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 .

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 .

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 .

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 ) .

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 ) .

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 .

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 .

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(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 O 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) 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 O 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 O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O 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 O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(d) Reduced 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 .

(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 O O O O O O O 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 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 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 ) .

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

(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 .

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

(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 .

O O 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

(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 ) .

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

(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 .

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

(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 ) )

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

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

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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

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

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

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 ) ,

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

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 ) .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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 .

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

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.


Extracellular flux measurement of siRNA - transfected Capan - 1 cells with glycolysis stress test assay ( 48 h post - transfection ) ; 2 - DG : 2 - deoxyglucose ; ECAR : extracellular acidification rate . n = 6 - 8 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 ) $ . Upper symbols : statistics for siTBL # 1 , P - values : 2 . 18 × 10 − 3 ( * ) time - point 8 , 9 . 83 × 10 − 3 ( * ) time - point 9 , 0 . 0102 ( * ) time - point 10 , 0 . 044 ( * ) time - point 11 ; 2 . 42 × 10 − 6 ( # ) time - point 8 , 1 . 47 × 10 − 4 ( # ) time - point 9 , 3 . 17 × 10 − 4 ( # ) time - point 10 , 0 . 0277 ( # ) time - point 11 ; 9 . 44 × 10 − 7 ( $ ) time - point 8 , 1 . 23 × 10 − 6 ( $ ) time - point 9 , 1 . 33 × 10 − 6 ( $ ) time - point 10 , 7 . 14 × 10 − 5 ( $ ) time - point 11 . Lower symbols : statistics for siTBL # 2 , P - values : 3 . 94 × 10 − 3 ( * ) time - point 5 , 0 . 0143 ( * ) time - point 6 , 0 . 0342 ( * ) time - point 7 , 1 . 45 × 10 − 3 ( * ) time - point 8 , 1 . 27 × 10 − 3 ( * ) time - point 9 , 2 . 38 × 10 − 3 ( * ) time - point 10 ; 0 . 01 ( # ) time - point 5 , 0 . 0362 ( # ) time - point 6 , 1 . 22 × 10 − 6 ( # ) time - point 8 , 1 . 28 × 10 − 5 ( # ) time - point 9 , 5 . 84 × 10 − 5 ( # ) time - point 10 ; 0 . 0118 ( $ ) time - point 5 , 0 . 0208 ( $ ) time - point 6 , 0 . 0291 ( $ ) time - point 7 , 4 . 52 × 10 − 7 ( $ ) time - point 8 , 6 . 04 × 10 − 8 ( $ ) time - point 9 , 1 . 43 × 10 − 7 ( $ ) time - point 10 , 4 . 83 × 10 − 3 ( $ ) time - point 11 . Two - way ANOVA with Bonferroni post - 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Extracellular flux measurement of siRNA-transfected Capan-1 cells with glycolysis stress test assay (48 h post-transfection); 2-DG: 2-deoxyglucose; ECAR: extracellular acidification rate. n = 6-8 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2) $. Upper symbols: statistics for siTBL#1, P-values: 2.18 × 10−3 (*) time-point 8, 9.83 × 10−3 (*) time-point 9, 0.0102 (*) time-point 10, 0.044 (*) time-point 11; 2.42 × 10−6 (#) time-point 8, 1.47 × 10−4 (#) time-point 9, 3.17 × 10−4 (#) time-point 10, 0.0277 (#) time-point 11; 9.44 × 10−7 ($) time-point 8, 1.23 × 10−6 ($) time-point 9, 1.33 × 10−6 ($) time-point 10, 7.14 × 10−5 ($) time-point 11. Lower symbols: statistics for siTBL#2, P-values: 3.94 × 10−3 (*) time-point 5, 0.0143 (*) time-point 6, 0.0342 (*) time-point 7, 1.45 × 10−3 (*) time-point 8, 1.27 × 10−3 (*) time-point 9, 2.38 × 10−3 (*) time-point 10; 0.01 (#) time-point 5, 0.0362 (#) time-point 6, 1.22 × 10−6 (#) time-point 8, 1.28 × 10−5 (#) time-point 9, 5.84 × 10−5 (#) time-point 10; 0.0118 ($) time-point 5, 0.0208 ($) time-point 6, 0.0291 ($) time-point 7, 4.52 × 10−7 ($) time-point 8, 6.04 × 10−8 ($) time-point 9, 1.43 × 10−7 ($) time-point 10, 4.83 × 10−3 ($) time-point 11. Two-way ANOVA with Bonferroni post-test.


