F , immunoblotting of NPC1L1 in MCs , MPCs and RCs of Du145TXR or MCF - 7ADR . G , immunoblotting of NPC1L1 in Du145TXR or MCF - 7ADR cells transfected with siNPC1L1 or siScramble .

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F, immunoblotting of NPC1L1 in MCs, MPCs and RCs of Du145TXR or MCF-7ADR. G, immunoblotting of NPC1L1 in Du145TXR or MCF-7ADR cells transfected with siNPC1L1 or siScramble.


H - I , colony formation assay and quantification of ( H ) Du145TXR or ( I ) MCF - 7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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H-I, colony formation assay and quantification of (H) Du145TXR or (I) MCF-7ADR cells transfected with siNPC1L1 or siScramble followed by treatment with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


A - C , flow cytometric analysis of ROS levels in Du145TXR or MCF - 7ADR cells treated with indicated agents for 12 and 36 hours . ( A ) Representative images and quantification of ROS in ( B ) Du145TXR or ( C ) MCF - 7ADR cells are shown . One - way ANOVA was used to analyze statistical differences . Mean with ± SD Data information : Results are representative of three independent experiments .

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

A-C, flow cytometric analysis of ROS levels in Du145TXR or MCF-7ADR cells treated with indicated agents for 12 and 36 hours. (A) Representative images and quantification of ROS in (B) Du145TXR or (C) MCF-7ADR cells are shown. One-way ANOVA was used to analyze statistical differences. Mean with ± SD Data information: Results are representative of three independent experiments.


D - E , cell viability of ( D ) Du145TXR or ( E ) MCF - 7ADR treated with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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D-E, cell viability of (D) Du145TXR or (E) MCF-7ADR treated with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


J - K , cell viability of ( J ) Du145TXR or ( K ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours . One - way ANOVA was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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

J-K, cell viability of (J) Du145TXR or (K) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 72 hours. One-way ANOVA was used to analyze statistical differences. Mean with ± SD . Data information: Results are representative of three independent experiments.


C - D , endocytosis analysis of ( C ) Du145TXR or ( D ) MCF - 7ADR cells treated with indicated agents for 8 hours , followed by staining with 250 μg / mL FITC - DEX for 4 hours . Scale bars , 10 μm . Data information : Results are representative of three independent experiments .

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

C-D, endocytosis analysis of (C) Du145TXR or (D) MCF-7ADR cells treated with indicated agents for 8 hours, followed by staining with 250 μg/mL FITC-DEX for 4 hours. Scale bars, 10 μm. Data information: Results are representative of three independent experiments.


E - F , phase contrast images of the morphological changes in ( E ) Du145TXR and ( F ) MCF - 7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours . Scale bars , 10 or 25 μm . The red arrows indicate vacuoles . Data information : Results are representative of three independent experiments .

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

E-F, phase contrast images of the morphological changes in (E) Du145TXR and (F) MCF-7ADR cells cultured in normal or serum free medium followed by treatment with indicated agents for 12 hours. Scale bars, 10 or 25 μm. The red arrows indicate vacuoles. Data information: Results are representative of three independent experiments.


I - J , colony formation assay and quantification of ( I ) Du145TXR or ( J ) MCF - 7ADR cells treated with indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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

I-J, colony formation assay and quantification of (I) Du145TXR or (J) MCF-7ADR cells treated with indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


C , immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours . Data information : Results are representative of three independent experiments .

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C, immunoblot analysis of NPC1L1 and NRF2 in Du145TXR or MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents for 24 hours. Data information: Results are representative of three independent experiments.


F - G , relative luciferase activities of wild - type ( WT ) or mutant ( MUT ) NPC1L1 promoter ( - 205 ~ - 215 , # 1 ) reporter constructs in ( F ) Du145TXR or ( G ) MCF - 7ADR cells treated with indicated agents . Student ' s t test was used to analyze statistical differences . Mean with ± SD . Data information : Results are representative of three independent experiments .

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

F-G, relative luciferase activities of wild-type (WT) or mutant (MUT) NPC1L1 promoter (-205~-215, #1) reporter constructs in (F) Du145TXR or (G) MCF-7ADR cells treated with indicated agents. Student's t test was used to analyze statistical differences. Mean with ± SD. Data information: Results are representative of three independent experiments.


J - K , colony formation assay and quantification of ( J ) Du145TXR or ( K ) MCF - 7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents . One - way ANOVA was used to analyze statistical differences . Data information : Results are representative of three independent experiments .

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

J-K, colony formation assay and quantification of (J) Du145TXR or (K) MCF-7ADR cells transfected with siNFE2L2 or siScramble followed by treatment with the indicated agents. One-way ANOVA was used to analyze statistical differences. Data information: Results are representative of three independent experiments.


immunohistochemical staining of Ki67 in indicated groups . ( D ) Representative images are shown . Scale bar , 50 μm . , immunohistochemical staining of cleaved - caspase 3 ( CC3 ) in indicated groups . ( F ) Representative images are shown . Scale bar , 50 μm .

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immunohistochemical staining of Ki67 in indicated groups. (D) Representative images are shown. Scale bar, 50 μm. , immunohistochemical staining of cleaved-caspase 3 (CC3) in indicated groups. (F) Representative images are shown. Scale bar, 50 μm.


( I ) Corresponding images and isolated tumors are shown . The red dotted line represents cured tumors .

(I) Corresponding images and isolated tumors are shown. The red dotted line represents cured tumors.


A ) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1 . Coimmunoprecipitation ( CoIP ) experiments using TAP - tagged Nrd1 ( either wt or ∆ CID ) as the bait . Representative gel of one out of two independent experiments .

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A) Deletion of the CID domain dramatically reduces the interaction of Nrd1 with Sen1. Coimmunoprecipitation (CoIP) experiments using TAP-tagged Nrd1 (either wt or ∆CID) as the bait. Representative gel of one out of two independent experiments.


C ) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1 . CoIP experiments using Nrd1 - TAP as the bait in a SEN1 or sen1 ∆ NIM background . Representative gel of one out of two independent experiments .

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

C) Deletion of the NIM decreases substantially the association of Nrd1 with Sen1. CoIP experiments using Nrd1-TAP as the bait in a SEN1 or sen1∆NIM background. Representative gel of one out of two independent experiments.


D ) CoIP experiments using HA - tagged Sen1 , either wt or ∆ NIM , as the bait . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA prior to immunoprecipitation . In these experiment , Sen1 could not be detected in the input extracts .

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

D) CoIP experiments using HA-tagged Sen1, either wt or ∆NIM, as the bait. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA prior to immunoprecipitation. In these experiment, Sen1 could not be detected in the input extracts.


A ) Deletion of the N - terminal of Sen1 is lethal . A ∆ sen1 strain ( YDL2767 ) covered by an URA3 - containing plasmid ( pFL38 ) expressing wt Sen1 was transformed with a TRP1 - plasmid ( pFL39 ) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left . After over - night growth in non - selective medium , cells were plated on minimal medium ( CSM ) containing 5 - fluorootic acid ( 5 - FOA ) to select those that have lost the URA3 plasmid . The absence of growth in 5 - FOA implies that the SEN1 version expressed in the TRP1 - plasmid does not support viability .

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

A) Deletion of the N-terminal of Sen1 is lethal. A ∆sen1 strain (YDL2767) covered by an URA3-containing plasmid (pFL38) expressing wt Sen1 was transformed with a TRP1-plasmid (pFL39) carrying either the wt or the mutant versions of SEN1 indicated in the scheme on the left. After over-night growth in non-selective medium, cells were plated on minimal medium (CSM) containing 5-fluorootic acid (5-FOA) to select those that have lost the URA3 plasmid. The absence of growth in 5-FOA implies that the SEN1 version expressed in the TRP1-plasmid does not support viability.


