A - G ) Slices through electron cryotomograms ( left panels ) , central slices of subtomogram averages ( middle panels ) , schematic representations ( right panels ) of MS - C - complexes ( arrows on slices ) of different mutants in H . pylori fliP * . green arrows in ( A D ) point to ~ 53 - nm ~ 67 - nm rings . Scale bars for cryotomogram slices are 50 nm , 20 nm for STAs . Empty panels indicate that there were not enough examples to produce a STA .

A-G) Slices through electron cryotomograms (left panels), central slices of subtomogram averages (middle panels), and schematic representations (right panels) of the MS- and C-complexes (white arrows on the slices) of different mutants in H. pylori fliP*. Light and dark green arrows in (A and D) point to the ~53-nm and ~67-nm cytoplasmic rings. Scale bars for cryotomogram slices are 50 nm, and 20 nm for STAs. Empty panels indicate that there were not enough examples to produce a STA.


MITOL - KO MEFs was vulnerable to ER stress . Control MEFs ( MITOLF / F ) MITOL - KO MEFs ( MITOL - / - ) were treated with DMSO as control , 0 . 8 µM thapsigargin ( Tg ) , 0 . 7 µg / mL tunicamycin ( Tu ) or 1 . 2 µg / mL brefeldin A ( Br ) for 24 hours . experiments using Tu below were performed at of 0 . 7 µg / mL . relative viability shows a between survival of MEFs in absence presence of ER stress inducers . Viable were detected by viability using counting kit - 8 .

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MITOL-KO MEFs was vulnerable to ER stress. Control MEFs (MITOLF/F) and MITOL-KO MEFs (MITOL-/-) were treated with DMSO as control, 0.8 µM thapsigargin (Tg), 0.7 µg/mL tunicamycin (Tu) or 1.2 µg/mL brefeldin A (Br) for 24 hours. All experiments using Tu below were performed at concentration of 0.7 µg/mL. The relative cell viability shows a ratio between the survival of MEFs in the absence and presence of ER stress inducers. Viable cells were detected by cell viability assay using cell counting kit-8.


MITOL KO increased ER stress - induced apoptosis . MEFs were treated with indicated ER stress inducers at same as Figure 1A for 18 hours stained with Annexin V - FITC for detection of apoptosis using flow cytometry .

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MITOL KO increased ER stress-induced apoptosis. MEFs were treated with indicated ER stress inducers at same concentration as Figure 1A for 18 hours and stained with Annexin V-FITC for the detection of apoptosis using flow cytometry.


MITOL re - expression in MITOL - KO MEFs rescued ER stress vulnerability . MEFs were transfected with MITOL - coding vector ( WT ) , MITOL C65 / 68S - coding vector ( CS ) , or empty vector ( Vec ) 24 hours prior to Tu treatment for 18 hours , followed by immunoblotting with indicated antibodies . caspase - 3 ( cC3 ) PARP ( cPARP ) were used as apoptosis markers .

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MITOL re-expression in MITOL-KO MEFs rescued ER stress vulnerability. MEFs were transfected with MITOL-coding vector (WT), MITOL C65/68S-coding vector (CS), or empty vector (Vec) 24 hours prior to Tu treatment for 18 hours, followed by immunoblotting with indicated antibodies. Cleaved caspase-3 (cC3) and cleaved PARP (cPARP) were used as apoptosis markers.


MITOL KO enhanced ER stress - induced Bax translocation cytochrome c release . Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours , followed by immunoblotting with indicated antibodies .

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MITOL KO enhanced ER stress-induced Bax translocation and cytochrome c release. Mitochondria or cytosol fraction was isolated from MEFs treated with Tu for 18 hours, followed by immunoblotting with indicated antibodies.


MITOL KO induced excessive mitochondrial depolarization by ER stress . MEFs transfected with indicated vectors were treated with Tu for 18 hours then stained with TMRM , followed by flow cytometric .

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MITOL KO induced excessive mitochondrial depolarization by ER stress. MEFs transfected with indicated vectors were treated with Tu for 18 hours and then stained with TMRM, followed by flow cytometric analysis.


IRE1α inhibition compensated MITOL - KO MEFs for vulnerability to ER stress . MEFs were transfected with scramble or siIRE1α ( # 1 , # 2 ) . After 24 hours , these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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IRE1α inhibition compensated MITOL-KO MEFs for vulnerability to ER stress. MEFs were transfected with scramble or siIRE1α (#1, #2). After 24 hours, these MEFs were exposed with Tu for 24 hours , followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MEFs were treated with 4µ8C ( 25 µM ) for 2 hours prior to Tu treatment for 24 hours ( B ) , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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MEFs were treated with 4µ8C (25 µM) for 2 hours prior to Tu treatment for 24 hours (B), followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MITOL KO did not affect autophosphorylation of IRE1α . MEFs were exposed with Tu for indicated periods phosphorylated IRE1α was detected by immunoblotting after Phos - tag SDS - PAGE .

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MITOL KO did not affect the autophosphorylation of IRE1α. MEFs were exposed with Tu for indicated periods and phosphorylated IRE1α was detected by immunoblotting after Phos-tag SDS-PAGE.


MITOL KO enhanced RIDD xbp1 splicing . MEFs were incubated with Tu for 4 hours . Expression of various UPR associated genes were determined by qRT -

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MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR


MITOL KO enhanced RIDD xbp1 splicing . MEFs were incubated with Tu for 4 hours . MEFs were transfected with either miR - 17 or miR - 34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours , followed by luciferase reporter ( F ) . Error bars represent SD ( n = 3 ) .

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MITOL KO enhanced RIDD and xbp1 splicing. MEFs were incubated with Tu for 4 hours. MEFs were transfected with either miR-17 or miR-34 luciferase reporter vector 24 hours prior to Tu treatment for 4 hours, followed by luciferase reporter assay (F). Error bars represent SD (n=3).


MEFs were incubated with Tu for 4 hours . Expression of various UPR associated genes were determined by qRT -

MEFs were incubated with Tu for 4 hours. Expression levels of various UPR associated genes were determined by qRT-PCR


IRE1α - mediated JNK phosphorylation was prolonged by MITOL KO . MEFs were incubated with Tu for indicated periods phosphorylated JNK was detected by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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IRE1α-mediated JNK phosphorylation was prolonged by MITOL KO. MEFs were incubated with Tu for indicated periods and phosphorylated JNK was detected by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


JNK inhibition reduced ER stress - induced apoptosis of MITOL - KO MEFs . MEFs were treated with 20 µM JNK inhibitor - II ( JNK inh ) 2 hours prior to Tu treatment for 24 hours , followed by immunoblotting with indicated antibodies . Error bars represent SD ( n = 3 ) .

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JNK inhibition reduced ER stress-induced apoptosis of MITOL-KO MEFs. MEFs were treated with 20 µM JNK inhibitor-II (JNK inh) 2 hours prior to Tu treatment for 24 hours, followed by immunoblotting with indicated antibodies. Error bars represent SD (n=3).


MITOL KO facilitated IRE1α oligomerization . MEFs were transfected with IRE1α - GFP expression vector for 24 hours prior to Tu treatment for indicated periods ( A ) . right panels show 5 - fold magnification of boxed regions . Percentages of with IRE1α - foci were calculated from 100 by visual inspection in each independent experiment ( A ) . To evaluate oligomerization level of endogenous IRE1α , without any transfection were solubilized separated by sucrose density - gradient centrifugation , followed by immunoblotting with anti - IRE1α antibody ( B ) .

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MITOL KO facilitated IRE1α oligomerization. MEFs were transfected with IRE1α-GFP expression vector for 24 hours prior to Tu treatment for indicated periods (A). The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment (A). To evaluate the oligomerization level of endogenous IRE1α, cells without any transfection were solubilized and separated by sucrose density-gradient centrifugation, followed by immunoblotting with anti-IRE1α antibody (B).


IRE1α oligomers were stabilized by MITOL KO . MEFs transfected with IRE1α - GFP were treated with Tu . After 4 hours , these were washed with PBS subsequently re - fed with fresh media for indicated periods . right panels show 5 - fold magnification of boxed regions . Percentages of with IRE1α - foci were calculated from 100 by visual inspection at indicated periods after wash out of Tu in each independent experiment . Scale bar represents 10 µm .