Gene expression microarray from Capan - 1 cells transfected with siRNA . Heatmap shows genes annotated to KEGG pathway hsa04110 " Cell Cycle " and / or gene ontology cluster GO : 0007049 " Cell Cycle " .

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

Gene expression microarray from Capan-1 cells transfected with siRNA. Heatmap shows genes annotated to KEGG pathway hsa04110 "Cell Cycle" and/or gene ontology cluster GO:0007049 "Cell Cycle".


Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice .

Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice.


Chromatin immunoprecipitation from Capan - 1 cells with or without siRNA - mediated knockdown of TBL1 . IgG and primers for PIK3CA coding region served as a negative control ; histone H3 served as a positive control .

Chromatin immunoprecipitation from Capan-1 cells with or without siRNA-mediated knockdown of TBL1. IgG and primers for PIK3CA coding region served as a negative control; histone H3 served as a positive control.


Proliferation time course assay in control and TBL1 shRNA - transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K . Cells were stained with crystal violet , and absorbance was measured at 595 nm . n = 9 cell culture wells per group ; statistically significant at P < 0 . 05 for indicated comparisons : shNC pBabe - puro vs . shNC pBabe - PI3K ( * ) , shTBL1 pBabe - puro vs . sbTBL1 pBabe - PI3K ( # ) , shNC pBabe - puro vs . shTBL1 pBabe - puro ( $ ) , shNC pBabe - PI3K vs . shTBL1 pBabe - PI3K ( † ) ; P - values : day 2 : 2 . 01 × 10 − 3 ( # ) , 4 . 11 × 10 − 9 ( $ ) , 5 . 77 × 10 − 3 ( † ) ; day 3 : 4 . 50 × 10 − 5 ( * ) , 1 . 01 × 10 − 9 ( # ) , 1 . 71 × 10 − 30 ( $ ) , 1 . 22 × 10 − 6 ( † ) ; day 4 : 5 . 61 × 10 − 22 ( * ) , 1 . 10 × 10 − 45 ( # ) , 6 . 65 × 10 − 62 ( $ ) ; two - way ANOVA with Bonferroni post - 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proliferation time course assay in control and TBL1 shRNA-transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K. Cells were stained with crystal violet, and absorbance was measured at 595 nm. n = 9 cell culture wells per group; statistically significant at P < 0.05 for indicated comparisons: shNC pBabe-puro vs. shNC pBabe-PI3K (*), shTBL1 pBabe-puro vs. sbTBL1 pBabe-PI3K (#), shNC pBabe-puro vs. shTBL1 pBabe-puro ($), shNC pBabe-PI3K vs. shTBL1 pBabe-PI3K (†); P-values: day 2: 2.01 × 10−3 (#), 4.11 × 10−9 ($), 5.77 × 10−3 (†); day 3: 4.50 × 10−5 (*), 1.01 × 10−9 (#), 1.71 × 10−30 ($), 1.22 × 10−6 (†); day 4: 5.61 × 10−22 (*), 1.10 × 10−45 (#), 6.65 × 10−62 ($); two-way ANOVA with Bonferroni post-test.


Matrigel invasion assay in same cells as in ( D ) . Invading cells per microscopy field were counted . n = 6 microscopy fields per group ; statistically significant at P < 0 . 05 ; P - values : 6 . 64 × 10 − 4 ( * ) , 1 . 35 × 10 − 7 ( # ) , 6 . 94 × 10 − 5 ( $ ) ; two - way ANOVA with Bonferroni post - 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 O O O O O O O O

Matrigel invasion assay in same cells as in (D). Invading cells per microscopy field were counted. n = 6 microscopy fields per group; statistically significant at P < 0.05; P-values: 6.64 × 10−4 (*), 1.35 × 10−7 (#), 6.94 × 10−5 ($); two-way ANOVA with Bonferroni post-test.


mRNA expression of PIK3CA in human patient samples ; significant difference ( P < 0 . 05 ) between tumor and normal tissue * P = 2 . 13 × 10 − 4 determined by paired two - tailed Student ' s t - test , n = 18 patients .

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

mRNA expression of PIK3CA in human patient samples; significant difference (P < 0.05) between tumor and normal tissue *P = 2.13 × 10−4 determined by paired two-tailed Student's t-test, n = 18 patients.


Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue ( normalized to 18S rRNA and relative to a pooled organ donor sample ) ; n = 17 patients ; P - value determined by testing for slope of regression line ≠ 0 .

