Expression data ( RNAseq ) for significantly differentially expressed genes ( q - value < 0 . 05 , FDR - adjusted p - value , n = 2 biological replicates for each condition ) . Scatter plot shows log2 ( fold change ) of gene expression data comparing endpoint to initial populations for Exp . 1 and Exp . 2 ( grey dots ) with the location of the gene in the reference genome as the x - axis . Those genes that are associated with AraC transcription units are highlighted ( red dots for Exp . 1 and blue dots for Exp . 2 ) . Above the plot , the transcription units are labeled green if AraC activates expression ( in the presence of arabinose ) or red if AraC represses expression of those genes .

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Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes.


Growth rate analysis of various weaned ( starting point of static phase ) and optimized ( endpoint of static phase ) strains with or without fucK or araB genes knocked out . Strains were grown in triplicate ( n = 3 ) on M9 minimal media with D - arabinose as the sole carbon source . The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation . The p - values reported were calculated using a two - sided Welch ' s t - test .

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Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.


A : Morphology on phase contrast microscopy for parental fibroblast lines ( upper panel ) and iPSCs ( lower panel ) from control Detroit 551 , WS5A and CP2A POLG patients ( scale bars , 50 µm ) .

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A: Morphology on phase contrast microscopy for parental fibroblast lines (upper panel) and iPSCs (lower panel) from control Detroit 551, WS5A and CP2A POLG patients (scale bars, 50 µm).


B : Immunofluorescence staining of stem cell markers POU5F1 ( green ) and SSEA4 ( red ) : upper panel - Detroit 551 control iPSCs , middle panel - WS5A iPSCs and lower panel - CP2A iPSCs ( Scale bar , 100 µm ) . Nuclei are stained with DAPI ( blue ) .

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B: Immunofluorescence staining of stem cell markers POU5F1 (green) and SSEA4 (red): upper panel - Detroit 551 control iPSCs, middle panel - WS5A iPSCs and lower panel - CP2A iPSCs (Scale bar, 100 µm). Nuclei are stained with DAPI (blue).


C : RT - qPCR quantification of gene expression for LIN28A , NANOG , and POU5F1 for all iPSCs from control Detroit 551 , WS5A and CP2A POLG patients ( n = 7 , technical replicates per line for ESCs ; n = 4 , technical replicates per clone for control , WS5A and CP2A iPSCs ) . The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1 .

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C: RT-qPCR quantification of gene expression for LIN28A, NANOG, and POU5F1 for all iPSCs from control Detroit 551, WS5A and CP2A POLG patients (n=7, technical replicates per line for ESCs; n=4, technical replicates per clone for control, WS5A and CP2A iPSCs). The gene expression of the individual clones is assessed with fold change using the comparative ΔΔCt method by normalizing iPSCs to ESC1.


D , E : Flow cytometric quantification of expression level of SSEA4 ( D , n = 9 , technical replicates per line for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) and POU5F1 ( E , n = 9 , technical replicates per line for ESCs ; n = 5 , technical replicates per clone for control iPSCs ; n = 3 , technical replicates per clone for WS5A iPSCs , n = 8 , technical replicates per clone for CP2A iPSCs ) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control , WS5A and CP2A fibroblasts .

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D, E: Flow cytometric quantification of expression level of SSEA4 (D, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) and POU5F1 (E, n=9, technical replicates per line for ESCs; n=5, technical replicates per clone for control iPSCs; n=3, technical replicates per clone for WS5A iPSCs, n=8, technical replicates per clone for CP2A iPSCs) for ESCs and iPSCs for both ESC control lines and iPSCs generated from Detroit 551 control, WS5A and CP2A fibroblasts.


A : Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN ( red ) and HNF4A ( green ) ( a ) ( scale bar , 100 µm ) , mesodermal derived cardiomyocytes with positive expression of TNNT2 ( red ) ( b ) ( scale bar , 100 µm ) and ectodermal derived dopaminergic neurons with positive expression of TH ( green ) and MAP2 ( red ) ( c ) ( scale bar , 10 µm ) . Nuclei are stained with DAPI ( blue ) .

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A: Representative confocal images of iPSC lineage specific differentiation into germ layers of endoderm derived hepatocytes with positive expression of ALBUMIN (red) and HNF4A (green) (a) (scale bar, 100 µm), mesodermal derived cardiomyocytes with positive expression of TNNT2 (red) (b) (scale bar, 100 µm) and ectodermal derived dopaminergic neurons with positive expression of TH (green) and MAP2 (red) (c) (scale bar, 10 µm). Nuclei are stained with DAPI (blue).


B : Confocal images of mitochondrial morphology for iPSC lines with co - staining of MTG ( upper panel ) and TMRE ( lower panel ) ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

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B: Confocal images of mitochondrial morphology for iPSC lines with co-staining of MTG (upper panel) and TMRE (lower panel) (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


Flow cytometric analysis of iPSCs generated from Detroit 551 , WS5A and CP2A fibroblasts for mitochondrial volume ( MTG ) ( C , n = 6 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A )

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Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for mitochondrial volume (MTG) (C, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A)


Flow cytometric analysis of iPSCs generated from Detroit 551 , WS5A and CP2A fibroblasts for total MMP ( TMRE ) ( D , n = 6 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A ) and specific MMP ( E , n = 6 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A ) calculated by dividing median fluorescence intensity ( MFI ) for total TMRE expression by MTG .

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Flow cytometric analysis of iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts for total MMP (TMRE) (D, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) and specific MMP (E, n=6, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG.


F : Intracellular ATP production in iPSCs generated from Detroit 551 , WS5A and CP2A fibroblasts ( n = 3 , technical replicates per clone for control and WS5A ; n = 4 , technical replicates per clone for CP2A ) .

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F: Intracellular ATP production in iPSCs generated from Detroit 551, WS5A and CP2A fibroblasts (n=3, technical replicates per clone for control and WS5A; n=4, technical replicates per clone for CP2A).


G - H : Flow cytometric analysis of TOMM20 expression level ( G , n = 4 , technical replicates per clone ) and specific TFAM level ( total TFAM / TOMM20 ) ( H , n = 4 , technical replicates per clone ) .

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G-H: Flow cytometric analysis of TOMM20 expression level (G, n=4, technical replicates per clone) and specific TFAM level (total TFAM/TOMM20) (H, n=4, technical replicates per clone).


I : Relative mtDNA copy number in Detroit 551 , WS5A and CP2A iPSCs by RT - qPCR analysis using ND1 and APP ( n = 5 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A and CP2A ) . Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP .

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I: Relative mtDNA copy number in Detroit 551, WS5A and CP2A iPSCs by RT-qPCR analysis using ND1 and APP (n=5, technical replicates per clone for control; n=5, technical replicates per clone for WS5A and CP2A). Values are presented as Log2 of the ratio between the expression values of ND1 in relation to APP.


B : Representative phase contrast images ( upper panel ) and immunostaining for specific stages during neural induction from iPSCs to NSCs . Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs ( a ) ; neuroepithelium with rosette - like structures ( b ) ; neurospheres with defined round shapes in suspension culture ( c ) and NSCs in monolayers ( d ) ( scale bars , 50 µm ) . Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel : iPSCs with positive staining of SSEA4 ( red ) and POU5F1 ( green ) ( e ) ( scale bar , 50 µm ) ; neuroepithelium with rosette - like structures with positive staining of PAX6 ( green ) and NESTIN ( red ) ( f ) ( scale bar , 50 µm ) ; neurospheres with positive staining of NESTIN ( red ) ( g ) ( scale bar , 100 µm ) ; NSCs with positive staining of PAX6 ( green ) ( h ) ( scale bar , 50 µm ) .

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B: Representative phase contrast images (upper panel) and immunostaining for specific stages during neural induction from iPSCs to NSCs. Upper panel displays the morphology in culture of different cell types during neural induction to NSCs from iPSCs including iPSCs (a); neuroepithelium with rosette-like structures (b); neurospheres with defined round shapes in suspension culture (c) and NSCs in monolayers (d) (scale bars, 50 µm). Lower panel demonstrates the representative phase contrast images for the immunostaining corresponding to the specific stages in the upper panel: iPSCs with positive staining of SSEA4 (red) and POU5F1 (green) (e) (scale bar, 50 µm); neuroepithelium with rosette-like structures with positive staining of PAX6 (green) and NESTIN (red) (f) (scale bar, 50 µm); neurospheres with positive staining of NESTIN (red) (g) (scale bar, 100 µm); NSCs with positive staining of PAX6 (green) (h) (scale bar, 50 µm).


C : Flow cytometric assessment of stemness marker POU5F1 and NSC markers NESTIN and PAX6 during neural induction from day 0 to day 5 ( n = 3 , technical replicates ) .