B ) Deletion of the N - terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo . Northern blot analyses of three well - characterized NNS - targets , the CUT NEL025c and the snoRNAs SNR13 and SNR33 , in a Sen1 - AID ( auxin - induced degron ) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1 . Sen1 - AID was depleted for 2h by the addition of 500 μM indole - 3 - acetic acid ( IAA ) to monitor the capacity of the plasmid - borne versions of SEN1 to induce transcription termination . The expected RNA species resulting from inefficient termination ( RT for readthrough species ) are indicated . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

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

B) Deletion of the N-terminal domain of Sen1 provokes dramatic defects in transcription termination in vivo. Northern blot analyses of three well-characterized NNS-targets, the CUT NEL025c and the snoRNAs SNR13 and SNR33, in a Sen1-AID (auxin-induced degron) strain carrying an empty vector or a plasmid expressing either the wt or the indicated versions of SEN1. Sen1-AID was depleted for 2h by the addition of 500 μM indole-3-acetic acid (IAA) to monitor the capacity of the plasmid-borne versions of SEN1 to induce transcription termination. The expected RNA species resulting from inefficient termination (RT for readthrough species) are indicated. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


C ) Overexpression of sen1 ∆ Nter restores viability . Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter ( pGAL ) in the presence of galactose .

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C) Overexpression of sen1∆Nter restores viability. Growth test of strains harboring the indicated version of SEN1 at the endogenous locus under the control of the GAL1 promoter (pGAL) in the presence of galactose.


D ) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N - terminal domain . Northern blot analysis of a typical NNS - target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose . assays were performed in a ∆ rrp6 background to detect the primary products of NNS - dependent termination and the U4 RNA is used as a loading control .

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D) Overexpression of SEN1 partially suppresses the termination defects associated with deletion of the N-terminal domain. Northern blot analysis of a typical NNS-target in strains expressing the indicated SEN1 versions from pGAL in the presence of galactose. assays were performed in a ∆rrp6 background to detect the primary products of NNS-dependent termination and the U4 RNA is used as a loading control.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . A ) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site ( PAS ) of mRNAs . Values on the y axis correspond to the median coverage . B ) Box - plot representation of the readthrough ( RT ) index as a readout of the termination efficiency at each mRNA . The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS , divided by the average signal from the TSS to the PAS of each mRNA . Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR ( where IQR is the interquartile distance or distance between the first and the third quartile ) . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . For each mRNA ( n = 5752 ) , the values correspond to the average of two independent biological replicates . Data populations were compared using a t - test ( bilateral distributions , unpaired data ) and p - values ≥ 0 . 05 are indicated as ns ( not significant ) .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. A) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site (PAS) of mRNAs. Values on the y axis correspond to the median coverage. B) Box-plot representation of the readthrough (RT) index as a readout of the termination efficiency at each mRNA. The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS, divided by the average signal from the TSS to the PAS of each mRNA. Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR (where IQR is the interquartile distance or distance between the first and the third quartile). The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. For each mRNA (n=5752), the values correspond to the average of two independent biological replicates. Data populations were compared using a t-test (bilateral distributions, unpaired data) and p-values≥ 0.05 are indicated as ns (not significant).


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . C - D ) IGV screenshots of two well - characterized protein - coding genes displaying substantial defects in premature termination in some mutants . The values correspond to the counts per million of counts ( CPM ) multiplied by 10 .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. C-D) IGV screenshots of two well-characterized protein-coding genes displaying substantial defects in premature termination in some mutants. The values correspond to the counts per million of counts (CPM) multiplied by 10.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . E ) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site ( TTS ) as the reference point . F ) Analysis of termination defects in the different mutants at CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS , divided by the average signal over a window of 100 nt downstream from the TSS ( CUT100 ) . For each CUT ( n = 329 ) , the values correspond to the average of two independent biological replicates . P - values < 0 . 001 are indicated by three asterisks .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. E) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site (TTS) as the reference point. F) Analysis of termination defects in the different mutants at CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS, divided by the average signal over a window of 100 nt downstream from the TSS (CUT100). For each CUT (n=329), the values correspond to the average of two independent biological replicates. P-values < 0.001 are indicated by three asterisks.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . G - H ) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. G-H) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs . Box - plots performed as in ( B ) . The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS , divided by the average signal over the mature snoRNA .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA.


RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid , upon depletion of the chromosomally - encoded version of Sen1 in a Sen1 - AID strain . Analysis of RNAPII occupancy around snoRNAs performed as for CUTs .

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

RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs.


A ) Deletion of Sen1 N - terminal domain does not prevent the interaction of Sen1 with RNAPII . CoIP experiments using Rbp3 - FLAG as the bait . Assays were performed in a Sen1 - AID strain harboring a plasmid expressing either SEN1 or sen1 ∆ Nter upon depletion of Sen1 - AID in the presence of IAA for 2h . An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1 ∆ Nter . Nrd1 is detected as a positive control . Representative gel of one out of two independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation .

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

A) Deletion of Sen1 N-terminal domain does not prevent the interaction of Sen1 with RNAPII. CoIP experiments using Rbp3-FLAG as the bait. Assays were performed in a Sen1-AID strain harboring a plasmid expressing either SEN1 or sen1∆Nter upon depletion of Sen1-AID in the presence of IAA for 2h. An asterisk denotes a major proteolytic Sen1 fragment detected in the extracts of roughly the size of sen1∆Nter. Nrd1 is detected as a positive control. Representative gel of one out of two independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.


B ) Deletion of the Sen1 N - terminal domain reduces the interaction of Sen1 with the S5P - CTD . CoIP experiments using TAP - Sen1 as the bait . Sen1 proteins were expressed from pGAL in the presence of galactose . Nab3 is detected as a positive control . Representative gel of one out of three independent experiments . Protein extracts were treated with RNaseA before immunoprecipitation .

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B) Deletion of the Sen1 N-terminal domain reduces the interaction of Sen1 with the S5P-CTD. CoIP experiments using TAP-Sen1 as the bait. Sen1 proteins were expressed from pGAL in the presence of galactose. Nab3 is detected as a positive control. Representative gel of one out of three independent experiments. Protein extracts were treated with RNaseA before immunoprecipitation.


C ) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability . Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1 - plasmid carrying the SEN1 versions indicated in the scheme on the left . The growth of the strain expressing the Nrd1 CID - sen1 ∆ Nter chimera in 5 - FOA implies that this gene can support viability .

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C) Replacing the Nter of Sen1 by the CID of Nrd1 restores viability. Growth test performed in the same conditions as in figure 3A but in the presence of a TRP1-plasmid carrying the SEN1 versions indicated in the scheme on the left. The growth of the strain expressing the Nrd1 CID-sen1∆Nter chimera in 5-FOA implies that this gene can support viability.


D ) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1 ∆ Nter mutant . Northern blot assays performed in a Sen1 - AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B . Experiments performed in a ∆ rrp6 background . Representative gel of one out of two independent experiments . The U4 RNA is used as a loading control .

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D) Substituting the Nter of Sen1 by Nrd1 CID but not Pcf11 CID partially suppresses the termination defects detected in the sen1∆Nter mutant. Northern blot assays performed in a Sen1-AID strain carrying an empty vector or a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in figure 3B. Experiments performed in a ∆rrp6 background. Representative gel of one out of two independent experiments.The U4 RNA is used as a loading control.


E ) Sen1 Nter interacts with the C - terminal domain ( Cter ) of Sen1 both in the presence and in the absence of the NIM in vitro . Pull - down experiments using either a wt or a ∆ NIM version of recombinant Sen1 Cter immobilized on glutathione - sepharose beads and a TAP - tagged version of Sen1 Nter expressed in yeast . Representative gel of one out of three independent experiments .

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E) Sen1 Nter interacts with the C-terminal domain (Cter) of Sen1 both in the presence and in the absence of the NIM in vitro. Pull-down experiments using either a wt or a ∆NIM version of recombinant Sen1 Cter immobilized on glutathione-sepharose beads and a TAP-tagged version of Sen1 Nter expressed in yeast. Representative gel of one out of three independent experiments.


A - C ) Comparison of the distribution of Sen1 and RNAPII ( Rpb1 ) at mRNAs and CUTs . Values on the y axis correspond to the median coverage . Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot .

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A-C) Comparison of the distribution of Sen1 and RNAPII (Rpb1) at mRNAs and CUTs. Values on the y axis correspond to the median coverage. Note that the scale of Sen1 and Rpb1 datasets have been modified to be able to visualize both in the same plot.


D - F ) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots .

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D-F) IGV screenshots of two mRNAs and one CUT exemplifying the trend observed in the metagene plots.


G ) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point . Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background .

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G) Metagene analysis of the distribution of the different Sen1 variants at mRNAs using either the TSS or the PAS as reference point. Values correspond to the median of Sen1 data normalized by the RNAPII signal obtained in the corresponding background.