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IRE1α oligomers were stabilized by MITOL KO. MEFs transfected with IRE1α-GFP were treated with Tu. After 4 hours, these cells were washed with PBS and subsequently re-fed with fresh media for indicated periods. The right panels show 5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection at indicated periods after wash out of Tu in each independent experiment. Scale bar represents 10 µm.


Sustained IRE1α autophosphorylation after wash out of Tu in MITOL - KO MEFs . MEFs were treated with Tu for 4 hours washed with PBS re - fed with fresh media for indicated periods , following by immunoblotting anti - IRE1α antibody after Phos - tag SDS - PAGE . Error bars represent SD ( n = 3 ) .

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Sustained IRE1α autophosphorylation after wash out of Tu in MITOL-KO MEFs. MEFs were treated with Tu for 4 hours and washed with PBS and re-fed with fresh media for indicated periods, following by immunoblotting anti-IRE1α antibody after Phos-tag SDS-PAGE. Error bars represent SD (n=3).


MITOL deletion promoted interaction between IRE1α BIM under ER stress . MITOL - depleted HEK293 ( KO ) were generated using CRISPR - Cas9 system . were co - transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours . were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

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MITOL deletion promoted the interaction between IRE1α and BIM under ER stress. MITOL-depleted HEK293 cells (KO) were generated using CRISPR-Cas9 system. Cells were co-transfected with indicated vectors 24 hours prior to Tu treatment for 12 hours. Cells were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Interaction between MITOL IRE1α . Lysates of HEK293 were subjected to immunoprecipitation with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies ( A ) .

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Interaction between MITOL and IRE1α. Lysates of HEK293 cells were subjected to immunoprecipitation with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies (A).


Interaction between MITOL IRE1α . Lysates of HEK293 co - transfected with indicated vectors were subjected to immunoprecipitation , followed by immunoblotting with indicated antibodies ( B ) .

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Interaction between MITOL and IRE1α. Lysates of HEK293 cells co-transfected with indicated vectors were subjected to immunoprecipitation, followed by immunoblotting with indicated antibodies (B).


Interaction between MITOL IRE1α . GST - pull - down of IRE1α or MITOL was performed on lysates of MEFs , followed by immunoblotting with indicated antibodies ( C , D ) . N - ter : 1 - 90 aa of MITOL , Loop : 158 - 210 aa of MITOL , C - ter : 241 - 278 aa of MITOL , 465 - 977 aa of IRE1α .

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Interaction between MITOL and IRE1α. GST-pull-down assay of IRE1α or MITOL was performed on lysates of MEFs, followed by immunoblotting with indicated antibodies (C, D). N-ter: 1-90 aa of MITOL, Loop: 158-210 aa of MITOL, C-ter: 241-278 aa of MITOL, 465-977 aa of IRE1α.


MITOL ubiquitylated IRE1α . Lysates of HEK293 immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

MITOL ubiquitylated IRE1α. Lysates of HEK293 cells immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL ubiquitylated IRE1α . Lysates of HEK293 transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

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MITOL ubiquitylated IRE1α. Lysates of HEK293 cells transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL ubiquitylated IRE1α . Lysates of MEFs ( G ) transfected with indicated vectors , were immunoprecipitated with indicated antibody , followed by immunoblotting with indicated antibodies .

MITOL ubiquitylated IRE1α. Lysates of MEFs (G) transfected with indicated vectors, were immunoprecipitated with indicated antibody, followed by immunoblotting with indicated antibodies.


MITOL adds lysine 63 - linked polyubiquitin chains to IRE1α . Lysates of HEK293 transfected with indicated vectors were immunoprecipitated with indicated antibody . IRE1α ubiquitylation was

MITOL adds lysine 63-linked polyubiquitin chains to IRE1α. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with indicated antibody. IRE1α ubiquitylation was detected


MITOL added polyubiquitin chains to K481 of IRE1α . Lysates of HEK293 transfected with each lysine mutant of IRE1α indicated vectors were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies . 481 : K481R , 545 : K545R , 568 : K568R .

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MITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R, 545: K545R, 568: K568R.


IRE1α K481R interacted with MITOL . Lysates of HEK293 transfected with indicated vectors were immunoprecipitated with anti - FLAG antibody , followed by immunoblotting with indicated antibodies .

IRE1α K481R interacted with MITOL. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with indicated antibodies.


Overexpression of IRE1α K481R induced apoptosis . MEFs were transfected with indicated vectors . After 24 hours , these were stained with Annexin V -

Overexpression of IRE1α K481R induced apoptosis. MEFs were transfected with indicated vectors. After 24 hours, these cells were stained with Annexin V-FITC


Overexpression of IRE1α K481R induced apoptosis . or subjected by immunoblotting with indicated antibodies ( D ) .

Overexpression of IRE1α K481R induced apoptosis. or subjected by immunoblotting with indicated antibodies (D).


Enhanced RIDD activity in IRE1α K481R . MEFs were transfected with indicated vectors 24 hours prior to ( E , F ) . RIDD activity was measured by qRT - PCR .

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Enhanced RIDD activity in IRE1α K481R. MEFs were transfected with indicated vectors 24 hours prior to analysis (E, F). The RIDD activity was measured by qRT-PCR.


Hyper - oligomerization of IRE1α K481R . MEFs were transfected with IRE1α - GFP or IRE1α K481R - GFP . After 24 hours , these were exposed with Tu for indicated periods GFP signals were observed . right panels show 5 . 5 - fold magnification of boxed regions . Percentages of with IRE1α - foci were calculated from 100 by visual inspection in each independent experiment .

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Hyper-oligomerization of IRE1α K481R. MEFs were transfected with IRE1α-GFP or IRE1α K481R-GFP. After 24 hours, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5-fold magnification images of the boxed regions. Percentages of cells with IRE1α-foci were calculated from 100 cells by visual inspection in each independent experiment.


Increased interaction of IRE1α K481R with BIM . transfected with indicated vectors 24 hours prior to immunoprecipitation , followed by immunoblotting with indicated antibodies .

Increased interaction of IRE1α K481R with BIM. Cells transfected with indicated vectors 24 hours prior to immunoprecipitation, followed by immunoblotting with indicated antibodies.


Accumulations of MITOL IRE1α in MAM fraction . Each organelle fraction was isolated from MEFs by percoll density - gradient centrifugation , followed by immunoblotting with indicated antibodies . mito : pure mitochondria , micro : microsome , cyto : pure cytosol .

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Accumulations of MITOL and IRE1α in the MAM fraction. Each organelle fraction was isolated from MEFs by percoll density-gradient centrifugation, followed by immunoblotting with indicated antibodies. mito: pure mitochondria, micro: microsome, cyto: pure cytosol.


High solubility of MITOL IRE1α in low dose digitonin . Raft - like structure including MAM was solubilized from crude mitochondrial fraction of MEFs with indicated of digitonin . S : supernatant , P : pellet .

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High solubility of MITOL and IRE1α in the low dose digitonin. Raft-like structure including MAM was solubilized from the crude mitochondrial fraction of MEFs with indicated concentration of digitonin. S: supernatant, P: pellet.


IRE1α regulation by MITOL occurred in MAM - rich mitochondrial fraction . Crude mitochondrial fraction containing MAM ( Mito plus MAM ) fraction containing ER ( Cyto plus ER ) were isolated from HEK293 transfected with indicated vector subsequently immunoprecipitation was performed to detect interaction between MITOL

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

IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the interaction between MITOL and IRE1α


IRE1α regulation by MITOL occurred in MAM - rich mitochondrial fraction . Crude mitochondrial fraction containing MAM ( Mito plus MAM ) fraction containing ER ( Cyto plus ER ) were isolated from HEK293 transfected with indicated vector subsequently immunoprecipitation was performed to detect level of IRE1α ubiquitylation ( D ) .