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C: Flow cytometric assessment of stemness marker POU5F1 and NSC markers NESTIN and PAX6 during neural induction from day 0 to day 5 (n=3, technical replicates).


D : Representative images of the immunofluorescent labeling for NSC markers SOX2 ( green ) and NESTIN ( red ) ( scale bar , 25 µm ) from Detroit 551 control iPSC - derived NSCs . Nuclei are stained with DAPI ( blue ) .

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D: Representative images of the immunofluorescent labeling for NSC markers SOX2 (green) and NESTIN (red) (scale bar, 25 µm) from Detroit 551 control iPSC-derived NSCs. Nuclei are stained with DAPI (blue).


A : Quantification of gene expression for NSC markers PAX6 , NESTIN and SOX2 for all NSCs from RT - qPCR analysis . The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs ( n = 3 , technical replicates per ESC line or iPSC clone ) .

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A: Quantification of gene expression for NSC markers PAX6, NESTIN and SOX2 for all NSCs from RT-qPCR analysis. The expression of the neural stem cell markers is assessed with fold change using the comparative ΔΔCt method by normalizing NSCs to iPSCs (n=3, technical replicates per ESC line or iPSC clone).


B , C : Quantification of protein expression level for PAX6 ( B , n = 6 , technical replicates per line for ESCs ; n = 7 , technical replicates per clone for control ; n = 8 , technical replicates per clone for WS5A ; n = 9 , technical replicates per clone for CP2A ) and NESTIN ( C , n = 6 , technical replicates per line for ESCs ; n = 7 , technical replicates per clone for control ; n = 8 , technical replicates per clone for WS5A ; n = 9 , technical replicates per clone for CP2A ) for iPSC - derived NSCs using flow cytometry .

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B, C: Quantification of protein expression level for PAX6 (B, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) and NESTIN (C, n=6, technical replicates per line for ESCs; n=7, technical replicates per clone for control; n=8, technical replicates per clone for WS5A; n=9, technical replicates per clone for CP2A) for iPSC-derived NSCs using flow cytometry.


D : Sequencing chromatogram showing the homozygous c . 2243G > C variation in POLG in WS5A iPSC - derived NSCs and the heterozygous c . 1399G > A and c . 2243G > C variation in POLG in CP2A iPSC - derived NSCs .

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D: Sequencing chromatogram showing the homozygous c.2243G>C variation in POLG in WS5A iPSC-derived NSCs and the heterozygous c.1399G>A and c.2243G>C variation in POLG in CP2A iPSC-derived NSCs.


E : Representative confocal images showing glial and neuronal lineages derived from NSCs . a , Immunostaining of NSC - derived astrocytes with GFAP ( red ) staining ( scale bar , 50 µm ) . b , Immunostaining of oligodendrocytes showing GALC ( red ) positive labeling ( scale bar , 50 µm ) . c , Dopaminergic neurons showing TH ( green ) and TUJ1 ( red ) positive staining ( scale bar , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

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E: Representative confocal images showing glial and neuronal lineages derived from NSCs. a, Immunostaining of NSC-derived astrocytes with GFAP (red) staining (scale bar, 50 µm). b, Immunostaining of oligodendrocytes showing GALC (red) positive labeling (scale bar, 50 µm). c, Dopaminergic neurons showing TH (green) and TUJ1 (red) positive staining (scale bar, 25 µm). Nuclei are stained with DAPI (blue).


A : Representative transmission electron microscopy images of mitochondrial structures in Detroit 551 control , WS5A and CP2A NSCs ( scale bar , 400 nm ) .

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A: Representative transmission electron microscopy images of mitochondrial structures in Detroit 551 control, WS5A and CP2A NSCs (scale bar, 400 nm).


Flow cytometric analysis of mitochondrial volume ( MTG ) ( B , n = 5 , technical replicates per clone ) , total MMP ( TMRE ) ( C , n = 5 , technical replicates per clone ) calculated by dividing median fluorescence intensity ( MFI ) for total TMRE expression by MTG in NSCs generated from control lines , WS5A and CP2A iPSCs .

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Flow cytometric analysis of mitochondrial volume (MTG) (B, n=5, technical replicates per clone), total MMP (TMRE) (C, n=5, technical replicates per clone) calculated by dividing median fluorescence intensity (MFI) for total TMRE expression by MTG in NSCs generated from control lines, WS5A and CP2A iPSCs.


Flow cytometric analysis of specific MMP ( D , n = 5 , technical replicates per clone ) , calculated by dividing median fluorescence intensity ( MFI ) in NSCs generated from control lines , WS5A and CP2A iPSCs .

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Flow cytometric analysis of specific MMP (D, n=5, technical replicates per clone), calculated by dividing median fluorescence intensity (MFI) in NSCs generated from control lines, WS5A and CP2A iPSCs.


E : Intracellular ATP production in NSCs ( n = 3 , technical replicates per clone ) .

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E: Intracellular ATP production in NSCs (n=3, technical replicates per clone).


Flow cytometric analysis of mitochondrial volume ( MTG ) ( F , n = 6 , technical replicates for control ; n = 5 , technical replicates for WS5A and CP2A ) , total MMP ( TMRE ) ( G , n = 5 , technical replicates for control ; n = 4 , technical replicates for WS5A and CP2A ) in Detroit 551 , WS5A and CP2A fibroblasts .

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Flow cytometric analysis of mitochondrial volume (MTG) (F, n=6, technical replicates for control; n=5, technical replicates for WS5A and CP2A), total MMP (TMRE) (G, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.


Flow cytometric analysis of specific MMP ( TMRE / MTG ) ( H , n = 5 , technical replicates for control ; n = 4 , technical replicates for WS5A and CP2A ) in Detroit 551 , WS5A and CP2A fibroblasts .

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Flow cytometric analysis of specific MMP (TMRE/MTG) (H, n=5, technical replicates for control; n=4, technical replicates for WS5A and CP2A) in Detroit 551, WS5A and CP2A fibroblasts.


I : Intracellular ATP production in Detroit 551 , WS5A and CP2A fibroblasts ( n = 6 , technical replicates for control ; n = 3 , technical replicates for WS5A and CP2A ) .

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I: Intracellular ATP production in Detroit 551, WS5A and CP2A fibroblasts (n=6, technical replicates for control; n=3, technical replicates for WS5A and CP2A).


A , B : Flow cytometric analysis of TOMM20 ( A , n = 3 , technical replicates per clone for control ; n = 4 , technical replicates per clone for WS5A and CP2A ) and TFAM protein expression ( B , n = 3 , technical replicates per clone for control and CP2A ; n = 5 , technical replicates per clone for WS5A ) in NSCs . Expressed as specific TFAM level ( total TFAM / TOMM20 ) .

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A, B: Flow cytometric analysis of TOMM20 (A, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and TFAM protein expression (B, n=3, technical replicates per clone for control and CP2A; n=5, technical replicates per clone for WS5A) in NSCs. Expressed as specific TFAM level (total TFAM/TOMM20).


C : Relative mtDNA copy number analyzed by RT - qPCR using primers and fluorogenic probes for regions of ND1 and nuclear gene APP in all NSCs ( n = 4 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 7 , technical replicates per clone for CP2A ) .

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C: Relative mtDNA copy number analyzed by RT-qPCR using primers and fluorogenic probes for regions of ND1 and nuclear gene APP in all NSCs (n=4, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=7, technical replicates per clone for CP2A).


D , E : Flow cytometric analysis of TOMM20 expression level ( D , n = 4 , technical replicates per clone ) and TFAM protein expression ( E , n = 4 , technical replicates for control and WS5A ; n = 5 , technical replicates for CP2A ) in fibroblasts . Expressed as specific TFAM level ( total TFAM / TOMM20 ) .

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D, E: Flow cytometric analysis of TOMM20 expression level (D, n=4, technical replicates per clone) and TFAM protein expression (E, n=4, technical replicates for control and WS5A; n=5, technical replicates for CP2A) in fibroblasts. Expressed as specific TFAM level (total TFAM/TOMM20).


F : Relative mtDNA copy number analyzed by RT - qPCR for regions of ND1 and nuclear gene APP in fibroblasts ( n = 3 , technical replicates for control ; n = 4 , technical replicates for WS5A and CP2A ) .

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F: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in fibroblasts (n=3, technical replicates for control; n=4, technical replicates for WS5A and CP2A).


G : Long - PCR for detection of mtDNA deletions in NSCs and their parental fibroblasts from WS5A and CP2A patients .

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G: Long-PCR for detection of mtDNA deletions in NSCs and their parental fibroblasts from WS5A and CP2A patients.