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

Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue (normalized to 18S rRNA and relative to a pooled organ donor sample); n = 17 patients; P-value determined by testing for slope of regression line ≠ 0.


Survival analysis in 105 patients with PDAC : High PIK3CA expression is significantly associated with shorter post - resection survival . P - value determined by log - rank test as described in Materials and Methods .

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

Survival analysis in 105 patients with PDAC: High PIK3CA expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.


A - D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6 / N mice . Six days later , tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA . ( A - C ) : Data plotted as mean ± SEM ; n = 4 animals in control ( shNC ) group and n = 3 animals in knockdown ( shTBL1 ) group ; significantly different ( P < 0 . 05 ) between control ( shNC ) and knockdown ( shTBL1 ) * . ( A ) Tumor volume determined by transcutaneous measurement with digital calipers ; P - values : 9 . 21 × 10 − 3 at day 15 , 6 . 02 × 10 − 4 at day 18 , 2 . 49 × 10 − 4 at day 20 ; two - way ANOVA with Bonferroni post - test . ( B ) Luminescence of tumors after intraperitoneal injection of firefly d - luciferin ; P = 0 . 0133 at day 20 ; two - way ANOVA with Bonferroni post - test . ( C ) Tumor mass at necropsy ; P = 0 . 0299 , one - sided Welch ' s t - test . ( D ) Luminescence images of one representative mouse in each group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6/N mice. Six days later, tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA. (A-C): Data plotted as mean ± SEM; n = 4 animals in control (shNC) group and n = 3 animals in knockdown (shTBL1) group; significantly different (P < 0.05) between control (shNC) and knockdown (shTBL1) *. (A) Tumor volume determined by transcutaneous measurement with digital calipers; P-values: 9.21 × 10−3 at day 15, 6.02 × 10−4 at day 18, 2.49 × 10−4 at day 20; two-way ANOVA with Bonferroni post-test. (B) Luminescence of tumors after intraperitoneal injection of firefly d-luciferin; P = 0.0133 at day 20; two-way ANOVA with Bonferroni post-test. (C) Tumor mass at necropsy; P = 0.0299, one-sided Welch's t-test. (D) Luminescence images of one representative mouse in each group.


E - G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6 / Nmice . Seven days later , mice were treated with 20 mg / kg gemcitabine delivered by intraperitoneal injection ; n = 4 animals in shNC + GEM group , n = 5 animals in all other groups ; significantly different ( P < 0 . 05 ) between groups : shNC vs . shNC + GEM ( * ) , shNC vs . shTBL1 ( # ) , shNC vs . shTBL1 + GEM ( $ ) , shNC + GEM vs . shTBL1 ( % ) , shNC + GEM vs . shTBL + GEM ( † ) ; shTBL1 vs . shTBL1 + GEM ( ‡ ) ; two - way ANOVA with Bonferroni post - test . ( E ) Tumor volume determined by transcutaneous measurement with digital calipers . P - values : day 17 : 8 . 48 × 10 − 3 ( # ) , 1 . 07 × 10 − 3 ( $ ) , day 20 : 3 . 07 × 10 − 3 ( * ) , 9 . 37 × 10 − 6 ( # ) , 1 . 56 × 10 − 8 ( $ ) , 0 . 0239 ( † ) . ( F ) Tumor volume relative to the volume at day 7 post - implantation ; P - values : day 17 : 3 . 94 × 10 − 3 ( $ ) , day 20 : 1 . 09 × 10 − 3 ( * ) , 1 . 39 × 10 − 7 ( $ ) , 0 . 01195 ( % ) , 3 . 12 × 10 − 6 ( ‡ ) . ( G ) Area under curve for relative tumor volume ; P = 0 . 0195 ( ‡ ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection; n = 4 animals in shNC+GEM group, n = 5 animals in all other groups; significantly different (P < 0.05) between groups: shNC vs. shNC+GEM (*), shNC vs. shTBL1 (#), shNC vs. shTBL1+GEM ($), shNC+GEM vs. shTBL1 (%), shNC+GEM vs. shTBL+GEM (†); shTBL1 vs. shTBL1+GEM (‡); two-way ANOVA with Bonferroni post-test. (E) Tumor volume determined by transcutaneous measurement with digital calipers. P-values: day 17: 8.48 × 10−3 (#), 1.07 × 10−3 ($), day 20: 3.07 × 10−3 (*), 9.37 × 10−6 (#), 1.56 × 10−8 ($), 0.0239 (†). (F) Tumor volume relative to the volume at day 7 post-implantation; P-values: day 17: 3.94 × 10−3 ($), day 20: 1.09 × 10−3 (*), 1.39 × 10−7 ($), 0.01195 (%), 3.12 × 10−6 (‡). (G) Area under curve for relative tumor volume; P = 0.0195 (‡).