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IRE1α regulation by MITOL occurred in the MAM-rich mitochondrial fraction. Crude mitochondrial fraction containing MAM (Mito plus MAM) and the cytosolic fraction containing ER (Cyto plus ER) were isolated from HEK293 cells transfected with indicated vector and subsequently immunoprecipitation assay was performed to detect the level of IRE1α ubiquitylation (D).


Knockdown of PACS2 impaired interaction of MITOL with IRE1α . HEK293 were transfected with either scramble or siPACS2 indicated vectors for 24 hours prior to immunoprecipitation .

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Knockdown of PACS2 impaired the interaction of MITOL with IRE1α. HEK293 cells were transfected with either scramble RNA or siPACS2 and indicated vectors for 24 hours prior to immunoprecipitation assay.


Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL . HEK293 were transfected with either scramble , siPACS2 indicated vectors 24 hours prior to immunoprecipitation .

Knockdown of PACS2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siPACS2 and indicated vectors 24 hours prior to immunoprecipitation assay.


Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL . HEK293 were transfected with either scramble , siMfn2 indicated vectors 24 hours prior to immunoprecipitation .

Knockdown of Mfn2 inhibited IRE1α ubiquitylation by MITOL. HEK293 cells were transfected with either scramble RNA, siMfn2 and indicated vectors 24 hours prior to immunoprecipitation assay.


MITOL ubiquitylated IRE1α in spinal cord of mice . Lysates of spinal cord in mice were immunoprecipitated with anti - IRE1α antibody , followed by immunoblotting with indicated antibodies . WT : MITOLF / F mice , MITOLnestin : MITOLF / F , Nestin - Cre mice .

MITOL ubiquitylated IRE1α in the spinal cord of mice. Lysates of the spinal cord in mice were immunoprecipitated with anti-IRE1α antibody, followed by immunoblotting with indicated antibodies. WT: MITOLF/F mice, MITOLnestin: MITOLF/F, Nestin-Cre mice.


Loss of MITOL led to hyper - oligomerization of IRE1α in spinal cord under ER stress . 3 month - old WT or MITOLnestin mice were treated with 1 mg / kg Tu for 24 hours . Spinal cord was solubilized with NP - 40 lysis buffer separated by sucrose - density gradient centrifugation .

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Loss of MITOL led to hyper-oligomerization of IRE1α in the spinal cord under ER stress. 3 month-old WT or MITOLnestin mice were treated with 1 mg/kg Tu for 24 hours. Spinal cord was solubilized with NP-40 lysis buffer and separated by sucrose-density gradient centrifugation.


MITOL deletion resulted in hyper - activation of IRE1α RNase in ER stress . WT or MITOLnestin mice were injected with Tu for 24 hours spinal cord was analyzed by qRT - PCR .

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MITOL deletion resulted in hyper-activation of IRE1α RNase in ER stress. WT or MITOLnestin mice were injected with Tu for 24 hours and spinal cord was analyzed by qRT-PCR.


Increased ER stress - induced apoptosis in MITOL - KO spinal cord . 3 month - old WT or MITOLnestin mice were treated with Tu for 24 hours each spinal cord was stained using TUNEL hoechst ( F ) Arrowheads indicate representative neurons .

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Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using TUNEL and hoechst (F) Arrowheads indicate the representative neurons.


Increased ER stress - induced apoptosis in MITOL - KO spinal cord . 3 month - old WT or MITOLnestin mice were treated with Tu for 24 hours each spinal cord was stained using cresyl violet ( G ) . Arrowheads indicate representative neurons .

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Increased ER stress-induced apoptosis in the MITOL-KO spinal cord. 3 month-old WT or MITOLnestin mice were treated with Tu for 24 hours and each spinal cord was stained using cresyl violet (G). Arrowheads indicate the representative neurons.


Dyf phenotype of ts - che - 3 mutant was confirmed , with grown at permissive ( 15ºC ) showing ability to uptake dye , while those raised at restrictive ( 25ºC ) are Dyf . Scale bar , 20

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The Dyf phenotype of the ts-che-3 mutant was confirmed, with animals grown at the permissive temperature (15ºC) showing the ability to uptake dye, while those raised at the restrictive temperature (25ºC) are Dyf. Scale bar, 20 µm


Using homodimeric kinesin OSM - 3 as a marker for IFT , proper localisation is seen in ts - che - 3 mutant at permissive , with strongest signal at basal ( bb ) . At restrictive , most of OSM - 3 signal is found within axoneme of short , bulbous cilia . Scale bar is 4

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Using the homodimeric kinesin OSM-3 as a marker for IFT, proper localisation is seen in the ts-che-3 mutant at the permissive temperature, with the strongest signal at the basal body (bb). At the restrictive temperature, most of the OSM-3 signal is found within the axoneme of the short, bulbous cilia. Scale bar is 4 µm


ts - che - 3 mutant shows normal osmotic avoidance at permissive but not at restrictive , indicating defective chemosensation . osm - 9 mutant is included as a positive control . n = 50 ; error bars are standard

The ts-che-3 mutant shows normal osmotic avoidance at permissive temperature but not at the restrictive temperature, indicating defective chemosensation. The osm-9 mutant is included as a positive control. n = 50 animals; error bars are standard error


Three TZ markers ( NPHP - 4 , CEP - 290 MKS - 6 ) are shown at both permissive ( 15ºC ) restrictive ( 25ºC ) temperatures in ts - che - 3 mutant . At permissive , three markers localise to TZ , while at restrictive exhibit ectopic localisation ( leakage ) distally into ciliary axoneme . While NPHP - 4 MKS - 6 show significant accumulations , CEP - 290 is grossly normal . At permissive , both IFT co - markers ( CHE - 13 XBX - 1 ) display strong localisation to basal ( bb ) along axoneme . At restrictive , IFT markers show accumulation in axoneme . Accumulations emphasized using

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

Three TZ markers (NPHP-4, CEP-290 and MKS-6) are shown at both the permissive (15ºC) and restrictive (25ºC) temperatures in the ts-che-3 mutant. At the permissive temperature, all three markers localise to the TZ, while at the restrictive temperature exhibit ectopic localisation (leakage) distally into the ciliary axoneme. While NPHP-4 and MKS-6 show significant accumulations, CEP-290 is grossly normal. At the permissive temperature, both IFT co-markers (CHE-13 and XBX-1) display strong localisation to the basal body (bb) and along the axoneme. At the restrictive temperature, the IFT markers show accumulation in the axoneme. Accumulations emphasized using asterisks


To determine if ciliary compartmentalisation could be restored , young ts - che - 3 mutant adults were transferred to permissive after being raised at restrictive . TZ reassembled at TZ , with loss of ectopic accumulations , IFT localisation was also restored - strongest at basal , similar to grown at permissive . ( c ) After 24 hours at restrictive , TZ appear to begin to accumulate distally within axoneme in ts - che - 3 mutants . This is seen , in small amounts , for NPHP - 4 CEP - 290 but with stronger ectopic localisation for MKS - 6 . IFT CHE - 13 XBX - 1 also show accumulations in axoneme after 24 hours at restrictive . Data information : For ( A ) , ( B ) , ( C ) , is shown for each marker at indicated in heat maps on right . Each point in plot represents one pixel along centre of basal ( BB ) , transition zone ( TZ ) middle segment ( MS ) regions . Dotted areas ( three pixels ) in MS were used to quantify ectopic localisation ( TZ markers ) or accumulation ( IFT markers ) for statistical analyses . n = 10 cilia ( 4 - 7 ) , Kruskal - Wallis , * P < 0 . 05 , * * P < 0 . 01 . tz , transition zone ; bb , basal ; scale bars are 4

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

To determine if ciliary compartmentalisation could be restored, young ts-che-3 mutant adults were transferred to the permissive temperature after being raised at the restrictive temperature. The TZ proteins reassembled at the TZ, with loss of the ectopic accumulations, and IFT protein localisation was also restored-strongest at the basal body, similar to animals grown at the permissive temperature. (c) After 24 hours at the restrictive temperature, TZ proteins appear to begin to accumulate distally within the axoneme in ts-che-3 mutants. This is seen, in small amounts, for NPHP-4 and CEP-290 but with stronger ectopic localisation for MKS-6. The IFT proteins CHE-13 and XBX-1 also show accumulations in the axoneme after 24 hours at the restrictive temperature. Data information: For (A), (B), and (C), fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (TZ markers) or accumulation (IFT markers) for statistical analyses. n = 10 cilia (4-7 animals), Kruskal-Wallis test, *P<0.05, **P<0.01. tz, transition zone; bb, basal body; scale bars are 4 µm