H : Representative images of confocal microscopy with immunofluorescence labeling of DA neuron - specific marker TH ( green ) and neuron - specific marker MAP2 ( red ) for iPSC - derived DA neurons ( scale bars , 25 µm ) . Nuclei are stained with DAPI ( blue ) .

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H: Representative images of confocal microscopy with immunofluorescence labeling of DA neuron-specific marker TH (green) and neuron-specific marker MAP2 (red) for iPSC-derived DA neurons (scale bars, 25 µm). Nuclei are stained with DAPI (blue).


I : Histogram of the positive cell population stained with DA marker TH in iPSC - derived DA neurons using flow cytometric analysis .

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I: Histogram of the positive cell population stained with DA marker TH in iPSC-derived DA neurons using flow cytometric analysis.


J : Flow cytometric measurement of specific TFAM level ( total TFAM / TOMM20 ) in iPSC - derived DA neurons from Detroit 551 control , WS5A and CP2A POLG patients ( n = 8 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 4 , technical replicates per clone for CP2A ) .

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J: Flow cytometric measurement of specific TFAM level (total TFAM/TOMM20) in iPSC-derived DA neurons from Detroit 551 control, WS5A and CP2A POLG patients (n=8, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=4, technical replicates per clone for CP2A).


K : Relative mtDNA copy number analyzed by RT - qPCR for regions of ND1 and nuclear gene APP in all iPSC - derived DA neurons ( n = 3 , technical replicates per clone ) .

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K: Relative mtDNA copy number analyzed by RT-qPCR for regions of ND1 and nuclear gene APP in all iPSC-derived DA neurons (n=3, technical replicates per clone).


A : Complex I immunohistochemistry in the occipital cortex of a neurologically healthy control ( a ) and two patients with POLG - disease ( b and c ) ( Scale bar , 20 µm ) . Patients have numerous complex - I negative neurons ( examples marked by arrows ) . ( d ) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases ( n = 5 ) and neurologically healthy controls ( n = 5 ) . Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons . For the purposes of comparison , a control sample has been arbitrarily set to one . The medians of the two groups are compared by Mann - Whitney U test . Data are presented as mean ( horizontal bars ) ± SEM ( vertical bars ) .

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A: Complex I immunohistochemistry in the occipital cortex of a neurologically healthy control (a) and two patients with POLG-disease (b and c) (Scale bar, 20 µm). Patients have numerous complex-I negative neurons (examples marked by arrows). (d) mtDNA relative quantification in microdissected neurons from the occipital cortex of patients with POLG diseases (n=5) and neurologically healthy controls (n=5). Each point represents the mean value of three technical replicates from a pooled sample of 10 neurons. For the purposes of comparison, a control sample has been arbitrarily set to one. The medians of the two groups are compared by Mann-Whitney U test. Data are presented as mean (horizontal bars) ± SEM (vertical bars).


B : Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 ( green ) and TOMM20 ( red ) in control , WS5A and CP2A NSCs ( scale bars , 50 µm ) . Nuclei are stained with DAPI ( blue ) .

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B: Representative confocal images of immunostaining for mitocondrial complex I subunit NDUFB10 (green) and TOMM20 (red) in control, WS5A and CP2A NSCs (scale bars, 50 µm). Nuclei are stained with DAPI (blue).


C - E : Flow cytometric measurements of mitochondrial complex I ( C , n = 4 , technical replicates per clone ) , II ( D , n = 3 , technical replicates per clone for control ; n = 4 , technical replicates per clone for WS5A and CP2A ) and IV ( E , n = 3 , technical replicates per clone for control and CP2A ; n = 4 , technical replicates per clone for WS5A ) protein level in iPSC - derived NSCs . Expressed as specific complex I , II and IV level ( total complex I , II , IV level / TOMM20 ) .

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C-E: Flow cytometric measurements of mitochondrial complex I (C, n=4, technical replicates per clone), II (D, n=3, technical replicates per clone for control; n=4, technical replicates per clone for WS5A and CP2A) and IV (E, n=3, technical replicates per clone for control and CP2A; n=4, technical replicates per clone for WS5A) protein level in iPSC-derived NSCs. Expressed as specific complex I, II and IV level (total complex I, II, IV level/TOMM20).


F - H : Flow cytometric measurements of mitochondrial complex I ( F , n = 4 , technical replicates per clone ) , II ( G , n = 4 , technical replicates per clone ) and IV ( H , n = 4 , technical replicates per clone ) protein level in iPSCs . Expressed as specific complex I , II and IV level .

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F-H: Flow cytometric measurements of mitochondrial complex I (F, n=4, technical replicates per clone), II (G, n=4, technical replicates per clone) and IV (H, n=4, technical replicates per clone) protein level in iPSCs. Expressed as specific complex I, II and IV level.


I - K : Flow cytometric measurements of mitochondrial complex I ( I , n = 4 , technical replicates ) , II ( J , n = 4 , technical replicates ) and IV ( K , n = 4 , technical replicates ) protein level in parental fibroblasts expressed as specific complex I , II and IV level .

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I-K: Flow cytometric measurements of mitochondrial complex I (I, n=4, technical replicates), II (J, n=4, technical replicates) and IV (K, n=4, technical replicates) protein level in parental fibroblasts expressed as specific complex I, II and IV level.


A - C : LCMS - based metabolomics for quantitative measurements of NAD + / NADH ratio ( A , n = 3 , technical replicates per clone ) , NAD + ( B , n = 3 , technical replicates per clone ) and NADH ( C , n = 3 , technical replicates per clone ) level in NSCs .

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A-C: LCMS-based metabolomics for quantitative measurements of NAD+/NADH ratio (A, n=3, technical replicates per clone), NAD+ (B, n=3, technical replicates per clone) and NADH (C, n=3, technical replicates per clone) level in NSCs.


D - F : LCMS - based metabolomics for quantitative measurement of NAD + / NADH ratio ( D , n = 3 , technical replicates per clone ) , NAD + ( E , n = 3 , technical replicates per clone ) and NADH ( F , n = 3 , technical replicates per clone ) level in iPSCs .

D-F: LCMS-based metabolomics for quantitative measurement of NAD+/NADH ratio (D, n=3, technical replicates per clone), NAD+ (E, n=3, technical replicates per clone) and NADH (F, n=3, technical replicates per clone) level in iPSCs.


G : Intracellular ROS production measurements of the specific ROS level calculated by total ROS / MTDR in control , WS5A and CP2A iPSC - derived NSCs using DCFDA and MTDR ( n = 6 , technical replicates per clone for control ; n = 5 , technical replicates per clone for WS5A ; n = 3 , technical replicates per clone for CP2A ) .

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G: Intracellular ROS production measurements of the specific ROS level calculated by total ROS/MTDR in control, WS5A and CP2A iPSC-derived NSCs using DCFDA and MTDR (n=6, technical replicates per clone for control; n=5, technical replicates per clone for WS5A; n=3, technical replicates per clone for CP2A).


H : Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551 , WS5A and CP2A iPSC - derived NSCs calculated by total ROS ( MitoSox Red ) / MTG ( n = 4 , technical replicates per clone for control and CP2A ; n = 3 , technical replicates per clone for WS5A ) .

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H: Flow cytometric measurements of mitochondrial ROS production at the specific ROS level in Detroit 551, WS5A and CP2A iPSC-derived NSCs calculated by total ROS (MitoSox Red)/MTG (n=4, technical replicates per clone for control and CP2A; n=3, technical replicates per clone for WS5A).


I - J : Representative images of senescence β - galactosidase staining ( scale bars , 20 µm ) ( I ) and quantification by calculating the percentage of positively stained cells by division of the negative cells from I ( J , n = 3 , technical replicates per clone ) .

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I-J: Representative images of senescence β-galactosidase staining (scale bars, 20 µm) (I) and quantification by calculating the percentage of positively stained cells by division of the negative cells from I (J, n=3, technical replicates per clone).


K - L : Representative images ( K ) and quantification ( L ) for western blotting with Phospho - SirT1 ( Ser47 ) , LC3B , UCP2 , PINK1 , Parkin , BNIP3 and β - ACTIN . Three independent experiments are included .

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K-L: Representative images (K) and quantification (L) for western blotting with Phospho-SirT1 (Ser47), LC3B, UCP2, PINK1, Parkin, BNIP3 and β-ACTIN. Three independent experiments are included.


a , Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( pseudo - coloured red ) , and Salmonella carrying mCherry - expressing vector ( red ) . The cis - Golgi ( green ) was detected by α - GM130 antibodies . Scale bar , 10 µm .

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a, Fluorescence microscopyHeLa cells infected with Shigella and Listeria visualized with 4′,6-diamidino-2-phenylindole (DAPI) (pseudo-coloured red), and Salmonella carrying mCherry-expressing vector (red). The cis-Golgi (green) was detected by α-GM130 antibodies. Scale bar, 10 µm.


b , Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA . The cis - Golgi ( GM130 , green ) and F - actin ( red ) are shown . Scale bar , 10 µm .