TBL1 - depletion leads to reduction in PI3 kinase p110α and cell cycle - associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6 / Nmice . Seven days later , mice were treated with 20 mg / kg gemcitabine delivered by intraperitoneal injection . After 21 days , mice were sacrificed , tumors were removed and proteins were extracted and immunoblotted .

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

TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection. After 21 days, mice were sacrificed, tumors were removed and proteins were extracted and immunoblotted.


DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( A ) The scatter plot shows the apparent Young ' s moduli obtained by AFM analysis . Bars represent the median values of the distributions . The immunoblot shows UBTD1 siRNA knock - down efficiency .

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

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (A) The scatter plot shows the apparent Young's moduli obtained by AFM analysis. Bars represent the median values of the distributions. The immunoblot shows UBTD1 siRNA knock-down efficiency.


DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( B ) Immunoblots ( left ) and quantification ( right ) show levels of GST - ROCK associated GTP - bound RhoA ( RhoA - GTP ) and total RhoA in lysates ( RhoA ) in confluent cells . Immunoblot of UBTD1 shows the level of siRNA depletion .

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

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (B) Immunoblots (left) and quantification (right) show levels of GST-ROCK associated GTP-bound RhoA (RhoA-GTP) and total RhoA in lysates (RhoA) in confluent cells. Immunoblot of UBTD1 shows the level of siRNA depletion.


DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( C ) Representative confocal images of focal adhesion ( paxillin ) and actin cytoskeleton ( phalloidin ) and outlines of focal adhesions using threshold - based image . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . ( D ) Cell area , number and area of focal adhesion were quantified ( ImageJ ) using the analysis as shown ( C ) .

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

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (C) Representative confocal images of focal adhesion (paxillin) and actin cytoskeleton (phalloidin) and outlines of focal adhesions using threshold-based image. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. (D) Cell area, number and area of focal adhesion were quantified (ImageJ) using the analysis as shown (C).


DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . Sub - confluent DU145 ( E ) cells were seeded on two matrices of different stiffnesses ( 4 and 12 kPa ) . Representative heat map ( left ) and quantification ( right ) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel .

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

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent DU145 (E) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.


A549 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . Sub - confluent A549 ( F ) cells were seeded on two matrices of different stiffnesses ( 4 and 12 kPa ) . Representative heat map ( left ) and quantification ( right ) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel .

A549 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. Sub-confluent A549 (F) cells were seeded on two matrices of different stiffnesses (4 and 12 kPa). Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 4kPa or 12kPa hydrogel.


DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( G ) Confluent DU145 cells were seeded on 12 kPa matrix . Representative heat map ( left ) and quantification ( right ) showing contractile forces generate by cells plated on 12kPa hydrogel .

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

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (G) Confluent DU145 cells were seeded on 12 kPa matrix. Representative heat map (left) and quantification (right) showing contractile forces generate by cells plated on 12kPa hydrogel.


DU145 cells were transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool or siUBTD1single1 or single2 ) for 24 h and plated on fibronectin - coated glass coverslips . ( H ) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness . DU145 cells were grown on collagen / acrylamide - coated glass coverslips ( soft ) or directly on glass coverslips ( stiff ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image .

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

DU145 cells were transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool or siUBTD1single1 or single2) for 24 h and plated on fibronectin-coated glass coverslips. (H) Representative confocal images of YAP and UBTD1 localization on two matrices of different stiffness. DU145 cells were grown on collagen/acrylamide-coated glass coverslips (soft) or directly on glass coverslips (stiff). Nuclei were stained with DAPI (Blue) on the MERGE image.


( A ) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20 % to 100 % confluence showing the increased expression of UBTD1 and β - catenin with cell confluency . E - cadherin was used as a positive control . Tubulin was used as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1 and β-catenin with cell confluency. E-cadherin was used as a positive control. Tubulin was used as a loading control.


, immunofluorescent labelling of UBTD1 and E - cadherin ( B ) at the cell - cell contact in confluent DU145 cells and analyzed by confocal microscopy . Right , representative histogram of a co - localization profile prepared with RGB profiler plugin of ImageJ .