In ts - che - 3 mutant grown at permissive ( 15ºC ) , both RPI - 2 / RP2 TRAM - 1 / TRAM1 are localised to PCMC , just proximal to ciliary axoneme . At restrictive , TRAM - 1 shows leakage into axoneme with its strongest localisation at PCMC . Intriguingly , RPI - 2 now displays strong localisation to axoneme , indicating that in absence of IFT , RPI - 2 may be targeted into cilium . When ts - che - 3 mutant grown at restrictive ( 25ºC ) are shifted at young adult to permissive ( 15ºC ) for 24 hours , both TRAM - 1 RPI - 2 exhibit restoration of their PCMC localisation . Consistent with other TZ markers , both MKS - 2 MKSR - 1 show ectopic localisation in axoneme when grown at restrictive compared to permissive . Also consistent with what was observed for other markers , after 24 hours at permissive , grown at restrictive show loss of ectopic localisation . is shown for each marker at indicated in heat maps on right . Each point in plot represents one pixel along centre of basal ( BB ) , transition zone ( TZ ) middle segment ( MS ) regions . Dotted areas ( three pixels ) in MS were used to quantify ectopic localisation ( MKS - 2 MKSR - 1 ) or accumulation ( RPI - 2 TRAM - 1 ) for statistical analyses . n = 10 cilia ( 4 - 6 ) , Kruskal - Wallis , * P < 0 . 05 , * * P < 0 . 01 ; scale bars are 4

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

In the ts-che-3 mutant grown at the permissive temperature (15ºC), both RPI-2/RP2 and TRAM-1/TRAM1 are localised to the PCMC, just proximal to the ciliary axoneme. At the restrictive temperature, TRAM-1 shows leakage into the axoneme with its strongest localisation at the PCMC. Intriguingly, RPI-2 now displays strong localisation to the axoneme, indicating that in the absence of IFT, RPI-2 may be targeted into the cilium. When ts-che-3 mutant animals grown at the restrictive temperature (25ºC) are shifted at young adult to the permissive (15ºC) for 24 hours, both TRAM-1 and RPI-2 exhibit restoration of their PCMC localisation. Consistent with other TZ markers, both MKS-2 and MKSR-1 show ectopic localisation in the axoneme when grown at the restrictive temperature compared to the permissive temperature. Also consistent with what was observed for other markers, after 24 hours at the permissive temperature, animals grown at the restrictive temperature show loss of the ectopic localisation. Fluorescence quantification is shown for each marker at the indicated temperature in the heat maps on the right. Each point in the plot represents one pixel along the centre of the basal body (BB), transition zone (TZ) and middle segment (MS) regions. Dotted areas (three pixels) in the MS were used to quantify ectopic localisation (MKS-2 and MKSR-1) or accumulation (RPI-2 and TRAM-1) for statistical analyses. n = 10 cilia (4-6 animals), Kruskal-Wallis test, *P<0.05, **P<0.01; scale bars are 4 µm


Loss of CHE - 3 function results in short , bulbous cilia containing electron dense accumulations abnormal - microtubule connections . Shown are transmission electron micrograph ( ) cross sections of an amphid channel cilium in wild - type che - 3 ( e1124 ) null mutant . Representative of wild - type cilia show intact middle segment ( containing doublet microtubules ) transition zone ( with Y - shaped links ) ( left top bottom panels ) regions . Representative of che - 3 ( e1124 ) cilia reveal apparently intact transition zones , with visible Y - link structures , but enlarged ciliary ends filled with electron - dense accumulations , which often appear vesicular . bulbous structures reveal doublet microtubules associated with , occasionally ectopic microtubule - to - connections , which sometimes appear similar to transition zone Y - links in region just distal to seemingly " normal " TZ structure . Schematics show longitudinal ( left ) cross section ( right ) representations of wild - type che - 3 ( e1124 ) cilia . tz , transition zone ; pcmc , periciliary compartment ; scale bars are as indicated in ( nm )

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

Loss of CHE-3 function results in short, bulbous cilia containing electron dense accumulations and abnormal membrane-microtubule connections. Shown are transmission electron micrograph (TEM) cross sections of an amphid channel cilium in wild-type and che-3(e1124) null mutant animals. Representative images of wild-type cilia show intact middle segment (containing doublet microtubules) and transition zone (with Y-shaped links) (left top and bottom panels) regions. Representative images of che-3(e1124) cilia reveal apparently intact transition zones, with visible Y-link structures, but enlarged ciliary ends filled with electron-dense accumulations, which often appear vesicular. The bulbous structures reveal doublet microtubules associated with the membrane, and occasionally ectopic microtubule-to-membrane connections, which sometimes appear similar to transition zone Y-links in the region just distal to the seemingly "normal" TZ structure. Schematics show longitudinal (left images) and cross section (right images) representations of wild-type and che-3(e1124) cilia. tz, transition zone; pcmc, periciliary membrane compartment; scale bars are as indicated in (nm) for


Using IFT marker BBS - 7 , cilia lengths were measured for wild - type ts - che - 3 mutant raised at permissive ( 15ºC ) shifted to restrictive ( 25ºC ) for three time points . After 12 hours some accumulations ( indicated by asterisks in ) are observed within cilia but there is not a significant reduction in cilium . After 18 24 hours , significant reduction in ciliary is observed large accumulations of BBS - 7 is seen within short bulbous cilia , similar to what is observed in ts - che - 3 raised at restrictive . n = 30 - 34 cilia , one per ; Kruskal - Wallis , * * P < 0 . 01 ; error bars are mean absolute deviation ; scale bars are 4

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

Using the IFT marker BBS-7, cilia lengths were measured for wild-type and the ts-che-3 mutant animals raised at the permissive (15ºC) and shifted to the restrictive temperature (25ºC) for three time points. After 12 hours some accumulations (indicated by asterisks in the fluorescence images) are observed within the cilia but there is not a significant reduction in cilium length. After 18 and 24 hours, significant reduction in ciliary length is observed and large accumulations of BBS-7 is seen within short bulbous cilia, similar to what is observed in ts-che-3 animals raised at the restrictive temperature. n = 30-34 cilia, one per animal; Kruskal-Wallis test, **P<0.01; error bars are mean absolute deviation; scale bars are 4 µm


IFT velocities were measured for wild - type or ts - che - 3 mutant ( grown at permissive ) imaged at permissive , restrictive or after 3 hours at restrictive . Compared to wild - type , anterograde velocities in ts - che - 3 dynein mutant are similar , while IFT retrograde rates are highly reduced for both middle distal ciliary segments . Interestingly , ts - che - 3 IFT dynein mutant appears to not only have slow retrograde IFT trains , but trains also appear much larger less frequent . Initially upon shift ( < 15min ) IFT rates are much faster for both wild - type ts - che - 3 dynein mutant , although retrograde remains much slower in mutant . After 3 hours at restrictive , IFT velocities are intermediate between rates seen at 15ºC those observed at initial shift to

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

IFT velocities were measured for wild-type or ts-che-3 mutant animals (grown at permissive temperature) and imaged at the permissive temperature, restrictive temperature or after 3 hours at the restrictive temperature. Compared to wild-type, anterograde velocities in the ts-che-3 dynein mutant are similar, while IFT retrograde rates are highly reduced for both the middle and distal ciliary segments. Interestingly, the ts-che-3 IFT dynein mutant appears to not only have slow retrograde IFT trains, but the trains also appear much larger and less frequent. Initially upon the temperature shift (<15min) IFT rates are much faster for both wild-type and ts-che-3 dynein mutant, although retrograde remains much slower in the mutant. After 3 hours at the restrictive temperature, IFT velocities are intermediate between the rates seen at 15ºC and those observed at the initial shift to 25ºC