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b, Fluorescence microscopy of HeLa cells transfected with either IpaJ or VirA. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.


c , Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains . Scale bar , 10 µm .

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c, Fluorescence microscopy of HeLa cells infected with indicated Shigella mutant strains. Scale bar, 10 µm.


d , Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild - type Shigella ( M90T ) or the indicated mutants . Error bars , means ± s . e . m . calculated from three independent experiments .

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d, Percentage of HeLa cells with disrupted Golgi morphology in 100 cells infected with either wild-type Shigella (M90T) or the indicated mutants. Error bars, means ± s.e.m. calculated from three independent experiments.


e , Number of recoverable c . f . u . per mouselung24 h after intranasal inoculation with 1 × 106 of wild - type M90T or each mutant Shigella strain as indicated . Limit of detection ( LOD ) for this assay is indicated by the dotted line . Error bars , geometrical means ± s . e . m . * * P < 0 . 001 .

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e, Number of recoverable c.f.u. per mouselung24 h after intranasal inoculation with 1 × 106 of wild-type M90T or each mutant Shigella strain as indicated. Limit of detection (LOD) for this assay is indicated by the dotted line. Error bars, geometrical means ± s.e.m. **P<0.001.


b , Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants . The cis - Golgi ( GM130 , green ) and F - actin ( red ) are shown . Scale bar , 10 µm .

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b, Fluorescence microscopy of HeLa cells transfected with the indicated IpaJ mutants. The cis-Golgi (GM130, green) and F-actin (red) are shown. Scale bar, 10 µm.


c , IpaJ or its catalytic mutants were expressed from a galactose - inducible promoter ( pGal413 vector ) and assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

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c, IpaJ or its catalytic mutants were expressed from a galactose-inducible promoter (pGal413 vector) and assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


e , Yeast strain harbouring a galactose - inducible IpaJ gene were transformed with a multi - copy vector containing the indicated genes . Yeast were assayed for a growth arrest phenotype on galactose or glucose ( control ) carbon source .

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e, Yeast strain harbouring a galactose-inducible IpaJ gene were transformed with a multi-copy vector containing the indicated genes. Yeast were assayed for a growth arrest phenotype on galactose or glucose (control) carbon source.


a - c , Mass spectra of purified ARF1 - strep in untreated ( a ) , IpaJ - treated ( b ) or IpaJ C64A - treated ( c ) cells , with the observed monoisotopic mass reported in daltons . * Observed 1 Da shift in molecular mass is accounted for in MS / MS data ( Supplementary Fig . 7 ) .

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a-c, Mass spectra of purified ARF1-strep in untreated (a), IpaJ-treated (b) or IpaJ C64A-treated (c) cells, with the observed monoisotopic mass reported in daltons. *Observed 1 Da shift in molecular mass is accounted for in MS/MS data (Supplementary Fig. 7).


f , In - gel fluorescence assay ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - His isolated from bacteria either not expressing ( lane 1 ) or expressing ( lane 2 ) NMTp . Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated . The expression levels of ARF1 - His were determined by Coomassie blue stain .

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f, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-His isolated from bacteria either not expressing (lane 1) or expressing (lane 2) NMTp. Bacterial cell lysates treated with recombinant IpaJ or IpaJ C64A as indicated. The expression levels of ARF1-His were determined by Coomassie blue stain.


g , In - gel fluorescence assay ( top panel ) visualizing Alexa Fluor 647 - labelled myristoylated ARF1 - strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated . The expressed amounts of ARF1 - strep are shown ( bottom panel ) .

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g, In-gel fluorescence assay (top panel) visualizing Alexa Fluor 647-labelled myristoylated ARF1-strep purified from HeLa cell lysates left untreated or incubated with IpaJ or its catalytic mutant as indicated. The expressed amounts of ARF1-strep are shown (bottom panel).


h , In - gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid , azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry . Arrows indicate proteins that are proteolytically demyristoylated by IpaJ .

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h, In-gel fluorescence assay visualizing protein extracts isolated from HeLa cells incubated with azide myristic acid, azide palmitic acid or geranylgeranyl alcohol azide and subsequently labelled with Alexa Fluor 647 alkyne by click chemistry. Arrows indicate proteins that are proteolytically demyristoylated by IpaJ.


b , In - gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant . The peptide - eGFP and IpaJ inputs are indicated . * Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide , potentially owing to reduced mobility in SDS - polyacrylamide gel electrophoresis caused by proteolytic reaction .

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b, In-gel fluorescence assay showing myristoylated peptides of the indicated proteins in cells expressing IpaJ or catalytic mutant. The peptide-eGFP and IpaJ inputs are indicated. *Demyristoylation of GRASP65 and hVPS15 resulted in slower mobility of the resulting peptide, potentially owing to reduced mobility in SDS-polyacrylamide gel electrophoresis caused by proteolytic reaction.


c , In - gel fluorescence assay showing myristoylated ARF1 T31N mutant ( a GDP - locked mutant ) or ARF1 Q71L ( a GTP - locked mutant ) after in cells co - expressing IpaJ or catalytic mutants as indicated . Equal loading of ARF1 ( middle panel ) and IpaJ ( lower panel ) was determined by western blot analysis .

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c, In-gel fluorescence assay showing myristoylated ARF1 T31N mutant (a GDP-locked mutant) or ARF1 Q71L (a GTP-locked mutant) after in cells co-expressing IpaJ or catalytic mutants as indicated. Equal loading of ARF1 (middle panel) and IpaJ (lower panel) was determined by western blot analysis.


d , Fluorescence microscopy showing ARF1 - mCherry and the Golgi apparatus ( GM130 ) after cellular microinjection of recombinant IpaJ or IpaJ C64A , over the indicated period . Scale bar , 10 µm .

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d, Fluorescence microscopy showing ARF1-mCherry and the Golgi apparatus (GM130) after cellular microinjection of recombinant IpaJ or IpaJ C64A, over the indicated period. Scale bar, 10 µm.


e , Fluorescence microscopy showing ARF1 - eGFP ( green ) and Golgi membranes ( blue , GM130 ) after infection with Shigella M90T and the indicated mutants ( red ; mCherry - expressing bacteria ) . Scale bar , 10µm .

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e, Fluorescence microscopy showing ARF1-eGFP (green) and Golgi membranes (blue, GM130) after infection with Shigella M90T and the indicated mutants (red; mCherry-expressing bacteria). Scale bar, 10µm.


( a ) PC12 cells stably expressing A53T mutant α - synuclein were induced for 24 h , after which expression was switched off by removing doxycycline from the medium for the next 24 h . Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period , as expression levels decay when synthesis is reduced or stopped . Cells were treated with EHNA or AMP - PNP or left untreated during the switch - off period . Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control .

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(a) PC12 cells stably expressing A53T mutant α-synuclein were induced for 24 h, after which expression was switched off by removing doxycycline from the medium for the next 24 h. Transgene product clearance can be inferred by assessing levels at various times after switching off expression after an initial induction period, as expression levels decay when synthesis is reduced or stopped. Cells were treated with EHNA or AMP-PNP or left untreated during the switch-off period. Blots were probed with antibody to HA to detect the transgene and antibody to actin as a loading control.


( b ) PC12 cells expressing Q74 - GFP were induced for 15 h and then treated with inhibitors for 48 h after switch - off . Band intensity quantification ( three independent experiments ) is shown .

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(b) PC12 cells expressing Q74-GFP were induced for 15 h and then treated with inhibitors for 48 h after switch-off. Band intensity quantification (three independent experiments) is shown.


( c ) Immunofluorescence images of cells expressing Q74 - GFP after 48 h of switch - off after treatment with ( EHNA ) or without ( Control ) EHNA .

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(c) Immunofluorescence images of cells expressing Q74-GFP after 48 h of switch-off after treatment with (EHNA) or without (Control) EHNA.


( d ) Quantification of aggregates and cell death in COS - 7 cells transiently transfected with Q74 - GFP either with ( EHNA ) or without ( Control ) EHNA ( added during the last 33 h before fixation ) .

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(d) Quantification of aggregates and cell death in COS-7 cells transiently transfected with Q74-GFP either with (EHNA) or without (Control) EHNA (added during the last 33 h before fixation).


( e ) Western - blot analysis of HeLa cells stably expressing GFP - UbG76V treated with lactacystin ( Lac ) , EHNA or AMP - PNP or left untreated ( Control ) using antibody to GFP . * * * P 0 . 0001 ; * * P 0 . 001 .

