.0001). Data information: Scale bars 10 μm.


C - E CLSM analysis of ATZ delivery to LAMP1 - positive endolysosomes in BafA1 - treated CRISPRWT MEF ( C ) ; in CRISPR17 MEF ( D ) ; in CRISPR8 MEF ( E ) . Scale bars : 10 μm .

O O O O O O O O O O O O O O O O O O O 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-E CLSM analysis of ATZ delivery to LAMP1-positive endolysosomes in BafA1-treated CRISPRWT MEF (C); in CRISPR17 MEF(D); in CRISPR8 MEF (E). Scale bars: 10 μm.


Quantification ( n = 10 , 10 , 11 cells , respectively ) . One - way ANOVA and Dunnett ' s multiple comparisons test , * * * * P < 0 . 0001 .

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Quantification (n=10, 10, 11 cells, respectively). One-way ANOVA and Dunnett's multiple comparisons test, **** P<0.0001.


G - I Distribution of gold - labelled ATZ - HA by IEM in CRISPRWT MEF exposed to BafA1 ( G ) ; in CRISPR17 ( H ) ; in CRISPR8 . ( I ) . ER , endoplasmic reticulum ; EV , ER - derived vesicles ; EL , endolysosomes . Scale bars : 1 μm .

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

G-I Distribution of gold-labelled ATZ-HA by IEM in CRISPRWT MEF exposed to BafA1 (G); in CRISPR17 (H); in CRISPR8. (I). ER, endoplasmic reticulum; EV, ER-derived vesicles; EL, endolysosomes. Scale bars: 1 μm.


GFP reconstitution in ATZ - HA transfected CRISPR17 MEF expressing FAM134B - T11 and GFP1 - 10 - LC3 . Scale bar : 10 μm . K Same as J by CLEM . ATZ - HA staining in red , LAMP1 - positive endolysosomes in cyan . FAM134B : LC - induce GFP reconstitution in green . FAM134B in gold is shown . Scale bar : 1 μm .

GFP reconstitution in ATZ-HA transfected CRISPR17 MEF expressing FAM134B-T11 and GFP1-10-LC3. Scale bar: 10 μm. K Same as J by CLEM. ATZ-HA staining in red, LAMP1-positive endolysosomes in cyan. FAM134B:LC-induce GFP reconstitution in green. FAM134B in gold is shown. Scale bar: 1 μm.


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

E. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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 O O O O O O 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. 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 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

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 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

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 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 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 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 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

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

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

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

B. TBK1 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 O O O O O O O O O O O O O O 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. 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O

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

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

B. 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 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 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 O O O O O O O O O O O O O


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

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

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

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 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 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 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 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 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 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 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours . Scale bar : 10µm Morphological parameters quantification within 48 hours after LPS

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Representative 3D microglia reconstructions showing morphological changes upon LPS injection within the first 48 hours. Scale bar: 10µm Morphological parameters quantification within 48 hours after LPS injection


Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection . Scale bar :

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Representative 3D microglia reconstructions from 5 and 15 months old mice showing microglia changes 2 and 10 days post LPS injection. Scale bar: 20µm


Morphological parameters quantification for 5 and 15 months old mice after LPS injection


Two - photon representative images of wild - type and Nlrp3 - / - mice ( 5 and 15 months old ) . Scale bar : 20µm . Quantification of morphological parameters for wild - type and Nlpr3 - / - mice after LPS

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Two-photon representative images of wild-type and Nlrp3-/- mice (5 and 15 months old). Scale bar: 20µm. Quantification of morphological parameters for wild-type and Nlpr3-/- mice after LPS injection


Representative cortical images of MXO4 staining for APP and APP / Nlrp3 - / - 15 months old mice . Scale bar : 50µm .

Representative cortical images of MXO4 staining for APP and APP/Nlrp3-/- 15 months old mice. Scale bar: 50µm.


Cortical amyloid plaque number and size quantification , and Amyloid - beta1 - 40 and 1 - 42 ELISA quantification for APP and APP / Nlrp3 - / - mice ( 5 and 15 month old )

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Cortical amyloid plaque number and size quantification, and Amyloid-beta1-40 and 1-42 ELISA quantification for APP and APP/Nlrp3-/- mice (5 and 15 month old)


Two - photon images of microglia ( e - GFP ) cells clustering around amyloid plaque for APP and APP / Nlrp3 - / - ( 5 and 15 months old ) . Scale bar : 20µm b ) Quantification of morphological parameters in ( a ) ( mean of 5 - 6 ± SEM ) .

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Two-photon images of microglia (e-GFP) cells clustering around amyloid plaque for APP and APP/Nlrp3-/- (5 and 15 months old). Scale bar: 20µm b) Quantification of morphological parameters in (a) (mean of 5-6±SEM).