H - I ) Screenshots of two well - characterized protein - coding genes that are regulated by NNS - dependent premature termination . Values correspond to unnormalized Sen1 occupancy .

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H-I) Screenshots of two well-characterized protein-coding genes that are regulated by NNS-dependent premature termination. Values correspond to unnormalized Sen1 occupancy.


J ) Profile of Sen1 binding at CUTs calculated as for mRNAs .

J) Profile of Sen1 binding at CUTs calculated as for mRNAs.


K - L ) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter . Plots correspond to unnormalized data .

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K-L) Examples of CUTs exhibiting differences in Sen1 occupancy upon deletion of the NIM or the Nter. Plots correspond to unnormalized data.


M - O ) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs .

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M-O) Analysis of the distribution of the different Sen1 versions around snoRNAs performed as for CUTs.


P ) Comparison of the total levels of the different Sen1 variants at mRNAs , CUTs and the termination region of snoRNAs ( i . e . a window of 200 nt downstream of the 3 ' end of the mature snoRNA , snoRNA - ter ) . Boxes in box - plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median . The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1 . 5 * IQR . The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1 . 5 * IQR . Datasets for the different Sen1 versions were compared using a t - test ( bilateral distributions , unpaired data ) . p - values ≥ 0 . 05 are indicated as ns ( not significant ) , one asterisk is used when 0 . 05 > p - value ≥ 0 . 01 , two asterisks when 0 . 01 > p - value ≥ 0 . 001 and three asterisks for p - value < 0 . 001 .

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P) Comparison of the total levels of the different Sen1 variants at mRNAs, CUTs and the termination region of snoRNAs (i.e. a window of 200 nt downstream of the 3' end of the mature snoRNA, snoRNA-ter). Boxes in box-plots include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR. The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. Datasets for the different Sen1 versions were compared using a t-test (bilateral distributions, unpaired data). p-values≥ 0.05 are indicated as ns (not significant), one asterisk is used when 0.05>p-value≥0.01, two asterisks when 0.01>p-value≥0.001 and three asterisks for p-value <0.001.


A ) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter . Northern blot assays performed in a Sen1 - AID , ∆ rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments . Where indicated , cells were treated with 50 mg / L of 6 - azauracile ( 6AU ) for 2h . Representative gel of one out of two independent biological replicates . The U4 RNA is used as a loading control .

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A) Decreasing the elongation rate alleviates the termination defects associated with deletion of Sen1 Nter. Northern blot assays performed in a Sen1-AID, ∆rrp6 strain carrying a plasmid expressing the indicated versions of SEN1 upon depletion of the endogenous Sen1 protein as in former experiments. Where indicated, cells were treated with 50 mg/L of 6-azauracile (6AU) for 2h. Representative gel of one out of two independent biological replicates.The U4 RNA is used as a loading control.


C . Cathepsin activity assay in WT and Tpl2 [ D270A ] BMDMs 0 . 5 h after uptake of latex beads . BMDMs were stained with the Magic Red cathepsin L substrate ( red ) . Average fluorescence intensity of the cathepsin probe per cell was quantified ( n = 40 - 51 cells ) . E . pH assay in WT and Tpl2 [ D270A ] BMDMs upon 0 . 5 h after incubation with latex beads . BMDMs were stained with the LysoTracker Red DND - 99 dye ( red ) . Average fluorescence intensity of the Lysotracker Red probe per cell was quantified ( n = 95 - 126 cells ) . Data information : One representative experiment out of three shown . Error bars and shaded areas represent SEM . * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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C. Cathepsin activity assay in WT and Tpl2[D270A] BMDMs 0.5 h after uptake of latex beads. BMDMs were stained with the Magic Red cathepsin L substrate (red). Average fluorescence intensity of the cathepsin probe per cell was quantified (n = 40-51 cells). E. pH assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the LysoTracker Red DND-99 dye (red). Average fluorescence intensity of the Lysotracker Red probe per cell was quantified (n = 95-126 cells). Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.


G . Reactive oxygen species assay in WT and Tpl2 [ D270A ] BMDMs upon 0 . 5 h after incubation with latex beads . BMDMs were stained with the ROS Deep Red dye . Average fluorescence intensity of the ROS Deep Red probe per cell was quantified ( n = 80 - 110 cells ) Data information : One representative experiment out of three shown . Error bars and shaded areas represent SEM . * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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G. Reactive oxygen species assay in WT and Tpl2[D270A] BMDMs upon 0.5 h after incubation with latex beads. BMDMs were stained with the ROS Deep Red dye. Average fluorescence intensity of the ROS Deep Red probe per cell was quantified (n = 80-110 cells) Data information: One representative experiment out of three shown. Error bars and shaded areas represent SEM. **** P < 0.0001. Student's unpaired t-test.


Experiments were performed using murine BMDMs . ( C ) Cell extracts from LPS - stimulated Nfkb1 [ SSAA ] and Nfkb1 [ SSAA ] / Tpl2 [ D270A ] BMDMs were immunoblotted for the indicated antigens .

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Experiments were performed using murine BMDMs. (C) Cell extracts from LPS-stimulated Nfkb1[SSAA] and Nfkb1[SSAA]/Tpl2[D270A] BMDMs were immunoblotted for the indicated antigens.


Experiments were performed using human primary monocyte - derived macrophages . ( K ) Cell extracts from LPS - stimulated , inhibitor - treated primary human macrophages were immunoblotted for p - ERK1 / 2 , ERK1 / 2 , p - p38 , p38 and HSP90 .

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Experiments were performed using human primary monocyte-derived macrophages. (K) Cell extracts from LPS-stimulated, inhibitor-treated primary human macrophages were immunoblotted for p-ERK1/2, ERK1/2, p-p38, p38 and HSP90.


C . Protein intensities of V - ATPase subunits from phagosomes purified from WT and Tpl2 [ D270A ] BMDMs , ( n = 3 biological replicates ) . Data information : Data were analysed by Student ' s t - test . Error bars represent SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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C. Protein intensities of V-ATPase subunits from phagosomes purified from WT and Tpl2[D270A] BMDMs, (n = 3 biological replicates). Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.


D . Protein intensities of RAB5 and LAMP - 1 from phagosome proteome analysis of WT and Tpl2 [ D270A ] BMDMs ( n = 3 biological replicates ) ( left ) . Immunoblot of isolated phagosomes from WT and Tpl2 [ D270A ] BMDMs probed for RAB5 , LAMP - 1 and vimentin . Phagosomal fractions of two biological replicates were pooled . One representative experiment out of two shown ( right ) ( n = 2 ) . Data information : Data were analysed by Student ' s t - test . Error bars represent SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D. Protein intensities of RAB5 and LAMP-1 from phagosome proteome analysis of WT and Tpl2[D270A] BMDMs (n = 3 biological replicates) (left). Immunoblot of isolated phagosomes from WT and Tpl2[D270A] BMDMs probed for RAB5, LAMP-1 and vimentin. Phagosomal fractions of two biological replicates were pooled. One representative experiment out of two shown (right) (n = 2). Data information: Data were analysed by Student's t-test. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.


A . TPL - 2 - dependent phosphoproteome following phagocytosis of latex beads ( 0 . 5 h ) was determined by TMT mass spectrometry . Volcano plot representing the significance ( - log10 P - values after Welch ' s t - test ) vs . phosphorylation fold change ( Welch difference ratios ) between WT and Tpl2 [ D270A ] BMDMs . Five biological replicates were analysed per genotype ( n = 5 ) . Three of the most highly and significantly downregulated phospho - sites in Tpl2 [ D270A ] BMDMs relative to WT , as well as unaltered DMXL1 phospho - sites , are shown .

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A. TPL-2-dependent phosphoproteome following phagocytosis of latex beads (0.5 h) was determined by TMT mass spectrometry. Volcano plot representing the significance (-log10 P-values after Welch's t-test) vs. phosphorylation fold change (Welch difference ratios) between WT and Tpl2[D270A] BMDMs. Five biological replicates were analysed per genotype (n = 5). Three of the most highly and significantly downregulated phospho-sites in Tpl2[D270A] BMDMs relative to WT, as well as unaltered DMXL1 phospho-sites, are shown.