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(e) Western-blot analysis of HeLa cells stably expressing GFP-UbG76V treated with lactacystin (Lac), EHNA or AMP-PNP or left untreated (Control) using antibody to GFP. ***P 0.0001; **P 0.001.


( a ) Stable PC12 cells expressing A53T α - synuclein were induced for 24 h , after which expression was switched off and cells were simultaneously transfected with p50 or dynamitin ( p50 ) , CC1 , CC2 , motor domain - deleted kinesin heavy chain ( ΔM - KHC ) or the tail region of kinesin heavy chain ( DN - KHC ) for the next 24 h . Dominant - negative constructs along with pEGFP - C1 ( 3 : 1 ratio of dominant - negative construct : pEGFP - C1 ) were used to transfect cells , and FACS was used to select transfected cells , which were analyzed by western blotting as in Figure 1a . Quantification of the band intensity from multiple experiments ( three independent experiments for dynein mutants and two for kinesin mutants ) is shown . * * * P 0 . 0001 ; * P 0 . 01 ; NS , not significant .

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(a) Stable PC12 cells expressing A53T α-synuclein were induced for 24 h, after which expression was switched off and cells were simultaneously transfected with p50 or dynamitin (p50), CC1, CC2, motor domain-deleted kinesin heavy chain (ΔM-KHC) or the tail region of kinesin heavy chain (DN-KHC) for the next 24 h. Dominant-negative constructs along with pEGFP-C1 (3:1 ratio of dominant-negative construct:pEGFP-C1) were used to transfect cells, and FACS was used to select transfected cells, which were analyzed by western blotting as in Figure 1a. Quantification of the band intensity from multiple experiments (three independent experiments for dynein mutants and two for kinesin mutants) is shown. ***P 0.0001; *P 0.01; NS, not significant.


( b ) COS - 7 cells were cotransfected with HA - Q74 or GFP - Q74 with empty vector ( pEGFP or pcDNA3 . 1 ) , p50 or dynamitin ( p50 ) , p62 subunit of dynactin complex ( p62 ) , the tail region of kinesin heavy chain ( DN - KHC ) , CC1 or CC2 for 48 h , after which the cells were fixed for immunofluorescence and immunocytochemistry ( using to antibody to HA ) . The percentage of GFP - positive cells with aggregates and cell death is shown . * * * P 0 . 0001 ; * * P 0 . 001 ; NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) COS-7 cells were cotransfected with HA-Q74 or GFP-Q74 with empty vector (pEGFP or pcDNA3.1), p50 or dynamitin (p50), p62 subunit of dynactin complex (p62), the tail region of kinesin heavy chain (DN-KHC), CC1 or CC2 for 48 h, after which the cells were fixed for immunofluorescence and immunocytochemistry (using to antibody to HA). The percentage of GFP-positive cells with aggregates and cell death is shown. ***P 0.0001; **P 0.001; NS, not significant.


( c ) Immunocytochemistry analysis ( using antibody to HA ) of COS - 7 cells cotransfected for 48 h with HA - Q74 ( red ) and pEGFP or p50 ( green ) . Panels on left are magnifications of boxed areas in second panels . The boxes are the same size . * * * P 0 . 0001 ; * * P 0 . 001 . Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z - VAD - fmk ( broad - spectrum caspase inhibitor ) are shown in Supplementary Figure 4 online .

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

(c) Immunocytochemistry analysis (using antibody to HA) of COS-7 cells cotransfected for 48 h with HA-Q74 (red) and pEGFP or p50 (green). Panels on left are magnifications of boxed areas in second panels. The boxes are the same size. ***P 0.0001; **P 0.001. Examples of fragmented nuclei in cells overexpressing p50 and nuclei in cells overexpressing p50 but treated with Z-VAD-fmk (broad-spectrum caspase inhibitor) are shown in Supplementary Figure 4 online.


( a ) Immunofluorescence analysis of COS - 7 cells cotransfected for 15 h with either empty vector ( control ) or CC1 and GFP - LC3 ( green ) , in a 3 : 1 ratio . Panels on right are magnifications of boxed areas .

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

(a) Immunofluorescence analysis of COS-7 cells cotransfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio. Panels on right are magnifications of boxed areas.


( b ) Western - blot analysis of COS - 7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin . Quantification of the band intensity from two independent experiments is shown .

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

(b) Western-blot analysis of COS-7 cells treated with EHNA or left untreated using antibodies to LC3 and to actin. Quantification of the band intensity from two independent experiments is shown.


( c ) NRK cells transiently transfected for 15 h with either empty vector ( control ) or CC1 and GFP - LC3 ( green ) , in a 3 : 1 ratio were immunostained with antibody to lgp120 , and the numbers of GFP - LC3 and lgp120 doubly stained vesicles in individual cells were scored .

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

(c) NRK cells transiently transfected for 15 h with either empty vector (control) or CC1 and GFP-LC3 (green), in a 3:1 ratio were immunostained with antibody to lgp120, and the numbers of GFP-LC3 and lgp120 doubly stained vesicles in individual cells were scored.


Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 ( gmrQ120 ) crossed to either a control stock ( w1118 ; W ) or to Dhc64C ( dynein heavy chain mutant ; Dhc ; a ) or Roblk ( dynein light chain mutant ; Robl ; b ) flies . P ≤ 0 . 0001 ( Mann - Whitney U 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 B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

Frequencies of ommatidia with different numbers of rhabdomeres are shown at 3 days after eclosion in progeny of flies expressing mutant huntingtin exon 1 (gmrQ120) crossed to either a control stock (w1118; W) or to Dhc64C (dynein heavy chain mutant; Dhc; a) or Roblk (dynein light chain mutant; Robl; b) flies. P ≤ 0.0001 (Mann-Whitney U test).


( c ) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 ( Q120 ) crossed with either control stock ( w1118 ; W ) or with Dhc64C ( Dhc ) flies . All fly experiments were repeated three times independently with similar significance levels . One representative experiment is shown .

O O O O O 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

(c) Ommatidia from the progeny of flies expressing mutant huntingtin exon 1 (Q120) crossed with either control stock (w1118; W) or with Dhc64C (Dhc) flies. All fly experiments were repeated three times independently with similar significance levels. One representative experiment is shown.


( a ) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation . At 7 weeks : HdhHD / + Dnchc1Loa / + n = 9 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 14 . At 9 weeks : HdhHD / + Dnchc1Loa / + n = 8 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 027 . At 11 weeks : HdhHD / + Dnchc1Loa / + n = 6 , HdhHD / + Dnchc1 + / + n = 9 , P = 0 . 001 . At 13 weeks : HdhHD / + Dnchc1Loa / + n = 5 , HdhHD / + Dnchc1 + / + n = 8 , P = 0 . 021 . At 15 weeks : HdhHD / + Dnchc1Loa / + n = 3 , HdhHD / + Dnchc1 + / + n = 5 , P = 0 . 021 .

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

(a) Tremor onset and severity in a mouse model of Huntington disease are enhanced by the Loa mutation. At 7 weeks: HdhHD/+ Dnchc1Loa/+ n = 9, HdhHD/+ Dnchc1+/+ n = 9, P = 0.14. At 9 weeks: HdhHD/+ Dnchc1Loa/+ n = 8, HdhHD/+ Dnchc1+/+ n = 9, P = 0.027. At 11 weeks: HdhHD/+ Dnchc1Loa/+ n = 6, HdhHD/+ Dnchc1+/+ n = 9, P = 0.001. At 13 weeks: HdhHD/+ Dnchc1Loa/+ n = 5, HdhHD/+ Dnchc1+/+ n = 8, P = 0.021. At 15 weeks: HdhHD/+ Dnchc1Loa/+ n = 3, HdhHD/+ Dnchc1+/+ n = 5, P = 0.021.


( b , c ) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease . HdhHD / + Dnchc1Loa / + , HdhHD / + Dnchc1 + / + and Hdh + / + Dnchc1Loa / + mice are shown . An average of six mice were used at every time point , with at least three mice per group after 13 weeks . ( b ) Grip strength . Overall effect for all time points between the three genotypes : P = 0 . 0024 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and HdhHD / + Dnchc1 + / + : P = 0 . 001 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and Hdh + / + Dnchc1Loa / + : P = 0 . 0157 . Effect at each time point between the three genotypes : 7 weeks , P = 0 . 023 ; 9 weeks , P = 0 . 001 ; 11 weeks , P 0 . 0001 ; 13 weeks , P = 0 . 004 ; 15 weeks , P = 0 . 336 ( most of the double mutants were dead by 15 weeks ) . ( c ) Accelerating rotarod . Overall effect for all time points between the three genotypes : P = 0 . 0367 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and HdhHD / + Dnchc1 + / + : P = 0 . 0245 . Overall pair comparison between HdhHD / + Dnchc1Loa / + and Hdh + / + Dnchc1Loa / + : P = 0 . 0553 . Effect at each time point between the three genotypes : 8 weeks , P 0 . 001 ; 10 weeks , P = 0 . 005 ; 12 weeks , P = 0 . 001 ; 14 weeks , P = 0 . 009 ; 16 weeks , P = 0 . 039 .