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

, immunofluorescent labelling of UBTD1 and E-cadherin (B) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.


immunofluorescent labelling of UBTD1 and β - catenin ( C ) at the cell - cell contact in confluent DU145 cells and analyzed by confocal microscopy . Right , representative histogram of a co - localization profile prepared with RGB profiler plugin of ImageJ .

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

immunofluorescent labelling of UBTD1 and β-catenin (C) at the cell-cell contact in confluent DU145 cells and analyzed by confocal microscopy. Right, representative histogram of a co-localization profile prepared with RGB profiler plugin of ImageJ.


Proximal ligation assay monitoring ( left ) and quantification ( right ) of UBTD1 association with E - cadherin or β - catenin in DU145 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image .

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

Proximal ligation assay monitoring (left) and quantification (right) of UBTD1 association with E-cadherin or β-catenin in DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). Nuclei were stained with DAPI (Blue) on the MERGE image.


Immunoblots of UBTD1 , β - catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA ( control , siCTRL ; UBTD1 : siUBTD1pool ) . EGFR , RhoGDI and Lamin A / C were used as controls for the membrane ( M ) , cytosol ( C ) and nucleus ( N ) compartments , respectively .

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

Immunoblots of UBTD1, β-catenin and YAP after subcellular fractionation of confluent DU145 cells transfected with the indicated siRNA (control, siCTRL; UBTD1: siUBTD1pool). EGFR, RhoGDI and Lamin A/C were used as controls for the membrane (M), cytosol (C) and nucleus (N) compartments, respectively.


Co - immunoprecipitation between endogenous β - catenin and UBTD1 was performed in confluent DU145 cells . IgG isotype was used as a negative control .

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

Co-immunoprecipitation between endogenous β-catenin and UBTD1 was performed in confluent DU145 cells. IgG isotype was used as a negative control.


( G ) Representative confocal images of β - catenin and UBTD1 in DU145 under resting ( Ctrl ) , after calcium chelator treatment ( EGTA ) and recovery ( Rec ) conditions . Nuclei were stained with DAPI ( Blue ) on the MERGE image .

O 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

(G) Representative confocal images of β-catenin and UBTD1 in DU145 under resting (Ctrl), after calcium chelator treatment (EGTA) and recovery (Rec) conditions. Nuclei were stained with DAPI (Blue) on the MERGE image.


( H ) Representative confocal images of β - catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment ( 24 h ) . Nuclei were stained with DAPI ( Blue ) on the MERGE image .

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

(H) Representative confocal images of β-catenin and UBTD1 in DU145 after cell scattering induced by HGF treatment (24 h). Nuclei were stained with DAPI (Blue) on the MERGE image.


Immunoblot of DU145 and A549 cells at various cell densities ranging from 20 % to 100 % confluence showing the increased expression of UBTD1 , p - YAP ( ser127 ) and YAP levels with the cell confluency . Tubulin was used as a loading control .

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

Immunoblot of DU145 and A549 cells at various cell densities ranging from 20% to 100% confluence showing the increased expression of UBTD1, p-YAP (ser127) and YAP levels with the cell confluency. Tubulin was used as a loading control.


DU145 cells were transfected for 48h with the indicated siRNA ( control , siCTRL ; UBTD1 , siUBTD1pool ) . ( B ) Immunoblots of p - YAP ( ser127 ) , YAP and UBTD1 ( left ) and quantification of YAP and p - YAP / YAP levels ( right ) in control or UBTD1 - depleted DU145 at low and high cell density . Immunoblot of UBTD1 shows the level of siRNA - mediated depletion . Actin was used as a loading controls . , immunoblots for the loading control ( actin ) and UBTD1 knock - down efficiency ( UBTD1 ) are duplicated because they belong to the same experiment shown in both panels .

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

DU145 cells were transfected for 48h with the indicated siRNA (control, siCTRL; UBTD1, siUBTD1pool). (B) Immunoblots of p-YAP (ser127), YAP and UBTD1 (left) and quantification of YAP and p-YAP/YAP levels (right) in control or UBTD1-depleted DU145 at low and high cell density. Immunoblot of UBTD1 shows the level of siRNA-mediated depletion. Actin was used as a loading controls. , immunoblots for the loading control (actin) and UBTD1 knock-down efficiency (UBTD1) are duplicated because they belong to the same experiment shown in both panels.