BMDMs of the indicated genotypes were infected with YFP - S . aureus ( MOI 10 ) for 1 h . ( B ) Representative images of YFP + BMDMs labelled with Magic Red cathepsin L substrate ( red ) and DAPI ( nuclear stain ) 1h post - infection . ( C ) Quantification of Magic Red cathepsin L staining from panel ( A ) . Average fluorescence intensity of Magic Red cathepsin L substrate per cell relative to fluorescence intensity of YFP - S . aureus ( n = 26 - 29 cells ) . ( D ) Quantification of Magic Red cathepsin L staining co - localised with YFP - S . aureus from panel ( A ) ( n = 26 - 29 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . P < 0 . 01 , * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (B) Representative images of YFP+ BMDMs labelled with Magic Red cathepsin L substrate (red) and DAPI (nuclear stain) 1h post-infection. (C) Quantification of Magic Red cathepsin L staining from panel (A). Average fluorescence intensity of Magic Red cathepsin L substrate per cell relative to fluorescence intensity of YFP-S.aureus (n = 26-29 cells). (D) Quantification of Magic Red cathepsin L staining co-localised with YFP-S. aureus from panel (A) (n = 26-29 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.


BMDMs of the indicated genotypes were infected with YFP - S . aureus ( MOI 10 ) for 1 h . ( E ) Representative images of YFP + BMDMs labelled with LysoTracker Red DND - 99 dye ( red ) and DAPI , 1h post - infection with YFP - S . aureus ( green ) . ( F ) Quantification of LysoTracker Red signal from panel ( D ) . Average fluorescence intensity of LysoTracker Red per cell relative to fluorescence intensity of YFP - S . aureus ( n = 66 - 98 cells ) . ( G ) Quantification of LysoTracker Red co - localisation with YFP - S . aureus from panel ( D ) ( n = 66 - 98 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . P < 0 . 01 , * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (E) Representative images of YFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with YFP-S. aureus (green).(F) Quantification of LysoTracker Red signal from panel (D). Average fluorescence intensity of LysoTracker Red per cell relative to fluorescence intensity of YFP-S. aureus (n = 66-98 cells). (G) Quantification of LysoTracker Red co-localisation with YFP-S. aureus from panel (D) (n = 66-98 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.


BMDMs of the indicated genotypes were infected with YFP - S . aureus ( MOI 10 ) for 1 h . ( H ) Representative images of BMDMs stained with an anti - EEA1 antibody ( red ) and DAPI , 1h post - infection with YFP - S . aureus ( green ) . ( I ) Quantification of EEA1 + S . aureus - containing phagosomes from panel ( G ) ( n = 27 cells ) . ( J ) Representative images of BMDMs stained with an anti - LAMP - 1 antibody ( red ) and DAPI , 1h post - infection with YFP - S . aureus ( green ) . ( K ) Quantification of LAMP - 1 + S . aureus phagosomes from panel ( I ) ( n = 25 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . P < 0 . 01 , * * * * P < 0 . 0001 . Student ' s unpaired t - test .

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BMDMs of the indicated genotypes were infected with YFP-S. aureus (MOI 10) for 1 h. (H) Representative images of BMDMs stained with an anti-EEA1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (I) Quantification of EEA1+ S. aureus-containing phagosomes from panel (G) (n = 27 cells). (J) Representative images of BMDMs stained with an anti-LAMP-1 antibody (red) and DAPI, 1h post-infection with YFP-S. aureus (green). (K) Quantification of LAMP-1+ S. aureus phagosomes from panel (I) (n = 25 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. P < 0.01, **** P < 0.0001. Student's unpaired t-test.


E . BMDMs of the indicated genotypes were infected with GFP - C . rodentium ( MOI 2 ) for 0 . 5h . C . rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0 . 5 h ( n = 12 ; 2 biological and 6 technical replicates per condition per experiment ) . As a control , WT cells were treated with bafilomycin A . Data information : One representative experiment out of three shown . Error bars represent SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , not significant ( ns ) . one - way ANOVA .

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E. BMDMs of the indicated genotypes were infected with GFP-C. rodentium (MOI 2) for 0.5h. C. rodentium CFU were assessed at 2 h after infection and normalised to CFU at 0.5 h (n = 12; 2 biological and 6 technical replicates per condition per experiment). As a control, WT cells were treated with bafilomycin A. Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). one-way ANOVA.


F . Representative images of GFP + BMDMs labelled with LysoTracker Red DND - 99 dye ( red ) and DAPI , 1h post - infection with GFP - C . rodentium ( green ) . G . Quantification of LysoTracker Red co - localisation with GFP - C . rodentium from panel ( F ) ( n = 54 - 63 cells ) . Data information : One representative experiment out of three shown . Error bars represent SEM . * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , not significant ( ns ) . P one - way ANOVA .

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F. Representative images of GFP+ BMDMs labelled with LysoTracker Red DND-99 dye (red) and DAPI, 1h post-infection with GFP-C. rodentium (green). G. Quantification of LysoTracker Red co-localisation with GFP- C. rodentium from panel (F) (n = 54-63 cells). Data information: One representative experiment out of three shown. Error bars represent SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001, not significant (ns). P one-way ANOVA.


( a , b ) Histograms of TDP43 ( WT ) - EGFP ( a ) and TDP43A315T - EGFP ( b ) levels in individual neurons . Increasing amounts of DNA significantly shifted the distribution of log - transformed expression levels toward higher values ( P 0 . 01 for all comparisons , two - sided Kolmogorov - Smirnov test ) . TDP43 ( WT ) - EGFP , n = 1 , 287 neurons , with 290 - 342 cells per group ; TDP43A315T - EGFP , n = 1 , 290 , with 315 - 332 neurons per group . Values were pooled from 12 wells per condition , with experiments performed in duplicate .

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(a,b) Histograms of TDP43(WT)-EGFP (a) and TDP43A315T-EGFP (b) levels in individual neurons. Increasing amounts of DNA significantly shifted the distribution of log-transformed expression levels toward higher values (P 0.01 for all comparisons, two-sided Kolmogorov-Smirnov test). TDP43(WT)-EGFP, n = 1,287 neurons, with 290-342 cells per group; TDP43A315T-EGFP, n = 1,290, with 315-332 neurons per group. Values were pooled from 12 wells per condition, with experiments performed in duplicate.


( c ) Automated fluorescence microscopy . Primary rodent cortical neurons were transfected with mApple and TDP43 - EGFP , and survival was determined by repeated imaging at regular intervals . The last time at which the cell was noted to be alive ( red arrows ) was used as the time of death . Cells that survive the entire length of the experiment ( cyan arrow ) were censored . Scale bar , 25 μm .

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(c) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (cyan arrow) were censored. Scale bar, 25 μm.


( d , e ) Cumulative risk of death over time for neurons transfected with EGFP , TDP43 ( WT ) - EGFP ( d ) or TDP43A315T - neurons ( e ) . Supplementary Table 1 lists the number of neurons , hazard ratios , 95 % CI and P values for each condition , as determined by Cox proportional hazards analysis . Results were pooled from 12 wells per condition , with experiments performed in duplicate .

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

(d,e) Cumulative risk of death over time for neurons transfected with EGFP, TDP43(WT)-EGFP (d) or TDP43A315T-neurons (e). Supplementary Table 1 lists the number of neurons, hazard ratios, 95% CI and P values for each condition, as determined by Cox proportional hazards analysis. Results were pooled from 12 wells per condition, with experiments performed in duplicate.


f , g ) Linear regression of calculated hazard ratio ( ± s . e . m . ) as a function of total TDP43 ( WT ) - EGFP ( R2 = 0 . 9353 ) ( f ) or TDP43A315T - EGFP ( R2 = 0 . 9574 ) ( g ) level . The total TDP43 level was determined by quantitative immunocytochemistry using TDP43 - specific antibodies ( Supplementary Fig . 3 ) and normalized to the amount of TDP43 in nontransfected neurons . Dashed lines , reference ( HR = 1 ) .

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

f,g) Linear regression of calculated hazard ratio (± s.e.m.) as a function of total TDP43(WT)-EGFP (R2 = 0.9353) (f) or TDP43A315T-EGFP (R2 = 0.9574) (g) level. The total TDP43 level was determined by quantitative immunocytochemistry using TDP43-specific antibodies (Supplementary Fig. 3) and normalized to the amount of TDP43 in nontransfected neurons. Dashed lines, reference (HR = 1).