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

(b,c) Dynein mutation reduces motor coordination and muscle function in a mouse model of Huntington disease. HdhHD/+ Dnchc1Loa/+, HdhHD/+ Dnchc1+/+ and Hdh+/+ Dnchc1Loa/+ mice are shown. An average of six mice were used at every time point, with at least three mice per group after 13 weeks. (b) Grip strength. Overall effect for all time points between the three genotypes: P = 0.0024. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.001. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0157. Effect at each time point between the three genotypes: 7 weeks, P = 0.023; 9 weeks, P = 0.001; 11 weeks, P 0.0001; 13 weeks, P = 0.004; 15 weeks, P = 0.336 (most of the double mutants were dead by 15 weeks). (c) Accelerating rotarod. Overall effect for all time points between the three genotypes: P = 0.0367. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and HdhHD/+ Dnchc1+/+: P = 0.0245. Overall pair comparison between HdhHD/+ Dnchc1Loa/+ and Hdh+/+ Dnchc1Loa/+: P = 0.0553. Effect at each time point between the three genotypes: 8 weeks, P 0.001; 10 weeks, P = 0.005; 12 weeks, P = 0.001; 14 weeks, P = 0.009; 16 weeks, P = 0.039.


( d ) Reduced lifespan of HdhHD / + Dnchc1Loa / + mice . HdhHD / + Dnchc1Loa / + ( red line ) n = 13 , HdhHD / + Dnchc1 + / + ( green line ) n = 9 , P = 0 . 038 . Mice were subjected to humane end points , and so the survival curve reflects the onset of the end points rather than the complete lifespan . Asterisks ( * ) represent mice that were found dead without reaching humane end points . No mortality of Hdh + / + Dnchc1Loa / + mice was observed .

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

(d) Reduced lifespan of HdhHD/+ Dnchc1Loa/+ mice. HdhHD/+ Dnchc1Loa/+ (red line) n = 13, HdhHD/+ Dnchc1+/+ (green line) n = 9, P = 0.038. Mice were subjected to humane end points, and so the survival curve reflects the onset of the end points rather than the complete lifespan. Asterisks (*) represent mice that were found dead without reaching humane end points. No mortality of Hdh+/+ Dnchc1Loa/+ mice was observed.


( a ) Brain sections from HdhHD / + Dnchc1 + / + or HdhHD / + Dnchc1Loa / + mice were stained with antibody to huntingtin ( EM48 ; green ) . No EM48 - stained aggregates were detected in brains of Hdh + / + Dnchc1Loa / + mice . Arrows indicate cells with aggregates .

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

(a) Brain sections from HdhHD/+ Dnchc1+/+ or HdhHD/+ Dnchc1Loa/+ mice were stained with antibody to huntingtin (EM48; green). No EM48-stained aggregates were detected in brains of Hdh+/+ Dnchc1Loa/+ mice. Arrows indicate cells with aggregates.


( b ) Western - blot analysis of mouse brain lysates from wild - type ( WT ) or Loa mutant mice using antibodies to LC3 and to actin .

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

(b) Western-blot analysis of mouse brain lysates from wild-type (WT) or Loa mutant mice using antibodies to LC3 and to actin.


( A ) Growth curves of S . pombe cultures in minimal medium without ( EMM ) or with antimycin A ( EMM + antA ) . Cultures were supplemented with either the complete amino - acid mix ( all AA mix , black ) , without any amino acids ( no AA , red ) , with arginine only ( R , blue ) , or with individual amino acids other than arginine ( individual AAs , grey ) .

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

(A) Growth curves of S. pombe cultures in minimal medium without (EMM) or with antimycin A (EMM + antA). Cultures were supplemented with either the complete amino-acid mix (all AA mix, black), without any amino acids (no AA, red), with arginine only (R, blue), or with individual amino acids other than arginine (individual AAs, grey).


( B ) Growth curves as in ( A ) for cultures supplemented with complete amino - acid mix ( black ) , without any amino acids ( red ) , or amino - acid mix without arginine ( green ) .

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

(B) Growth curves as in (A) for cultures supplemented with complete amino-acid mix (black), without any amino acids (red), or amino-acid mix without arginine (green).


( C ) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine ( EMM + R ) ( Dataset file 1 ) . Mutants that passed our filtering ( Methods ) and whose colony - size ratios were 30 % bigger when supplemented with arginine are shown in red ; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles .

O 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 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

(C) Scatter plot of normalised colony sizes of cells grown on EMM against normalised colony sizes of cells grown on EMM supplemented with arginine (EMM+R) (Dataset file 1). Mutants that passed our filtering (Methods) and whose colony-size ratios were 30% bigger when supplemented with arginine are shown in red; mutants encoding known genes of the arginine biosynthesis pathway are shown with blue circles.


( E ) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants ( grey ) and respiratory deficient mutants ( orange , based on previous screens ( Malecki & Bähler , 2016 ) ( Welch ' s t - test pval * * < 0 . 002 ) . The same analysis was performed for mutants grown on EMM ( left ; 4 biological repeats , Dataset file 1 ) or on EMM supplemented with antimycin A ( right ; 2 biological repeats , Dataset file 2 ) . Boxplots were created using the R boxplot command with default settings . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box plot of mean colony size ratios of mutants grown with vs without arginine for all mutants (grey) and respiratory deficient mutants (orange, based on previous screens (Malecki & Bähler, 2016) (Welch's t-test pval** < 0.002). The same analysis was performed for mutants grown on EMM (left; 4 biological repeats, Dataset file 1) or on EMM supplemented with antimycin A (right; 2 biological repeats, Dataset file 2). Boxplots were created using the R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( A ) Normalised concentration values of 16 amino acids in cells grown in EMM without ( grey ) or with ( blue ) antimycin A . For each condition , four independent samples were analysed . Concentrations are presented relative to the mean value for the given amino acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . Boxplots were created using R boxplot command with default settings . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

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

(A) Normalised concentration values of 16 amino acids in cells grown in EMM without (grey) or with (blue) antimycin A. For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. Boxplots were created using R boxplot command with default settings. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( B ) Amino - acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM . Left : heat map showing concentration changes ( log2 fold change ) of each measured amino acid during the time course relative to time point 0 ( before antimycin A addition ) . Right : simplified scheme of amino - acid biosynthetic pathways . Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow . * Ornithine ( Orn ) is abbreviated as O .

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

(B) Amino-acid concentrations before and after addition of antimycin A to cells exponentially growing in EMM. Left: heat map showing concentration changes (log2 fold change) of each measured amino acid during the time course relative to time point 0 (before antimycin A addition). Right: simplified scheme of amino-acid biosynthetic pathways. Amino acids for which any biosynthetic enzyme has an annotated mitochondrial location are highlighted in yellow. *Ornithine (Orn) is abbreviated as O.


( C ) Left panels : cell growth in EMM without ( red ) or with ( blue ) arginine supplementation for five selected respiratory mutants as indicated ( graphs selected from Fig EV3 ) . Right panels : normalised intracellular amino - acid concentrations as in ( A ) for the same 5 respiratory mutants ( blue ) and wild - type control cells ( grey ) . For each mutant and the wild type strain , four independent samples were analysed . * Ornithine ( Orn ) is abbreviated as O . Boxplot features as in ( A ) .

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

(C) Left panels: cell growth in EMM without (red) or with (blue) arginine supplementation for five selected respiratory mutants as indicated (graphs selected from Fig EV3). Right panels: normalised intracellular amino-acid concentrations as in (A) for the same 5 respiratory mutants (blue) and wild-type control cells (grey). For each mutant and the wild type strain, four independent samples were analysed. *Ornithine (Orn) is abbreviated as O. Boxplot features as in (A).


( A ) Normalised amino - acid concentrations in cells grown in EMM without ( grey ) or with arginine ( red ) . For each condition , four independent samples were analysed . Concentrations are presented relative to the mean value for the given amino acid in EMM medium . * Ornithine ( Orn ) is abbreviated as O . The central band indicates the median , the upper and lower limits of the box indicate first and third quartiles , and the whiskers show the most extreme data , limited to 1 . 5 - time interquartile range .