Flow cytometry plots from APP and APP / Nlrp3 - / - mice ( 15 months old ) , cells were gated on CD11b and MXO4 after microglia isolation . d ) Relative Aβ microglia uptake

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Flow cytometry plots from APP and APP/Nlrp3-/- mice (15 months old), cells were gated on CD11b and MXO4 after microglia isolation. d) Relative Aβ microglia uptake quantification


Two - photon microglia ( e - GFP ) images from APP and APP / Nlrp3 - / - in areas free of plaque ( 5 and 15 months old mice ) . Scale bar : 20µm Quantification of APP and APP / Nlrp3 - / - morphological parameters for microglia in areas free of

Two-photon microglia (e-GFP) images from APP and APP/Nlrp3-/- in areas free of plaque (5 and 15 months old mice). Scale bar: 20µm Quantification of APP and APP/Nlrp3-/- morphological parameters for microglia in areas free of plaque


Iba - 1 ( green ) , CD169 ( red ) and MXO4 staining in plaque - associated areas ( cortex and hippocampus ) of 5 and 15 months old of APP and APP / Nlrp3 - / - . Note the colocalization between Iba - 1 and CD169 ( white arrows ) in APP 15 - month - old mice 2 days post LPS injection . Scale bar :

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Iba-1 (green), CD169 (red) and MXO4 staining in plaque-associated areas (cortex and hippocampus) of 5 and 15 months old of APP and APP/Nlrp3-/-. Note the colocalization between Iba-1 and CD169 (white arrows) in APP 15-month-old mice 2 days post LPS injection. Scale bar: 20µm


Iba - 1 , Ki67 and DAPI staining in the cortex and hippocampus of wild - type and Nlrp3 - / - ( 5 and 15 months old ) . Microglia proliferates upon LPS injection ( white arrows ) . Non - microglia cells proliferation was observed as well ( yellow arrows ) Scale bar : 20µm . Quantification of microglial proliferation in cortex ( left panel ) and hippocampus ( right panel ) ( mean of 5 ± SEM ; two - way ANOVA followed by Tukey ' s post hoc test , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) .

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Iba-1, Ki67 and DAPI staining in the cortex and hippocampus of wild-type and Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows) Scale bar: 20µm. Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel) (mean of 5±SEM; two-way ANOVA followed by Tukey's post hoc test, *p<0.05, **p<0.01, ***p<0.001).


Iba - 1 , Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP / Nlrp3 - / - ( 5 and 15 months old ) . Microglia proliferates upon LPS injection ( white arrows ) . Non - microglia cells proliferation was observed as well ( yellow arrows ) . Scale bar : 20µm Quantification of microglial proliferation in cortex ( left panel ) and hippocampus ( right panel )

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Iba-1, Ki67 and MXO4 staining in the cortex and hippocampus of APP and APP/Nlrp3-/- (5 and 15 months old). Microglia proliferates upon LPS injection (white arrows). Non-microglia cells proliferation was observed as well (yellow arrows). Scale bar: 20µm Quantification of microglial proliferation in cortex (left panel) and hippocampus (right panel)


( C ) Relative mRNA expression from real - time PCR analysis of the GnRH transcript in GFP - positive cells , in comparison with GFP - negative cells . The GnRH transcript appears to be selectively expressed in GFP - positive cells . Mann - Whitney U test , n = 4 mice from 2 litters .

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(C) Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann-Whitney U test, n=4 mice from 2 litters.


( D ) Relative mRNA expression from real - time PCR analysis of Neuropilin - 1 ( Nrp1 ) , Nrp2 , Plexin A1 ( PlxnA1 ) , and PlxnA4 transcripts in FACS - isolated GFP - positive GnRH neurons from control ( Nrp1loxP / loxP ; grey ) and mutant ( Gnrh : : cre ; Nrp1loxP / loxP ; red ) P0 mice . Unpaired t - test , n = 5 to 6 mice per group from 3 litters .

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(D) Relative mRNA expression from real-time PCR analysis of Neuropilin-1 (Nrp1), Nrp2, Plexin A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; grey) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n=5 to 6 mice per group from 3 litters.


( E ) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1 - immunoreactive vomeronasal / terminal nerve fibers ( empty arrowheads ) at the nose - brain junction in sagittal slices from E14 . 5 control and mutant embryos . GnRH neurons ( green ) themselves express Nrp1 ( red , white arrows ) in control Nrp1loxP / loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh : : cre ; Nrp1loxP / loxP littermates ( black arrows ) . Scale bar : 50μm .

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

(E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.


( A ) Quantification of total number of GnRH neurons during embryogenesis , at E12 . 5 , E14 . 5 and E18 . 5 . More GnRH neurons were found in E14 . 5 and E18 . 5 mutant embryos . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 4 to 6 mouse embryos from at least 3 litters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5 and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 to 6 mouse embryos from at least 3 litters.