( a ) Primary neurons were labeled with 35S - labeled methionine , and TDP43 was immunoprecipitated using anti - TDP43 antibodies ( IP - TDP43 ) or nonspecific IgG antibodies ( IP - IgG ) . ( b ) Log - normal plot of normalized TDP43 levels . TDP43 half - life was determined by fitting a first - order exponential curve to the data ( R2 = 0 . 8984 ) . Values were pooled from five independent experiments .

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

(a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R2 = 0.8984). Values were pooled from five independent experiments.


( c ) Potential confounders in half - life analyses . Top , total protein ideally decreases over time in a predictable manner ( top right panel ) . Middle , toxicity reduces the amount of measured protein , potentially shortening the calculated half - life ( middle right panel ) . Bottom , protein aggregation might also shorten half - life estimations if the protein cannot be immunoprecipitated ( black arrows , bottom right panel ) or lengthen it if the protein is more stable and successfully immunoprecipitated ( gray arrows , bottom right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Potential confounders in half-life analyses. Top, total protein ideally decreases over time in a predictable manner (top right panel). Middle, toxicity reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Bottom, protein aggregation might also shorten half-life estimations if the protein cannot be immunoprecipitated (black arrows, bottom right panel) or lengthen it if the protein is more stable and successfully immunoprecipitated (gray arrows, bottom right panel).


( d ) OPL . Primary cortical neurons were transfected with EGFP and TDP43 ( WT ) - Dendra2 , photoconverted then imaged by AFM . Dead cells ( arrows ) were excluded at the time of death . Pre , before photoconversion . ( e ) The half - life of TDP43 ( WT ) - Dendra2 was determined by fitting a first - order exponential curve to the data ( R2 = 0 . 9558 ) . ( f ) Including cells with aggregates prolonged TDP43 ( WT ) - Dendra2 half - life ( pink line , R2 = 0 . 9003 ; * P 0 . 0001 , F 15 . 34 , extra sum - of - squares F - test ) . Excluding cells that died over the 36 - h experiment also prolonged half - life ( cyan line , R2 = 0 . 7171 ; * * P 0 . 0001 , extra sum - of - squares F - test ) . Error bars represent s . e . m . Values were pooled from eight wells per condition , with experiments performed in quadruplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) OPL. Primary cortical neurons were transfected with EGFP and TDP43(WT)-Dendra2, photoconverted then imaged by AFM. Dead cells (arrows) were excluded at the time of death. Pre, before photoconversion. (e) The half-life of TDP43(WT)-Dendra2 was determined by fitting a first-order exponential curve to the data (R2 = 0.9558). (f) Including cells with aggregates prolonged TDP43(WT)-Dendra2 half-life (pink line, R2 = 0.9003; *P 0.0001, F 15.34, extra sum-of-squares F-test). Excluding cells that died over the 36-h experiment also prolonged half-life (cyan line, R2 = 0.7171; **P 0.0001, extra sum-of-squares F-test). Error bars represent s.e.m. Values were pooled from eight wells per condition, with experiments performed in quadruplicate.


( a ) Scatter plot depicting half - life measurements for individual cells expressing TDP43 ( WT ) - Dendra2 ( cyan , n = 206 ) and TDP43A315T - Dendra2 ( red , n = 251 ) . * P = 0 . 0029 , U = 11 , 797 , Mann - Whitney test .

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

(a) Scatter plot depicting half-life measurements for individual cells expressing TDP43(WT)-Dendra2 (cyan, n = 206) and TDP43A315T-Dendra2 (red, n = 251). *P = 0.0029, U = 11,797, Mann-Whitney test.


b ) Probability density plot for single - cell half - life measurements , demonstrating a reduced half - life of TDP43A315T - Dendra2 ( red , n = 251 ) , compared to TDP43 ( WT ) - Dendra2 ( cyan , n = 206 ; * * P = 0 . 0007 , two - sided Kolmogorov - Smirnov test ) . Values were pooled from eight wells per condition , with experiments performed in quadruplicate .

O O O O O O O O O O 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

b) Probability density plot for single-cell half-life measurements, demonstrating a reduced half-life of TDP43A315T-Dendra2 (red, n = 251), compared to TDP43(WT)-Dendra2 (cyan, n = 206; **P = 0.0007, two-sided Kolmogorov-Smirnov test). Values were pooled from eight wells per condition, with experiments performed in quadruplicate.


( c , d ) Probability density plots of single - cell half - life measurements for TDP43 ( WT ) - Dendra2 ( c ) and TDP43A315T - Dendra2 ( d ) . At 0 . 5 μM , all three of the compounds significantly reduced TDP43 ( WT ) - Dendra2 half - life compared to vehicle control ( DMSO , n = 110 ; FPZ , n = 96 , f * P = 1 × 10 − 4 ; MTM , n = 129 , m * P = 2 × 10 − 7 ; NCP , n = 111 , n * P = 2 × 10 − 5 , two - sided Kolmogorov - Smirnov test ) . The compounds also reduced TDP43A315T - Dendra2 half - life , in comparison to vehicle control ( DMSO , n = 120 ; FPZ , n = 155 , f * P = 2 × 10 − 9 ; MTM , n = 152 , m * P = 0 . 004 ; NCP , n = 111 , n * P = 0 . 005 , two - sided Kolmogorov - Smirnov test ) . Colored hash marks represent values from individual neurons in b - d . Values were obtained from eight wells per condition , with experiments performed in triplicate .

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

(c,d) Probability density plots of single-cell half-life measurements for TDP43(WT)-Dendra2 (c) and TDP43A315T-Dendra2 (d). At 0.5 μM, all three of the compounds significantly reduced TDP43(WT)-Dendra2 half-life compared to vehicle control (DMSO, n = 110; FPZ, n = 96, f* P = 1 × 10−4; MTM, n = 129, m* P = 2 × 10−7; NCP, n = 111, n* P = 2 × 10−5, two-sided Kolmogorov-Smirnov test). The compounds also reduced TDP43A315T-Dendra2 half-life, in comparison to vehicle control (DMSO, n = 120; FPZ, n = 155, f* P = 2 × 10−9; MTM, n = 152, m* P = 0.004; NCP, n = 111, n* P = 0.005, two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons in b-d. Values were obtained from eight wells per condition, with experiments performed in triplicate.


( a ) Each of the compounds induced LC3 - II in primary neurons , as determined by western blotting . At 5 μM but not 0 . 5 μM , the compounds significantly increased LC3 - II / GAPDH ( b ) and LC3 - II / LC3 - I ( c ) ratios ( nonsignificant ( NS ) , P > 0 . 05 ; 0 . 05 * P 0 . 01 , * * P 0 . 01 ; F = 13 . 74 ( in b ) and 9 . 74 ( in c ) , one - way ANOVA with Dunnett ' s post - test ) . Error bars represent s . e . m . Values were pooled from five independent experiments .

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

(a) Each of the compounds induced LC3-II in primary neurons, as determined by western blotting. At 5 μM but not 0.5 μM, the compounds significantly increased LC3-II/GAPDH (b) and LC3-II/LC3-I (c) ratios (nonsignificant (NS), P > 0.05; 0.05 *P 0.01, **P 0.01; F = 13.74 (in b) and 9.74 (in c), one-way ANOVA with Dunnett's post-test). Error bars represent s.e.m. Values were pooled from five independent experiments.


( d ) Optical pulse labeling of LC3 - Dendra2 in rodent primary cortical neurons . Scale bars , 25 μm .

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(d) Optical pulse labeling of LC3-Dendra2 in rodent primary cortical neurons. Scale bars, 25 μm.


( e ) The change in red fluorescence intensity over time was used to calculate LC3 - Dendra2 half - life , as in Figure 2 ( DMSO , R2 = 0 . 7333 ; Beclin , R2 = 0 . 6968 , FPZ , R2 = 0 . 8752 ; MTM , R2 = 0 . 8447 ; NCP , R2 = 0 . 8357 ; * P 0 . 0001 for global trend , F = 12 . 6 , extra sum - of - squares F - test ) . Error bars represent s . e . m . (

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(e) The change in red fluorescence intensity over time was used to calculate LC3-Dendra2 half-life, as in Figure 2 (DMSO, R2 = 0.7333; Beclin, R2 = 0.6968, FPZ, R2 = 0.8752; MTM, R2 = 0.8447; NCP, R2 = 0.8357; *P 0.0001 for global trend, F = 12.6, extra sum-of-squares F-test). Error bars represent s.e.m. (


. ( f ) The median single - cell half - life of LC3 - Dendra2 was significantly reduced by beclin expression or treatment with 0 . 5 μM NCP , MTM or FPZ ( * P 0 . 01 , * * P 0 . 001 , Kruskal Wallis statistic = 52 . 56 , Kruskal - Wallis with Dunn ' s test ) .