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 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

(A) Normalised amino-acid concentrations in cells grown in EMM without (grey) or with arginine (red). For each condition, four independent samples were analysed. Concentrations are presented relative to the mean value for the given amino acid in EMM medium. *Ornithine (Orn) is abbreviated as O. The central band indicates the median, the upper and lower limits of the box indicate first and third quartiles, and the whiskers show the most extreme data, limited to 1.5-time interquartile range.


( C ) Growth curves of strains deleted for car1 , car2 or car3 , and a wild - type control . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine ( light blue ) or EMM with both arginine and antimycin A ( dark blue ) as indicated .

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

(C) Growth curves of strains deleted for car1, car2 or car3, and a wild-type control. Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine (light blue) or EMM with both arginine and antimycin A (dark blue) as indicated.


( D ) Growth curves of strains deleted for car2 ( top ) or car3 ( bottom ) . Strains were grown in EMM ( black ) , EMM with antimycin A ( red ) , EMM with arginine and antimycin A ( dark blue ) , EMM with mix of all amino acids and antimycin A ( bright blue ) , and EMM with antimycin A and each individual amino acid from the mix ( grey ) .

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(D) Growth curves of strains deleted for car2 (top) or car3 (bottom). Strains were grown in EMM (black), EMM with antimycin A (red), EMM with arginine and antimycin A (dark blue), EMM with mix of all amino acids and antimycin A (bright blue), and EMM with antimycin A and each individual amino acid from the mix (grey).


( E ) Growth curves of wild - type control ( WT ) and car2 deletion strain ( Δcar2 ) . Strains were grown in EMM media with antimycin A and with either a mix of all amino acids ( + AA mix ) or different combinations of lysine ( K ) , glutamine ( Q ) , glutamic acid ( E ) , or arginine ( R ) as indicated . Dashed line : time point where wild - type strain saturates in EMM with amino - acid mix .

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(E) Growth curves of wild-type control (WT) and car2 deletion strain (Δcar2). Strains were grown in EMM media with antimycin A and with either a mix of all amino acids (+AA mix) or different combinations of lysine (K), glutamine (Q), glutamic acid (E), or arginine (R) as indicated. Dashed line: time point where wild-type strain saturates in EMM with amino-acid mix.


( A ) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without ( EMM ) or with arginine ( EMM + R ) . Transcripts annotated as up - or down - regulated Core Environmental Stress Response ( CESR ) are indicated in red and blue , respectively . All data is normalised to EMM time point 0 .

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(A) Changes in transcript abundance 30 and 120 min after addition of antimycin A to exponentially growing cells without (EMM) or with arginine (EMM+R). Transcripts annotated as up- or down-regulated Core Environmental Stress Response (CESR) are indicated in red and blue, respectively. All data is normalised to EMM time point 0.


( B ) Graphs as in ( A ) showing expression profiles of transcripts encoding enzymes for arginine catabolism ( blue ) or arginine biosynthesis ( red ) .

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(B) Graphs as in (A) showing expression profiles of transcripts encoding enzymes for arginine catabolism (blue) or arginine biosynthesis (red).


( C ) Heat map of expression changes for transcripts repressed over 2 - fold at 30 min in cells supplemented with arginine ( EMM + R ) relative to cells without supplementation ( EMM ) . Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM , EMM with arginine and ssp1 deletion mutant grown in EMM . The data are normalised to EMM timepoint 0 .

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(C) Heat map of expression changes for transcripts repressed over 2-fold at 30 min in cells supplemented with arginine (EMM +R) relative to cells without supplementation (EMM). Values for timepoints 0 and 30 min after antimycin A addition are shown for cells grown in EMM, EMM with arginine and ssp1 deletion mutant grown in EMM. The data are normalised to EMM timepoint 0.


( D ) Heat map of expression changes of the same genes as in ( B ) after one hour of inhibiting TOR kinase with torin1 or caffeine / rapamycin treatment relative to cell before treatment ; cells were grown in rich media - YES ( data from ( Rodríguez - López et al . , 2019 ) )

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(D) Heat map of expression changes of the same genes as in (B) after one hour of inhibiting TOR kinase with torin1 or caffeine/rapamycin treatment relative to cell before treatment; cells were grown in rich media - YES (data from (Rodríguez-López et al., 2019))


A , B Immunoblot analyses of the protein abundance of the METTL3 / METTL14 / WTAP heterotrimer in PBMCs ( A ) and BMDMs ( B ) upon challenge with VSV - eGFP , poly ( I : C ) , HSV - 1 or HSV60 at the indicated time points .

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A, B Immunoblot analyses of the protein abundance of the METTL3/METTL14/WTAP heterotrimer in PBMCs (A) and BMDMs (B) upon challenge with VSV-eGFP, poly(I:C), HSV-1 or HSV60 at the indicated time points.


E A549 cells were stimulated with poly ( I : C ) for 16 hrs and then treated with PR - 171 ( 10 μM ) or PS - 341 ( 10 μM ) for additional 4 hrs and 8 hrs . The cell lysates were analyzed by immunoblotting . Representative images from 3 independent biological experiments are shown ( left ) , and the relative levels of WTAP are presented as the mean ± SD ( right ) . F A549 cells were stimulated with poly ( I : C ) for 16 hrs and then treated with CQ ( 50 mM ) or Baf A1 ( 0 . 2 mM ) for additional 4 hrs and 8 hrs . The cell lysates were analyzed by immunoblotting . LC3B served as a good marker of the autophagy process . Representative images from 3 independent biological experiments are shown ( left ) , and the relative levels of WTAP are presented as the mean ± SD ( right ) .

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

E A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with PR-171 (10 μM) or PS-341 (10 μM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right). F A549 cells were stimulated with poly (I:C) for 16 hrs and then treated with CQ (50 mM) or Baf A1 (0.2 mM) for additional 4 hrs and 8 hrs. The cell lysates were analyzed by immunoblotting. LC3B served as a good marker of the autophagy process. Representative images from 3 independent biological experiments are shown (left), and the relative levels of WTAP are presented as the mean ± SD (right).


H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG - tagged WTAP , and HA - tagged K48 - linked , K63 - linked or wild - type ubiquitin and treated with DMSO or PR171 ( 10 μM ) .

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H Coimmunoprecipitation and immunoblot analyses of extracts of 293T cells transfected with various combinations of plasmids encoding FLAG-tagged WTAP, and HA-tagged K48-linked, K63-linked or wild-type ubiquitin and treated with DMSO or PR171 (10 μM).


C qRT - PCR analyses to detect the mRNA abundances of IFNB1 , RANTES , DDX58 , and ISG15 in WT and WTAP - / - 293T cells followed by treatments with intracellular poly ( I : C ) , poly ( dA : dT ) , or VSV - eGFP .

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C qRT-PCR analyses to detect the mRNA abundances of IFNB1, RANTES, DDX58, and ISG15 in WT and WTAP-/- 293T cells followed by treatments with intracellular poly(I:C), poly(dA:dT), or VSV-eGFP.


I , J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly ( I : C ) - LMW ( I ) or poly ( dA : dT ) ( J ) at different time points with the indicated antibodies .

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I, J Immunoblot analyses to detect the protein expression of ISGs in shCtrl and shWTAP A549 cells that were stimulated with poly(I:C)-LMW (I) or poly(dA:dT) (J) at different time points with the indicated antibodies.


A , B Immunoblot analyses of total and phosphorylated ( p - ) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP - specific siRNA , followed by stimulation with poly ( I : C ) ( A ) or poly ( dA : dT ) ( B ) at different time points .

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A, B Immunoblot analyses of total and phosphorylated (p-) TBK1 and IRF3 in A549 cells transfected with Ctrl or WTAP-specific siRNA, followed by stimulation with poly (I:C) (A) or poly (dA:dT) (B) at different time points.


E , F Immunoblot analyses of total and phosphorylated ( p - ) STAT1 in shCtrl and shWTAP A549 cells , followed by stimulation with IFN - α ( E ) or - β ( F ) at different time points .

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E, F Immunoblot analyses of total and phosphorylated (p-) STAT1 in shCtrl and shWTAP A549 cells, followed by stimulation with IFN-α (E) or -β (F) at different time points.


A , B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN - I and Jak - STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP - specific siRNAs for 48 hrs .

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A, B Immunoblot analyses to detect the protein expression of critical adaptors in the IFN-I and Jak-STAT signaling pathway in A549 cells after transfection with Ctrl or WTAP-specific siRNAs for 48 hrs.


F , G Abundance of IRF3 ( F ) and IFNAR1 ( G ) transcripts ( detection site - 1 and - 2 ) among mRNA immunoprecipitated with anti - m6A antibody from shCtrl and shWTAP A549 cells . n = 3 independent biological replicates , and error bars represent standard deviations .