To assess effects of IFT restoration on TZ assembly , ts - che - 3 mutant were shifted from restrictive to permissive at different time points during their development ( days 1 , 4 8 of adulthood ) . TZ markers MKS - 6 ( a ) NPHP - 4 ( b ) show strong ectopic ciliary accumulations when raised at restrictive until day 1 of adulthood . After a shift to permissive for 24 hours , day 2 adults show " repaired " TZ localisation resembling that of wild - type . For both markers , Day 4 adults ( maintained at restrictive ) show less ectopic TZ localisation than Day 1 adults . correction of ectopic localisation following shift to permissive for 24 hours was reduced in day 4 ( to day 5 ) of adulthood compared to that observed in day 1 ( to day 2 ) adults . For MKS - 6 , but not NPHP - 4 , day 8 adults ( maintained at restrictive ) also display less ectopic accumulation than day 1 adults . correction of ectopic TZ localisation in shifted to permissive after 8 days of adulthood is also much smaller in magnitude for both markers , indicating that this repair is most effective in day 1 adults . In summary , as adults , ectopic TZ localisation is reduced , while ability to correct this phenotype by restoring IFT function

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

To assess the effects of IFT restoration on TZ assembly, ts-che-3 mutant animals were shifted from the restrictive to the permissive temperature at different time points during their development (days 1, 4 and 8 of adulthood). The TZ markers MKS-6 (a) and NPHP-4 (b) show strong ectopic ciliary accumulations when raised at the restrictive temperature until day 1 of adulthood. After a shift to the permissive temperature for 24 hours, day 2 adults show "repaired" TZ protein localisation resembling that of wild-type animals. For both markers, Day 4 adults (maintained at the restrictive temperature) show less ectopic TZ protein localisation than Day 1 adults. The correction of ectopic localisation following shift to the permissive temperature for 24 hours was reduced in day 4 (to day 5) of adulthood compared to that observed in day 1 (to day 2) adults. For MKS-6, but not NPHP-4, day 8 adults (maintained at the restrictive temperature) also display less ectopic accumulation than day 1 adults. The correction of ectopic TZ protein localisation in animals shifted to the permissive temperature after 8 days of adulthood is also much smaller in magnitude for both markers, indicating that this repair is most effective in day 1 adults. In summary, as the adults age, ectopic TZ protein localisation is reduced, while the ability to correct this phenotype by restoring IFT function al


A . Colony formation ( CFA ) with imatinib with representative of colonies formed by each within panel . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - unpaired Student ' s t - ( H1915 P = 0 . 0004 , H2110 P = 0 . 0001 ) .

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

A. Colony formation assay (CFA) with imatinib with representative images of colonies formed by each cell line within the panel. Error bars represent mean ±S.E.M., n=9, *P < 0.001 by two-tailed unpaired Student's t-test (H1915 P= 0.0004, H2110 P=0.0001).


B . Western blot after 1 hour treatment with imatinib .

B. Western blot analysis after 1 hour treatment with imatinib.


C . MTT for NSCLC lines Beas - 2B with dasatinib or DMSO . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - unpaired Student ' s t - ( H1915 P = 1 . 09 × 10 - 5 , H2110 P = 0 . 009 A549 P = 6 × 10 - 6 ) .

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

C. MTT assay for NSCLC cell lines and Beas-2B cells with dasatinib or DMSO. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (H1915 P=1.09×10-5, H2110 P=0.009 and A549 P=6×10-6).


D . Western blot after 1 h treatment with dasatinib .

D. Western blot analysis after 1 h treatment with dasatinib.


A - C . Drug - resistant ABL1 - T315I rescues dasatinib - imatinib - induced cytotoxicity . Western blot of ( A ) transient ( B ) tetracycline - inducible overexpression of ABL1 - T315I in H1915 treated with dasatinib or imatinib .

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

A -C. Drug-resistant ABL1-T315I rescues dasatinib- and imatinib-induced cytotoxicity. Western blot analysis of (A) transient and (B) tetracycline-inducible overexpression of ABL1-T315I in H1915 cells treated with dasatinib or imatinib.


C . CFA with dasatinib or imatinib for H1915 with or without tetracycline - inducible expression of ABL1 - T315I . Error bars represent mean ± S . E . M . , n = 9 , * P < 0 . 001 by two - unpaired Student ' s t - ( control , DMSO vs dasatinib P = 4 . 3 × 10 - 12 vs imatinib P = 6 × 10 - 6 ; dasatinib , control vs tetracycline , P = 9 × 10 - 7 ; imatinib , control vs tetracycline P = 9 . 7 × 10 - 5 ) .

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

C. CFA with dasatinib or imatinib for H1915 cells with or without tetracycline-inducible expression of ABL1-T315I. Error bars represent mean ±S.E.M., n=9, *P<0.001 by two-tailed unpaired Student's t-test (control, DMSO vs dasatinib P=4.3×10-12 vs imatinib P=6×10-6; dasatinib, control vs tetracycline, P=9×10-7; imatinib, control vs tetracycline P=9.7×10-5).


D - F . Knockdown of mutated ABL1 reduces viability of lung cancer . Top , western blot of ABL1 knockdown in ( D ) Beas - 2B , ( E ) H1915 ( F ) H2110 upon transfection with three ABL1 siRNAs , negative control ( scrambled ) CyclophilinB positive control . Bottom , MTT showing viability of ( D ) Beas - 2B , ( E ) H1915 ( F ) H2110 in response to ABL1 knockdown . Error bars represent mean ± S . E . M , n = 9 , * P < 0 . 005 , * * P < 0 . 001 by two - unpaired Student ' s t - ( Beas - 2B , scrambled vs siABL1 - 3 P = 0 . 002 ; H1915 , scrambled vs siABL1 - 1 P = 0 . 004 , vs siABL1 - 2 P = 0 . 001 , vs siABL1 - 3 P = 0 . 002 ; H2110 scrambled vs siABL1 - 1 P = 5 × 10 - 6 , vs siABL1 - 2 P = 2 × 10 - 5 , vs siABL1 - 3 P = 4 . 2 × 10 - 6 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F. Knockdown of mutated ABL1 reduces viability of lung cancer cells. Top, western blot analysis of ABL1 knockdown in (D) Beas-2B, (E) H1915 and (F) H2110 cells upon transfection with three ABL1 siRNAs, negative control (scrambled) and CyclophilinB positive control. Bottom, MTT assay showing viability of (D) Beas-2B, (E) H1915 and (F) H2110 cells in response to ABL1 knockdown. Error bars represent mean ±S.E.M, n=9, *P<0.005, **P<0.001 by two-tailed unpaired Student's t-test (Beas-2B, scrambled vs siABL1-3 P=0.002; H1915, scrambled vs siABL1-1 P=0.004, vs siABL1-2 P=0.001, vs siABL1-3 P=0.002; H2110 scrambled vs siABL1-1 P=5×10-6, vs siABL1-2 P=2×10-5, vs siABL1-3 P=4.2×10-6).


A - C . Average volumes of xenografts derived from H1915 ( A ) , H650 ( B ) ( C ) H1915 with doxycycline inducible ABL1 gate - keeper mutation ( H1915 - ABL1T315I ) in mice treated with either vehicle or imatinib ( 100mg / kg ) . Error bars represent ± S . D . , n = 6 - 10 mice per group , * P = 0 . 0016 by Mann - Whitney U .

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

A-C. Average tumor volumes of xenografts derived from H1915 (A), H650 (B) and (C) H1915 cell line with doxycycline inducible ABL1 gate-keeper mutation (H1915-ABL1T315I) in mice treated with either vehicle or imatinib (100mg/kg). Error bars represent ±S.D., n=6-10 mice per group, *P=0.0016 by Mann-Whitney U test.


A C . Root mean square deviation ( RMSD ) of movement of ( A ) αC - helix ( C ) DFG motif in active inactive conformation of ABL1 kinase with R351W mutation ( orange ) compared to wild type ( WT ) kinase ( black ) . B D . Time - lapse show movement within αC - helix ( B ) DFG motif ( D ) of inactive structure of WT ( black outline ) R351W ( orange outline ) constructs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and C. Root mean square deviation (RMSD) analysis of the movement of (A) αC-helix and (C) DFG motif in the active and inactive conformation of ABL1 kinase with R351W mutation (orange) compared to the wild type (WT) kinase (black). B and D. Time-lapse images show movement within the αC-helix (B) and DFG motif (D) of the inactive structure of the WT (black outline) and R351W (orange outline) constructs.