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

. (f) The median single-cell half-life of LC3-Dendra2 was significantly reduced by beclin expression or treatment with 0.5 μM NCP, MTM or FPZ (*P 0.01, **P 0.001, Kruskal Wallis statistic = 52.56, Kruskal-Wallis with Dunn's test).


( g ) Probability density plot of LC3 - Dendra2 half - life measurements from individual neurons treated with vehicle control ( DMSO , n = 140 ) , NCP ( n = 128 ) , MTM ( n = 91 ) , FPZ ( n = 77 ) or those expressing beclin ( n = 101 ) . n * , P = 6 × 10 − 4 ; m * , P = 1 × 10 − 10 ; f * , P = 5 × 10 − 9 ; b * , P = 2 × 10 − 3 ; two - sided Kolmogorov - Smirnov test ) . Colored hash marks represent values from individual neurons . Values were pooled from eight wells per condition , with experiments performed in triplicate .

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

(g) Probability density plot of LC3-Dendra2 half-life measurements from individual neurons treated with vehicle control (DMSO, n = 140), NCP (n = 128), MTM (n = 91), FPZ (n = 77) or those expressing beclin (n = 101). n*, P = 6 × 10−4; m*, P = 1 × 10−10; f*, P = 5 × 10−9; b*, P = 2 × 10−3; two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons. Values were pooled from eight wells per condition, with experiments performed in triplicate.


( a ) FPZ ( n = 490 ) and MTM ( n = 580 ) but not NCP ( n = 523 ) lowered total TDP43 ( WT ) - EGFP levels in comparison to vehicle ( DMSO , n = 1 , 007 ) .

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(a) FPZ (n = 490) and MTM (n = 580) but not NCP (n = 523) lowered total TDP43(WT)-EGFP levels in comparison to vehicle (DMSO, n = 1,007).


( b ) Histogram of normalized , log - transformed TDP43 ( WT ) - EGFP levels .

(b) Histogram of normalized, log-transformed TDP43(WT)-EGFP levels.


( c ) Each compound also reduced TDP43A315T - EGFP levels ( DMSO , n = 983 ; FPZ , n = 550 ; MTM , n = 542 ; NCP , n = 493 ) .

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(c) Each compound also reduced TDP43A315T-EGFP levels (DMSO, n = 983; FPZ, n = 550; MTM, n = 542; NCP, n = 493).


( d ) Histogram of single - cell TDP43 ( A315T ) - EGFP levels . Asterisks in b and d denote median values for cells exposed to vehicle ( gray ) , FPZ ( green ) , MTM ( blue ) or NCP ( purple ) . For FPZ , MTM and NCP versus DMSO in a and d , P 1 × 10 − 3 by two - sided Kolmogorov - Smirnov test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in b and d denote median values for cells exposed to vehicle (gray), FPZ (green), MTM (blue) or NCP (purple). For FPZ, MTM and NCP versus DMSO in a and d, P 1 × 10−3 by two-sided Kolmogorov-Smirnov test.


e ) No compound reduced TDP43 ( WT ) - EGFP inclusions 48 h after transfection ( DMSO , n = 360 ; FPZ , n = 194 ; MTM , n = 234 ; NCP , n = 203 ) .

O O O O O O O O O O O O O O O O O O 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) No compound reduced TDP43(WT)-EGFP inclusions 48 h after transfection (DMSO, n = 360; FPZ, n = 194; MTM, n = 234; NCP, n = 203).


( f ) FPZ and MTM but not NCP reduced TDP43A315T - EGFP inclusions at 48 h ( DMSO , n = 327 ; FPZ , n = 226 ; MTM , n = 262 ; NCP , n = 253 ) . ( g ) No compound affected TDP43 ( WT ) - EGFP localization 24 h after transfection ( DMSO , n = 256 ; FPZ , n = 212 ; MTM , n = 250 ; NCP , n = 241 ) . ( h , i ) Each compound ( h ) prevented TDP43A315T - EGFP cytoplasmic mislocalization ( DMSO , n = 233 ; FPZ , n = 268 ; MTM , n = 263 ; NCP , n = 270 ) and reduced cytoplasmic TDP43 ( A315T ) - EGFP levels ( DMSO , n = 125 ; FPZ , n = 102 ; MTM , n = 121 ; NCP , n = 144 . * P 0 . 05 ; NS , P > 0 . 05 ) ( i ) . For a , c and e - i , * P 0 . 05 ; nonsignificant ( NS ) , P > 0 . 05 , one - way ANOVA with Dunnett ' s test . Error bars represent s . e . m . Values in a - d were pooled from 8 - 12 wells per condition from six experiments , values in e - h were pooled from eight wells per condition from three experiments , and values in i were pooled from five wells per condition from two experiments .

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

(f) FPZ and MTM but not NCP reduced TDP43A315T-EGFP inclusions at 48 h (DMSO, n = 327; FPZ, n = 226; MTM, n = 262; NCP, n = 253). (g) No compound affected TDP43(WT)-EGFP localization 24 h after transfection (DMSO, n = 256; FPZ, n = 212; MTM, n = 250; NCP, n = 241). (h,i) Each compound (h) prevented TDP43A315T-EGFP cytoplasmic mislocalization (DMSO, n = 233; FPZ, n = 268; MTM, n = 263; NCP, n = 270) and reduced cytoplasmic TDP43(A315T)-EGFP levels (DMSO, n = 125; FPZ, n = 102; MTM, n = 121; NCP, n = 144. *P 0.05; NS, P > 0.05) (i). For a, c and e-i, *P 0.05; nonsignificant (NS), P > 0.05, one-way ANOVA with Dunnett's test. Error bars represent s.e.m. Values in a-d were pooled from 8-12 wells per condition from six experiments, values in e-h were pooled from eight wells per condition from three experiments, and values in i were pooled from five wells per condition from two experiments.


( a , b ) Primary neurons transfected with TDP43 ( WT ) - EGFP ( a ) or TDP43A315T - EGFP ( b ) were treated with autophagy inducers and survival determined by AFM . Data were obtained from eight wells per condition , with experiments performed in triplicate . Supplementary Table 2 lists the number of neurons per condition , hazard ratios , 95 % CI and P values , as determined by Cox hazards analysis . ( c ) Autophagic induction improved survival in TDP43M337V HB9 - positive human iPSC - derived MNs . ( d ) FPZ and MTM also reduced the risk of death in MAP2 - positive human iPSC - derived MNs Values in c and d were pooled from 18 wells per condition , with experiments performed in triplicate . ( e ) Astrocytes differentiated from human iPSCs exhibit mutant TDP43 - related toxicity that is mitigated by FPZ and MTM . Values were pooled from 18 wells per condition , performed in triplicate . Supplementary Table 3 lists the number of human iPSC - derived neurons and astrocytes per condition , hazard ratios , 95 % CI and P values , as determined by Cox hazards analysis .

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

(a,b) Primary neurons transfected with TDP43(WT)-EGFP (a) or TDP43A315T-EGFP (b) were treated with autophagy inducers and survival determined by AFM. Data were obtained from eight wells per condition, with experiments performed in triplicate. Supplementary Table 2 lists the number of neurons per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis. (c) Autophagic induction improved survival in TDP43M337V HB9-positive human iPSC-derived MNs. (d) FPZ and MTM also reduced the risk of death in MAP2-positive human iPSC-derived MNs Values in c and d were pooled from 18 wells per condition, with experiments performed in triplicate. (e) Astrocytes differentiated from human iPSCs exhibit mutant TDP43-related toxicity that is mitigated by FPZ and MTM. Values were pooled from 18 wells per condition, performed in triplicate. Supplementary Table 3 lists the number of human iPSC-derived neurons and astrocytes per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis.


a ) EGF enhances colocalization of phosphorylated ( P ) - bRAF , P - MEK and P - ERK with LC3 . Immunofluorescence ( IF ) showing colocalization of P - bRAF ( green ) , P - MEK ( green ) and P - ERK ( green ) with LC3 ( red ) in NIH / 3T3 cells in presence or absence of EGF ( 10 min as shown in scheme ) . The bars represent mean ± s . e . m . * * P0 . 01 , * * * P0 . 001 ; Student ' s t - test , 60 cells analysed from two experiments . Scale bars , 10 μm .