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F, G Abundance of IRF3 (F) and IFNAR1 (G) transcripts (detection site-1 and -2) among mRNA immunoprecipitated with anti-m6A antibody from shCtrl and shWTAP A549 cells. n = 3 independent biological replicates, and error bars represent standard deviations.


A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly ( I : C ) or poly ( dA : dT ) at the indicated time points . B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV - eGFP at the indicated time points .

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A Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 cells stimulated with poly (I:C) or poly (dA:dT) at the indicated time points. B Immunoblot analyses to detect IRF3 and IFNAR1 protein expression in A549 and L929 cells infected with VSV-eGFP at the indicated time points.


H , Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs and then treated with PR - 171 or DMSO for additional 4 and 8 hrs . Representative images from 3 independent biological experiments are shown ( H ) ,

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H, Immunoblot analysis of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. Representative images from 3 independent biological experiments are shown (H),


protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly ( I : C ) - LMW or infected with VSV - GFP for 16 hrs and then treated with PR - 171 or DMSO for additional 4 and 8 hrs . the relative levels of IFNAR1 ( left ) and IRF3 ( right ) are presented as the mean ± SD ( I ) .

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protein abundance of IRF3 and IFNAR1 in A549 cells transfected with poly (I:C)-LMW or infected with VSV-GFP for 16 hrs and then treated with PR-171 or DMSO for additional 4 and 8 hrs. the relative levels of IFNAR1 (left) and IRF3 (right) are presented as the mean ± SD (I).


mRNA expression of individual patients normalized to 18S - rRNA and relative to a pooled organ donor sample . Lines between dots connect samples from the same patient . CP : chronic pancreatitis ; PDAC : pancreatic ductal adenocarcinoma ; significant difference ( P < 0 . 05 ) between tumor and normal tissue P = 9 . 39 × 10 − 5 determined by paired two - tailed Student ' s t - test , n = 17 patients .

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mRNA expression of individual patients normalized to 18S-rRNA and relative to a pooled organ donor sample. Lines between dots connect samples from the same patient. CP: chronic pancreatitis; PDAC: pancreatic ductal adenocarcinoma; significant difference (P < 0.05) between tumor and normal tissue P = 9.39 × 10−5 determined by paired two-tailed Student's t-test, n = 17 patients.


Association of microarray - based TBL1 expression and survival in 105 patients with PDAC : High TBL1 expression is significantly associated with shorter post - resection survival . P - value determined by log - rank test as described in Materials and Methods .

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Association of microarray-based TBL1 expression and survival in 105 patients with PDAC: High TBL1 expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.


Immunohistochemistrystaining for TBL1 in human paraffin - embedded tissue sections . 1 , 2 : healthy tissue with acini and duct showing no or only very weak staining . 3 , 4 : PanIN - 1 lesions with occasional nuclear and cytoplasmicstaining . 5 , 6 : PanIN - 2 lesion with nuclear and occasional cytoplasmicstaining . 7 - 10 : PanIN - 3 lesion with nuclear and increased occurrence and intensity of cytoplasmaticstaining . 11 , 12 : invasive pancreatic ductal adenocarcinoma cells with nuclear and cytoplasmicstaining . Scale bars : 100 μm . Images are representative of 12 patient samples .

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Immunohistochemistrystaining for TBL1 in human paraffin-embedded tissue sections. 1, 2: healthy tissue with acini and duct showing no or only very weak staining. 3, 4: PanIN-1 lesions with occasional nuclear and cytoplasmicstaining. 5, 6: PanIN-2 lesion with nuclear and occasional cytoplasmicstaining. 7-10: PanIN-3 lesion with nuclear and increased occurrence and intensity of cytoplasmaticstaining. 11, 12: invasive pancreatic ductal adenocarcinoma cells with nuclear and cytoplasmicstaining. Scale bars: 100 μm. Images are representative of 12 patient samples.


Immunohistochemistrystaining for TBL1 in paraffin - embedded tissue sections of wild - type or p48 + / Cre ; Kras + / LSL - G12Dmice . 1 , 2 : healthy tissue with acini , islet of Langerhans , and duct ( zoom - in in panel 2 ) showing occasional weak nuclearstaining . 3 , 4 : PanIN - 1 lesions with nuclearstaining . 5 , 6 : PanIN - 2 lesions with nuclearstaining . 7 , 8 : PanIN - 3 lesion with nuclearstaining . 9 - 12 : invasive pancreatic ductal adenocarcinoma cells with nuclear and occasional faint cytoplasmicstaining . Scale bars : 100 μm . Images are representative of 32 wild - type and 29 p48 + / Cre ; Kras + / LSL - G12Dmice .

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Immunohistochemistrystaining for TBL1 in paraffin-embedded tissue sections of wild-type or p48+/Cre; Kras+/LSL-G12Dmice. 1, 2: healthy tissue with acini, islet of Langerhans, and duct (zoom-in in panel 2) showing occasional weak nuclearstaining. 3, 4: PanIN-1 lesions with nuclearstaining. 5, 6: PanIN-2 lesions with nuclearstaining. 7, 8: PanIN-3 lesion with nuclearstaining. 9-12: invasive pancreatic ductal adenocarcinoma cells with nuclear and occasional faint cytoplasmicstaining. Scale bars: 100 μm. Images are representative of 32 wild-type and 29 p48+/Cre; Kras+/LSL-G12Dmice.


Left panel : EdU incorporation in siRNA - transfected Capan - 1 cells ( 72 h post - transfection ) , n = 6 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 $ ; P - values : siTBL1 # 1 8 . 24 × 10 − 4 ( * ) , 5 . 43 × 10 − 4 ( # ) , 7 . 93 × 10 − 6 ( $ ) ; siTBL1 # 2 1 . 66 × 10 − 11 ( * ) , 1 . 27 × 10 − 11 ( # ) , 8 . 79 × 10 − 13 ( $ ) , one - way ANOVA with Bonferroni post - test . Right panel : EdU incorporation of Panc02 cells with stable expression of shRNA ; n = 6 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( shNC ) # P = 0 . 0398 ; Welch ' s t - test .

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Left panel: EdU incorporation in siRNA-transfected Capan-1 cells (72 h post-transfection), n = 6 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: siTBL1#1 8.24 × 10−4 (*), 5.43 × 10−4 (#), 7.93 × 10−6 ($); siTBL1 #2 1.66 × 10−11 (*), 1.27 × 10−11 (#), 8.79 × 10−13 ($), one-way ANOVA with Bonferroni post-test. Right panel: EdU incorporation of Panc02 cells with stable expression of shRNA; n = 6 cell culture wells per group; significantly different (P < 0.05) from control (shNC) #P = 0.0398; Welch's t-test.


Cellgrowth of siRNA - transfected Capan - 1 or Panc8680 cells ; n = 3 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 $ ; P - values : Capan - 1 cells 48 h time - point : 0 . 0408 ( * ) for siTBL1 # 1 / 0 . 0464 for siTBL1 # 2 , Capan - 1 cells 72 h time - point : 8 . 60 × 10 − 5 ( * ) , 1 . 82 × 10 − 3 ( # ) , 3 . 24 × 10 − 5 ( $ ) for siTBL1 # 1 / 2 . 39 × 10 − 10 ( * ) , 3 . 34 × 10 − 9 ( # ) , 1 . 08 × 10 − 10 ( $ ) for siTBL1 # 2 ; Panc8680 72 h time - point : 1 . 55 × 10 − 4 ( * ) , 2 . 73 × 10 − 5 ( $ ) ; two - way ANOVA with Bonferroni post - test .

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Cellgrowth of siRNA-transfected Capan-1 or Panc8680 cells; n = 3 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: Capan-1 cells 48 h time-point: 0.0408 (*) for siTBL1 #1 / 0.0464 for siTBL1 #2, Capan-1 cells 72 h time-point: 8.60 × 10−5 (*), 1.82 × 10−3 (#), 3.24 × 10−5 ($) for siTBL1 #1 / 2.39 × 10−10 (*), 3.34 × 10−9 (#), 1.08 × 10−10 ($) for siTBL1 #2; Panc8680 72 h time-point: 1.55 × 10−4 (*), 2.73 × 10−5 ($); two-way ANOVA with Bonferroni post-test.


Uptake of radio - labeled 3H - 2 - deoxyglucose in siRNA - transfected Capan - 1 cells ( 48 h post - transfection ) ; n = 6 cell culture wells per group ; statistically different ( P < 0 . 05 ) from control ( siNC # 2 ) $ P = 0 . 03 ; Welch ' s t - test .

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Uptake of radio-labeled 3H-2-deoxyglucose in siRNA-transfected Capan-1 cells (48 h post-transfection); n = 6 cell culture wells per group; statistically different (P < 0.05) from control (siNC #2) $P = 0.03; Welch's t-test.