E . left , western blot of transient overexpression of wild type ABL1 ( WT ) ABL1 - R351W , compared to empty vector control ( EV ) in HEK293T . Right , of p - CRKL signal on densitometry of blots from 6 independent experiments . Error bars represent ± S . D . , n = 6 , * P = 0 . 0004 by two - unpaired Student ' s t - .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. left, western blot analysis of transient overexpression of wild type ABL1 (WT) and ABL1-R351W, compared to empty vector control (EV) in HEK293T cells. Right, quantification of p-CRKL signal based on densitometry analysis of blots from 6 independent experiments. Error bars represent ±S.D., n=6, *P=0.0004 by two-tailed unpaired Student's t-test.


F . Overexpression of mutant ABL1 constructs with mutations identified in selected lung tumors ( Ding et al , 2008 ) in H157 . Values indicate quantified band for p - CRKL , relative to α - tubulin HA - tagged ABL1 .

O O O O O O O O O O O O O O O O O O O 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. Overexpression of mutant ABL1 constructs with mutations identified in selected primary lung tumors (Ding et al, 2008) in H157 cells. Values indicate quantified band intensity for p-CRKL, relative to α-tubulin and HA-tagged ABL1.


A . Immunofluorescence imaging of endogenous ABL1 in panel of NSCLC lines ; scale bars = 20 μm . B C of versus localization of ( B ) endogenous ABL1 in NSCLC lines ( C ) tetracycline - inducible expression of ABL1 - WT , ABL1 - R351W ABL1 - G340L in Beas - 2B . Error bars represent ± S . D . , n = 6 - 10 , * P < 0 . 005 by two - unpaired Student ' s t - ( Beas - 2B vs H1915 P = 6 . 6 × 10 - 5 , vs H2110 P = 0 . 0001 ; WT vs R351W P = 0 . 003 , vs G340L P = 0 . 002 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 imaging of endogenous ABL1 in the panel of NSCLC cell lines; scale bars=20 μm. B and C Quantification of nuclear versus cytoplasmic localization of (B) endogenous ABL1 in NSCLC cell lines and (C) tetracycline-inducible expression of ABL1-WT, ABL1-R351W and ABL1-G340L in Beas-2B cells. Error bars represent ±S.D., n=6-10 images, *P<0.005 by two-tailed unpaired Student's t-test (Beas-2B vs H1915 P=6.6×10-5, vs H2110 P=0.0001; WT vs R351W P=0.003, vs G340L P=0.002).


of total signal attributed to phosphopeptides ( blue bars ; 11 unique peptide ions ) non - phosphorylated peptides ( grey bars ; 25 unique peptide ions ) following SAX fractionation at ( A ) pH 6 . 0 , ( B ) pH 6 . 8 ( C ) pH 8 . 0 , with later fractions consisting of up to 100 % phosphopeptide ion signal . Representative pHis - containing myoglobin peptide ( LFTGHPETLEK ; solid ) , its non - phosphorylated counterpart ( LFTGHPETLEK ; dashed ) , quantified across 16 SAX fractions ; SAX was performed at pH 6 . 0 ( red ) , pH 6 . 8 ( blue ) or pH 8 . 0 ( green ) . % of total peak of each peptide across gradient is plotted for each fraction . In order to assess pHis stability effects of SAX separation in a mixture ( black ) , SAX was also repeated at pH 6 . 8 with phosphorylated myoglobin spiked into a human lysate prior to digestion . Data for five pHis - containing myoglobin peptides is shown in Fig . EV3 .

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

Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3.


siRNA - mediated knockdown of PHPT1 in HeLa ( 24 h ) with reference to GAPDH loading control . NT - non - targeting siRNA control .

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siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control.


Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) .

Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm).


Representative SAX profile of trypsin digested HeLa lysate ( Abs280 nm ) . Base peak chromatograms are shown for select SAX fractions following high - resolution LC - MS / MS using an Orbitrap Fusion mass spectrometer : ( D ) fraction 3 ( green ) ; ( E ) fraction 6 ( red ) ; ( F ) fraction 10 ( grey ) . Peptides were fragmented by HCD , with neutral loss of 98 amu from precursor ions triggering EThCD ( Ferries et al , 2017 ) . Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot . ptmRS node was used for phosphosite localisation . was performed on three independent biological replicates for each condition ( NT or PHPT1 siRNA ) .

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

Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).


Comparison of triplet score ( ≥ 5 % S / N ) following HCD of unique phosphopeptides ( 5 % FDR ) identified from UPAX separated tryptic digests of HeLa derived phosphopeptides . Site of phosphorylation ( ptmRS score ≥ 0 . 90 ) is indicated . % of phosphopeptides exhibiting either no neutral loss ( Triplet = 0 ) , or neutral loss of any 1 ( Triplet = 1 ) , 2 ( Triplet = 2 ) , or 3 ions from precursor ( ∆ 80 , ∆ 98 / or ∆ 116 amu ) . Distribution of triplet score following HCD fragmentation as a function of site localisation confidence ( ptmRS score ≥ 0 . 75 , ≥ 0 . 90 , ≥ 0 . 95 , ≥ 0 . 99 ) for unique singly phosphorylated peptide as a function of residue phosphorylated . Numbers of phosphopeptides ( ptmRS ≥ 0 . 90 ) exhibiting different combinations of three neutral loss species ( ∆ 80 , ∆ 98 / or ∆ 116 amu from precursor ) for each of phosphorylated residues .

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

Comparison of triplet score (≥5% S/N) following HCD of unique phosphopeptides (5% FDR) identified from UPAX separated tryptic digests of HeLa cell derived phosphopeptides. Site of phosphorylation (ptmRS score ≥ 0.90) is indicated. % of phosphopeptides exhibiting either no neutral loss (Triplet =0), or neutral loss of any 1 (Triplet = 1), 2 (Triplet =2), or 3 ions from the precursor (∆80, ∆98 and/or ∆116 amu). Distribution of triplet score following HCD fragmentation as a function of site localisation confidence (ptmRS score ≥ 0.75, ≥ 0.90, ≥ 0.95, ≥ 0.99) for unique singly phosphorylated peptide as a function of the residue phosphorylated. Numbers of phosphopeptides (ptmRS ≥ 0.90) exhibiting different combinations of the three neutral loss species (∆80, ∆98 and/or ∆116 amu from precursor) for each of the phosphorylated residues.


( B ) RAW 264 . 7 transfected for 48 h with control ( sc ) , MTMR6 or Jumpy siRNA , were pretreated for 30 min with 100 nM Bafilomycin A1 ( BafA1 ) , 10 μg / ml E64d 10 μg / ml pepstatin , then incubated for 30 min in full or starvation media in presence of BafA1 , E64d pepstatin . were lysed analysed by immunoblotting with anti - LC3 or anti ‐ actin . Densitometric LC3 ‐ II / actin ratios are shown underneath blot . Graph : LC3 ‐ II / actin for Jumpy siRNA in full medium is equal to or exceeds LC3 ‐ II / actin for control siRNA ( sc ) in starvation medium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RAW 264.7 cells transfected for 48 h with control (sc), MTMR6 or Jumpy siRNA, were pretreated for 30 min with 100 nM Bafilomycin A1 (BafA1), 10 μg/ml E64d and 10 μg/ml pepstatin, then incubated for 30 min in full or starvation media in the presence of BafA1, E64d and pepstatin. Cells were lysed and analysed by immunoblotting with anti-LC3 or anti‐actin. Densitometric LC3‐II/actin ratios are shown underneath the blot. Graph: LC3‐II/actin ratio for Jumpy siRNA in full medium is equal to or exceeds LC3‐II/actin ratio for control siRNA (sc) in starvation medium.