O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR 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) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. **P0.01, ***P0.001; Student's t-test, 60 cells analysed from two experiments. Scale bars, 10 μm.


( b ) P - ERK colocalizes with pre - autophagosomal and autophagosomal structures . IF depicting colocalization of P - ERK ( green or red ) with LC3 ( red ) , ATG5 - ATG12 ( red ) , ATG16 ( green ) , vps34 ( red ) , WIPI1 ( red ) , WIPI2 ( red ) and P - ULK1 ( red ) in EGF - treated NIH / 3T3 cells . Scale bars , 10 μm . The bars represent mean ± s . e . m . 50 cells analysed from two experiments . ( c ) Growth factors in serum maintain P - ERK / LC3 colocalization . IF depicting colocalization of P - ERK ( green ) with LC3 ( red ) in 2 h serum - fed NIH / 3T3 cells .

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

(b) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5-ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. (c) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells.


( a - c ) Native merged images or images with colocalization highlighted in white pixels are shown . Nuclei are blue ( DAPI ) . Scale bar , 10 μm .

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(a-c) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.


( a ) P - and total MEK and ERK are enriched in APh in vivo . Immunoblots for indicated proteins in homogenate ( Hom ) , APh , APL and Lys fractions from livers of fed mice , n = 4 .

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(a) P- and total MEK and ERK are enriched in APh in vivo. Immunoblots for indicated proteins in homogenate (Hom), APh, APL and Lys fractions from livers of fed mice, n=4.


( b ) EGF enhances enrichment of P - MEK and ERK in APh fractions in vitro . Immunoblots for indicated proteins in Hom , APh and APL fractions from NIH / 3T3 cells in presence or absence of EGF ( 10 min ) . The bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 ; Student ' s t - test , n = 3 .

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

(b) EGF enhances enrichment of P-MEK and ERK in APh fractions in vitro. Immunoblots for indicated proteins in Hom, APh and APL fractions from NIH/3T3 cells in presence or absence of EGF (10 min). The bars represent mean±s.e.m. *P0.05, **P0.01; Student's t-test, n=3.


( c ) P - and total MEK and ERK localize on to the cytoplasmic / extra - luminal face of APh in vivo . Left ; model depicting localization of MEK and ERK on the extra - luminal face of autophagic vesicles and the ability of trypsin to degrade extra - luminal MEK and ERK , and right ; immunoblots for indicated proteins in APh fractions from mice livers untreated ( − ) or treated with increasing amounts of trypsin for 15 min . The bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 compared with corresponding trypsin - untreated value ; ANOVA - Bonferroni post hoc test , n = 3 . The p44 , p42 forms of ERK , LC3 - I and membrane - associated LC3 - II , and 37 kDa and 25 kDa forms of cathepsin ( Cath ) B are indicated .

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

(c) P- and total MEK and ERK localize on to the cytoplasmic/extra-luminal face of APh in vivo. Left; model depicting localization of MEK and ERK on the extra-luminal face of autophagic vesicles and the ability of trypsin to degrade extra-luminal MEK and ERK, and right; immunoblots for indicated proteins in APh fractions from mice livers untreated (−) or treated with increasing amounts of trypsin for 15 min. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with corresponding trypsin-untreated value; ANOVA-Bonferroni post hoc test, n=3. The p44, p42 forms of ERK, LC3-I and membrane-associated LC3-II, and 37 kDa and 25 kDa forms of cathepsin (Cath) B are indicated.


( a ) EGF enhances nuclear LC3 - II content in hepatocytes . Immunofluorescence ( IF ) depicting nuclear LC3 - II in RALA hepatocytes in presence or absence of EGF ( 10 min ) . Native / inverted images are shown . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with control ( Con ) ; Student ' s t - test , 50 cells from two experiments .

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(a) EGF enhances nuclear LC3-II content in hepatocytes. Immunofluorescence (IF) depicting nuclear LC3-II in RALA hepatocytes in presence or absence of EGF (10 min). Native/inverted images are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with control (Con); Student's t-test, 50 cells from two experiments.


( b ) EGF enhances nuclear P - ERK / LC3 - II colocalization . IF depicting P - ERK ( green ) / LC3 - II ( red ) colocalization in untreated ( Con ) or EGF - treated NIH / 3T3 cells . Native images ( top ) / images with colocalization in white pixels ( bottom ) are shown . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , 50 cells from two experiments .

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

(b) EGF enhances nuclear P-ERK/LC3-II colocalization. IF depicting P-ERK (green)/LC3-II (red) colocalization in untreated (Con) or EGF-treated NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, 50 cells from two experiments.


( c ) Adipogenic differentiation increases nuclear LC3 - II . Images depict nuclear LC3 - II in 3T3 - L1 preadipocytes in maintenance or differentiation medium ( 2 h ) . Scale bar , 5 μm . Bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - test , 50 cells from two experiments .

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(c) Adipogenic differentiation increases nuclear LC3-II. Images depict nuclear LC3-II in 3T3-L1 preadipocytes in maintenance or differentiation medium (2 h). Scale bar, 5 μm. Bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, 50 cells from two experiments.


( d ) Nuclear P - ERK / LC3 - II colocalization in serum - fed cells . IF showing P - ERK ( green ) / LC3 ( red ) colocalization in 2 h serum - fed NIH / 3T3 cells . Native images ( top ) / images with colocalization in white pixels ( bottom ) are shown . Scale bar , 5 μm .

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(d) Nuclear P-ERK/LC3-II colocalization in serum-fed cells. IF showing P-ERK (green)/LC3 (red) colocalization in 2 h serum-fed NIH/3T3 cells. Native images (top)/images with colocalization in white pixels (bottom) are shown. Scale bar, 5 μm.


( e ) LC3 interacts with ERK in vivo . Immunoblots showing co - immunoprecipitation of LC3 with ERK , MEK and bRAF in homogenate ( Hom ) ( e , top ) , and of LC3 with P - and total ERK in nuclear fractions from mouse livers ( e , bottom ) .

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

(e) LC3 interacts with ERK in vivo. Immunoblots showing co-immunoprecipitation of LC3 with ERK, MEK and bRAF in homogenate (Hom) (e, top), and of LC3 with P- and total ERK in nuclear fractions from mouse livers (e, bottom).


( f ) Blocking nuclear transport decreases EGF - induced increase in nuclear LC3 - II . LC3 IF ( red ) in EGF - treated NIH / 3T3 cells pre - exposed ( 30 min ) or not to WGA . Bars represent mean ± s . e . m . * * * P0 . 001 compared to with ; Student ' s t - test , 60 cells from n = 3 . Scale bar , 10 μm .

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

(f) Blocking nuclear transport decreases EGF-induced increase in nuclear LC3-II. LC3 IF (red) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared to with; Student's t-test, 60 cells from n=3. Scale bar, 10 μm.


( g ) Blocking nuclear transport decreases nuclear ERK content . ERK IF ( green ) in EGF - treated NIH / 3T3 cells pre - exposed ( 30 min ) or not to WGA . Bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test .

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(g) Blocking nuclear transport decreases nuclear ERK content. ERK IF (green) in EGF-treated NIH/3T3 cells pre-exposed (30 min) or not to WGA. Bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test.


( h ) Blocking nuclear transport does not modify P - ERK / LC3 - II colocalization . IF depicting nuclear P - ERK ( green ) / LC3 ( red ) colocalization in EGF - treated NIH / 3T3 cells pre - exposed or not to WGA . For ( g ) and ( h ) : Scale bar , 10 μm , bars are mean ± s . e . m . 50 cells from n = 2 . Nuclei are blue ( DAPI ) . Arrows indicate LC3 puncta , ERK or colocalization .

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(h) Blocking nuclear transport does not modify P-ERK/LC3-II colocalization. IF depicting nuclear P-ERK (green)/LC3 (red) colocalization in EGF-treated NIH/3T3 cells pre-exposed or not to WGA. For (g) and (h): Scale bar, 10 μm, bars are mean±s.e.m. 50 cells from n=2. Nuclei are blue (DAPI). Arrows indicate LC3 puncta, ERK or colocalization.


( a ) Atg7 − / − livers display decreased ERK phosphorylation . Immunoblots for the indicated proteins in liver homogenate ( Hom ) and cytosolic ( Cyt ) fractions of control ( Con ) and liver - specific Atg7 − / − mice are shown . The bars represent mean ± s . e . m . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 compared with Con ; Student ' s t - test , n = 4 .

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(a) Atg7−/− livers display decreased ERK phosphorylation. Immunoblots for the indicated proteins in liver homogenate (Hom) and cytosolic (Cyt) fractions of control (Con) and liver-specific Atg7−/− mice are shown. The bars represent mean±s.e.m. *P0.05, **P0.01, ***P0.001 compared with Con; Student's t-test, n=4.


( b ) Atg7 − / − livers display decreased ERK2 dimers . Immunoblots show the indicated proteins in Hom from Con and Atg7 − / − livers . The bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared to Con ; Student ' s t - test , n = 4 - 5 . Arrows indicate p42 monomers and dimers .

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(b) Atg7−/− livers display decreased ERK2 dimers. Immunoblots show the indicated proteins in Hom from Con and Atg7−/− livers. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared to Con; Student's t-test, n=4-5. Arrows indicate p42 monomers and dimers.


( c ) Atg7 − / − brown adipose tissues ( BAT ) display decreased ERK phosphorylation . Immunoblots for indicated proteins in Hom from Con and Atg7 − / − BAT . The bars represent mean ± s . e . m . * * P0 . 01 compared with Con ; Student ' s t - test , n = 4 - 6 .

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(c) Atg7−/− brown adipose tissues (BAT) display decreased ERK phosphorylation. Immunoblots for indicated proteins in Hom from Con and Atg7−/−BAT. The bars represent mean±s.e.m. **P0.01 compared with Con; Student's t-test, n=4-6.


( d ) Atg7 − / − livers exhibit decreased nuclear ERK phosphorylation . Immunoblots show the indicated proteins in homogenate ( Hom , lane 1 ) and nuclear fractions ( lanes 2 - 5 ) from Con and Atg7 − / − livers . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 4 - 5 .

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(d) Atg7−/− livers exhibit decreased nuclear ERK phosphorylation. Immunoblots show the indicated proteins in homogenate (Hom, lane 1) and nuclear fractions (lanes 2-5) from Con and Atg7−/− livers. The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=4-5.


( e ) LC3 C terminus glycine - deleted ( ΔG ) mutants exhibit decreased ERK phosphorylation . Immunoblots for indicated proteins in total lysates from CFP - LC3 - and CFP - LC3ΔG - transfected NIH / 3T3 cells exposed or not to EGF ( 10 min ) . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 8 .

O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR 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 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) LC3 C terminus glycine-deleted (ΔG) mutants exhibit decreased ERK phosphorylation. Immunoblots for indicated proteins in total lysates from CFP-LC3- and CFP-LC3ΔG-transfected NIH/3T3 cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=8.


( a ) Atg5 − / − MEFs display reduced ERK phosphorylation during nutrient deprivation . Quantification of P - ERK levels normalized to total ERK ( detected by immunoblotting ) in WT and Atg5 − / − MEFs cultured in the absence of serum for indicated times . The bars represent mean ± s . e . m . * P0 . 05 compared with corresponding WT value ; * * * P0 . 001 , * * * * P0 . 0001 compared with 10 min serum - starved Atg5 − / − MEFs ; ANOVA - Bonferroni post hoc test , n = 3 .

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

(a) Atg5−/− MEFs display reduced ERK phosphorylation during nutrient deprivation. Quantification of P-ERK levels normalized to total ERK (detected by immunoblotting) in WT and Atg5−/− MEFs cultured in the absence of serum for indicated times. The bars represent mean±s.e.m. *P0.05 compared with corresponding WT value; ***P0.001, ****P0.0001 compared with 10 min serum-starved Atg5−/− MEFs; ANOVA-Bonferroni post hoc test, n=3.


( b ) Serum - deprived Atg5 − / − MEFs display decreased ERK and p90RSK phosphorylation . Immunoblots for the indicated proteins in total lysates from 2 h serum - deprived WT and Atg5 − / − MEFs . The bars represent mean ± s . e . m . * P0 . 05 , * * * P0 . 001 compared with Con ; Student ' s t - test , n = 3 .

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(b) Serum-deprived Atg5−/− MEFs display decreased ERK and p90RSK phosphorylation. Immunoblots for the indicated proteins in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs. The bars represent mean±s.e.m. *P0.05, ***P0.001 compared with Con; Student's t-test, n=3.


( c ) Atg5 − / − MEFs display decreased nuclear ERK activity . Immunoblots for phosphorylated HA ( haemagglutinin ) - tagged Elk1 in total lysates from 2 h serum - deprived WT and Atg5 − / − MEFs cells exposed or not to EGF ( 10 min ) .

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(c) Atg5−/− MEFs display decreased nuclear ERK activity. Immunoblots for phosphorylated HA (haemagglutinin)-tagged Elk1 in total lysates from 2 h serum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min).


( d ) Atg5 − / − MEFs display decreased Elk1 - driven gene expression . Elk1 - driven ZFP36 mRNA levels from 2 hserum - deprived WT and Atg5 − / − MEFs cells exposed or not to EGF ( 10 min ) . The bars represent mean ± s . e . m . * * * P0 . 001 compared with Con ; Student ' s t - test , n = 3 . (

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

(d) Atg5−/− MEFs display decreased Elk1-driven gene expression. Elk1-driven ZFP36 mRNA levels from 2 hserum-deprived WT and Atg5−/− MEFs cells exposed or not to EGF (10 min). The bars represent mean±s.e.m. ***P0.001 compared with Con; Student's t-test, n=3. (


( e ) Decreased ERK phosphorylation in Atg5 − / − MEFs occurs independently of changes in ERK phosphatases . Immunoblots for the indicated proteins in 2 h serum - deprived WT MEFs transfected with scrambled siRNA ( scr ) , and Atg5 − / − MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF . The bars represent mean ± s . e . m . , n = 3 .

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(e) Decreased ERK phosphorylation in Atg5−/− MEFs occurs independently of changes in ERK phosphatases. Immunoblots for the indicated proteins in 2 h serum-deprived WT MEFs transfected with scrambled siRNA (scr), and Atg5−/− MEFs transfected with scr or siRNAs against MKP3 or PP2A in the presence or absence of EGF. The bars represent mean±s.e.m., n=3.


( a ) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5 - ATG12 , LC3 and WIPI1 . Immunofluorescence ( IF ) showing colocalization ( depicted as white pixels by ' colocalization finder application ' ) of WT - ERK2 - HA , FRS ERK2 mutants ( L198A - , L232A - , L198A / L232A - , Y261A - ERK2 - HA ) or common docking ( CD ) mutant ( D319N - ERK2 - HA ) with ATG5 - ATG12 ( panels 1 - 6 ) , LC3 ( panels 7 - 12 ) or WIPI1 ( panels 13 - 18 ) in EGF - treated NIH / 3T3 cells . ERK2 is stained in red , and autophagy proteins are stained in green . Scale bar , 10 μm . The bars represent mean ± s . e . m . * * P0 . 01 , * * * * P0 . 0001 compared with WT - ERK2 - transfected cells ; Student ' s t - test , 50 cells analysed from n = 2 . (

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

(a) Mutations in FRS on ERK2 decrease colocalization of ERK2 with ATG5-ATG12, LC3 and WIPI1. Immunofluorescence (IF) showing colocalization (depicted as white pixels by 'colocalization finder application') of WT-ERK2-HA, FRS ERK2 mutants (L198A-, L232A-, L198A/L232A-, Y261A-ERK2-HA) or common docking (CD) mutant (D319N-ERK2-HA) with ATG5-ATG12 (panels 1-6), LC3 (panels 7-12) or WIPI1 (panels 13-18) in EGF-treated NIH/3T3 cells. ERK2 is stained in red, and autophagy proteins are stained in green. Scale bar, 10 μm. The bars represent mean±s.e.m. **P0.01, ****P0.0001 compared with WT-ERK2-transfected cells; Student's t-test, 50 cells analysed from n=2. (