( C , D ) RAW 264 . 7 were transfected for 36 h with control ( scramble ) or Jumpy siRNA , transfected once more with corresponding siRNA mRFP ‐ GFP ‐ LC3 construct overnight incubated for 2 h in full or starvation media . were fixed LC3 analysed by confocal microscopy . ( C ) Representative confocal of RAW 264 . 7 in full or starvation media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . Red yellow arrows indicate GFP − RFP + GFP + RFP + , respectively . ( D ) Quantitation of number of LC3 per , total per ( GFP + RFP + GFP − RFP + ) GFP + RFP + per GFP − RFP + per . Data , mean ± s . e . m . n = 5 independent experiments , 30 per experiments . * P0 . 05 , * * P0 . 01 , * * * P0 . 001 ( t ‐ ) . Scale bars , 5 μm .

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

(C, D) RAW 264.7 cells were transfected for 36 h with control (scramble) or Jumpy siRNA, transfected once more with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct overnight and incubated for 2 h in full or starvation media. Cells were fixed and LC3 puncta analysed by confocal fluorescence microscopy. (C) Representative confocal images of RAW 264.7 cells in full or starvation media after transfection with control (scramble) or Jumpy siRNA (siJumpy). Red and yellow arrows indicate GFP−RFP+ and GFP+RFP+ puncta, respectively. (D) Quantitation of number of LC3 puncta per cell, total puncta per cell (GFP+RFP+ and GFP−RFP+ puncta) GFP+RFP+ puncta per cell and GFP−RFP+ puncta per cell. Data, mean±s.e.m. n=5 independent experiments, 30 cells per experiments. *P0.05, **P0.01, ***P0.001 (t‐test). Scale bars, 5 μm.


( E , F ) C2C12 myoblasts were transfected for 48 h with control ( scramble ) or Jumpy siRNA , followed by a second transfection overnight with corresponding siRNA mRFP ‐ GFP ‐ LC3 construct . were fixed LC3 were counted by confocal microscopy . ( E ) Representative confocal of C2C12 in full media after transfection with control ( scramble ) or Jumpy siRNA ( siJumpy ) . ( F ) of number of LC3 per . Data , mean ± for n = 3 ( independent experiments ) , 30 per experiments . * P 0 . 05 ( t - ) . Scale bars , 10 μm .

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(E, F) C2C12 myoblasts were transfected for 48 h with control (scramble) or Jumpy siRNA, followed by a second transfection overnight with corresponding siRNA and mRFP‐GFP‐LC3 DNA construct. Cells were fixed and LC3 puncta were counted by confocal fluorescence microscopy. (E) Representative confocal images of C2C12 cells in full media after transfection with control (scramble) or Jumpy siRNA (siJumpy). (F) Quantification of the number of LC3 puncta per cell. Data, mean±SEM for n=3 (independent experiments), 30 cells per experiments. *P 0.05 (t-test). Scale bars, 10 μm.


( G , H ) C2C12 were transfected for 48 h with control ( sc ) ( lanes 1 , 2 , 5 , 6 ) or Atg5 siRNA ( lanes 3 , 4 , 6 , 7 ) followed by a second transfection for 48 h with same siRNA control ( lanes 1 , 3 , 5 , 7 ) or Jumpy siRNA ( lanes 2 , 4 , 6 , 8 ) . were incubated for 1 h with or without 100 nM Baf A1 , 10 μg / ml E64d 10 μg / ml pepstatin in full media , lysed analysed by immunoblotting with anti ‐ LC3 or anti ‐ actin ( G ) . ( H ) Densitometric LC3 ‐ II / actin ratios for samples treated with BafA1 protease inhibitors from G ( lanes 5 - 8 ) . Inset shows Atg5 knock ‐ down by immunoblotting .

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

(G, H) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2, 5, 6) or Atg5 siRNA (lanes 3, 4, 6, 7) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3, 5, 7) or Jumpy siRNA (lanes 2, 4, 6, 8). Cells were incubated for 1 h with or without 100 nM Baf A1, 10 μg/ml E64d and 10 μg/ml pepstatin in full media, lysed and analysed by immunoblotting with anti‐LC3 or anti‐actin (G). (H) Densitometric LC3‐II/actin ratios for samples treated with BafA1 and protease inhibitors from G (lanes 5-8). Inset shows Atg5 knock‐down by immunoblotting.


( A ) Proteolysis of long ‐ lived in C2C12 myoblasts . C2C12 were transfected with control ( scramble ) or Jumpy siRNA ( siJumpy ) , labelled overnight in media containing [ 3H ] leucine , washed , incubated for 2 h in complete media ( containing cold leucine ) incubated for 4 h in full or starvation media . Leucine release was calculated from radioactivity in tricarboxylic acid ‐ soluble form relative to total radioactivity . Results shown represent mean ± s . e . m . for combined data from three independent experiments .

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

(A) Proteolysis of long‐lived proteins in C2C12 myoblasts. C2C12 cells were transfected with control (scramble) or Jumpy siRNA (siJumpy), labelled overnight in media containing [3H] leucine, washed, incubated for 2 h in complete media (containing cold leucine) and incubated for 4 h in full or starvation media. Leucine release was calculated from radioactivity in the tricarboxylic acid‐soluble form relative to total cell radioactivity. Results shown represent mean±s.e.m. for combined data from three independent experiments.


( B ) Quantitation of p62 . C2C12 were transfected for 48 h with control ( sc ) or Jumpy siRNA p62 analysed by immunoblotting , quantitated by densitometry represented as a of control . Data are mean ± s . e . m . ( n = 4 independent experiments ) .

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

(B) Quantitation of p62 protein levels. C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA and p62 levels analysed by immunoblotting, quantitated by densitometry and represented as a percentage of control. Data are mean±s.e.m. (n=4 independent experiments).


( C ) C2C12 were transfected for 48 h with control ( sc ) or Jumpy siRNA , incubated for 4 h with or without 100 nM Bafilomycin A1 in full media , lysed , probed for endogenous p62 actin by immunoblotting of p62 were quantitated ( mean ± s . e . m . , n = 3 ) .

O O O 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

(C) C2C12 cells were transfected for 48 h with control (sc) or Jumpy siRNA, incubated for 4 h with or without 100 nM Bafilomycin A1 in full media, lysed, probed for endogenous p62 and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=3).


( D ) C2C12 were transfected for 48 h with control ( sc ) ( lanes 1 , 2 ) or Beclin siRNA ( lanes 3 , 4 ) followed by a second transfection for 48 h with same siRNA control ( lanes 1 , 3 ) or Jumpy siRNA ( lanes 2 , 4 ) . were lysed , probed for p62 , Beclin actin by immunoblotting of p62 were quantitated ( mean ± s . e . m . , n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C2C12 cells were transfected for 48 h with control (sc) (lanes 1, 2) or Beclin siRNA (lanes 3, 4) followed by a second transfection for 48 h with same siRNA and control (lanes 1, 3) or Jumpy siRNA (lanes 2, 4). Cells were lysed, probed for p62, Beclin and actin by immunoblotting and percentage of p62 were quantitated (mean±s.e.m., n=5).


( E - M ) C2C12 were transfected for 48 h with GFP , GFP ‐ Jumpy ( Jumpy ) , GFP ‐ MTM1 ( MTM1 ) or GFP ‐ MTMR2 ( MTMR2 ) , fixed immunostained with anti ‐ p62 antibody ( red ) . p62 were quantitated by confocal microscopy . Representative confocal of GFP ( E ) , GFP ‐ Jumpy ( F ) , GFP ‐ MTM1 ( G ) , GFP ‐ MTMR2 ( H ) transfected , immunostained for p62 ( I ) , ( J ) , ( K ) ( L ) , respectively . Scale bars , 5 μm . lines represent outline of . Quantitation of p62 per ( M ) . Bars , ( n = 3 independent experiments , with an average of 30 per experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-M) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of GFP (E), GFP‐Jumpy (F), GFP‐MTM1 (G), GFP‐MTMR2 (H) transfected cells, immunostained for p62 (I), (J), (K) and (L), respectively. Scale bars, 5 μm. White lines represent outline of the cells. Quantitation of p62 puncta per cell (M). Bars, SEM (n=3 independent experiments, with an average of 30 cells per experiments).


( N , O ) C2C12 were transfected for 48 h with GFP , GFP ‐ Jumpy ( Jumpy ) , GFP ‐ MTM1 ( MTM1 ) or GFP ‐ MTMR2 ( MTMR2 ) , lysed , analysed for p62 , GFP actin by immunoblotting ( N ) of p62 were quantitated ( mean ± s . e . m . , n = 3 ) ( O ) .

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

(N, O) C2C12 cells were transfected for 48 h with GFP, GFP‐Jumpy (Jumpy), GFP‐MTM1 (MTM1) or GFP‐MTMR2 (MTMR2), lysed, analysed for p62, GFP and actin by immunoblotting (N) and percentage of p62 were quantitated (mean±s.e.m., n=3) (O).


( P , Q ) C2C12 were transfected for 48 h with GFP or GFP ‐ Jumpy ( Jumpy ) , incubated with or without 100 nM Bafilomycin A1 ( BafA1 ) for 4 h , lysed , analysed for p62 , GFP actin by immunoblotting ( P ) of p62 were quantitated ( mean ± s . e . m . , n = 4 ) ( Q ) . * P0 . 05 , * * * P0 . 001 , † ns ( t ‐ ) .

O O O 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 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

(P, Q) C2C12 cells were transfected for 48 h with GFP or GFP‐Jumpy (Jumpy), incubated with or without 100 nM Bafilomycin A1 (BafA1) for 4 h, lysed, analysed for p62, GFP and actin by immunoblotting (P) and percentage of p62 were quantitated (mean±s.e.m., n=4) (Q). *P0.05, ***P0.001, †ns (t‐test).


( A ) Transient association of Jumpy WT with LC3 + organelles . Time lapse of C2C12 myoblasts transfected for 24 h with GFP ‐ Jumpy WT Cherry ‐ LC3 analysed by live confocal microscopy in a 5LIVE Zeiss microscope . EBSS was added to z ‐ stacks collected at 3 ‐ min intervals for a total of 45 min . collected were processed to generate a maximum projection ( collapsing a 3D image into an x - y projection ) for each time point . Arrows indicate colocalization of GFP ‐ Jumpy WT with LC3 . Graph : Plots of relative ( GFP pixel ) over time of GFP ‐ Jumpy WT colocalizing with Cherry ‐ LC3 + ‐ positive indicated by arrows .

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(A) Transient association of Jumpy WT with LC3+ organelles. Time lapse sequence of C2C12 myoblasts transfected for 24 h with GFP‐Jumpy WT and Cherry‐LC3 and analysed by live confocal microscopy in a 5LIVE Zeiss microscope. EBSS was added to the cells and z‐stacks collected at 3‐min intervals for a total of 45 min. The collected images were processed to generate a maximum projection (collapsing a 3D image into an x-y projection) for each time point. Arrows indicate colocalization of GFP‐Jumpy WT with LC3. Graph: Plots of relative fluorescence intensity (GFP fluorescence pixel intensity) over time of GFP‐Jumpy WT colocalizing with Cherry‐LC3+‐positive puncta indicated by arrows.


( B - I ) Association of Jumpy CS mutant with autophagic organelles . C2C12 were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) tdTomato ‐ LC3 ( LC3 ) , fixed immunostained with anti ‐ G58K ( Alexa 648 ‐ labelled secondary antibody ) . Jumpy CS ( green ) , LC3 ( red ) G58K ( in D , H or blue in E , I ) . Boxed areas ( B - E ) are shown at higher magnification in corresponding panel below ( F - I ) . Scale bars , 2 μm . arrows indicate colocalization between Jumpy CS , LC3 but not G58K , blue arrow indicates colocalization between Jumpy CS G58K but not LC3 .

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(B-I) Association of Jumpy CS mutant with autophagic organelles. C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐G58K (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and G58K (white in D, H or blue in E, I). Boxed areas (B-E) are shown at higher magnification in the corresponding panel below (F-I). Scale bars, 2 μm. White arrows indicate colocalization between Jumpy CS, LC3 but not G58K, blue arrow indicates colocalization between Jumpy CS and G58K but not LC3.


( A - H ) C2C12 were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) tdTomato ‐ LC3 ( LC3 ) , fixed immunostained with anti ‐ Atg16 ( Alexa 648 ‐ labelled secondary antibody ) . Jumpy CS ( green ) , LC3 ( red ) Atg16 ( in B F or blue in D H ) . Boxed areas ( A - D ) are shown at higher magnification in corresponding panel below ( E - H ) . arrows indicate colocalization among Jumpy CS , LC3 Atg16 , yellow arrow indicates colocalization between Jumpy CS LC3 but not Atg16 , blue arrow indicates colocalization between Jumpy CS Atg16 but not LC3 .

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(A-H) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg16 (Alexa 648‐labelled secondary antibody). Jumpy CS (green), LC3 (red) and Atg16 (white in B and F or blue in D and H). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization among Jumpy CS, LC3 and Atg16, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg16, blue arrow indicates colocalization between Jumpy CS and Atg16 but not LC3.


( I , J ) C2C12 were transfected for 24 h with GFP ( I ) , fixed immunostained with anti ‐ Atg16 ( J ) .

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(I, J) C2C12 cells were transfected for 24 h with GFP (I), fixed and immunostained with anti‐Atg16 (J).


( K - R ) C2C12 were transfected for 24 h with GFP ‐ Jumpy C330S ( Jumpy CS ) tdTomato ‐ LC3 ( LC3 ) , fixed immunostained with anti ‐ Atg12 ( M ) antibody . Jumpy CS ( green ) , LC3 ( red ) Atg12 ( in L , P or blue in N , R ) . Boxed areas ( K - N ) are shown at higher magnification in corresponding panel below ( O - R ) . arrows indicate colocalization among Jumpy CS , LC3 Atg12 , yellow arrow indicates colocalization between Jumpy CS LC3 but not Atg12 , blue arrow indicates colocalization between Jumpy CS Atg12 but not LC3 .

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

(K-R) C2C12 cells were transfected for 24 h with GFP‐Jumpy C330S (Jumpy CS) and tdTomato‐LC3 (LC3), fixed and immunostained with anti‐Atg12(M) antibody. Jumpy CS (green), LC3 (red) and Atg12 (white in L, P or blue in N, R). Boxed areas (K-N) are shown at higher magnification in the corresponding panel below (O-R). White arrows indicate colocalization among Jumpy CS, LC3 and Atg12, yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg12, blue arrow indicates colocalization between Jumpy CS and Atg12 but not LC3.


( S - ZA ) C2C12 were transfected for 24 h with Jumpy CS Cherry ‐ LC3 ( LC3 ) , fixed immunostained with anti ‐ lamp1 . Jumpy CS ( green ) , LC3 ( red ) Lamp1 ( in T , X or blue in V , ZA ) . Boxed areas ( S - V ) are shown at higher magnification in corresponding panel below ( W - ZA ) . Yellow arrows indicate colocalization between Jumpy CS LC3 but not Lamp1 . Scale bars , 2 μm .

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

(S-ZA) C2C12 cells were transfected for 24 h with Jumpy CS and Cherry‐LC3 (LC3), fixed and immunostained with anti‐lamp1. Jumpy CS (green), LC3 (red) and Lamp1 (white in T, X or blue in V, ZA). Boxed areas (S-V) are shown at higher magnification in the corresponding panel below (W-ZA). Yellow arrows indicate colocalization between Jumpy CS and LC3 but not Lamp1. Scale bars, 2 μm.


( A - C ) C2C12 were transfected for 48 h with Jumpy siRNA , followed by a second transfection overnight with Jumpy siRNA GFP ‐ WIPI ‐ 1 , incubated for 4 h in starvation media , fixed immunostained with anti ‐ Atg16 antibody ( red ) . GFP ‐ WIPI1 ‐ transfected ( A ) , endogenous Atg16 immunostaining ( B ) , colocalization of GFP ‐ WIPI ‐ 1 Atg16 is indicated in yellow in merge image ( C ) . Boxed areas are shown at higher magnification as inset . Scale bars , 10 5 μm in insets .

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

(A-C) C2C12 were transfected for 48 h with Jumpy siRNA, followed by a second transfection overnight with Jumpy siRNA and GFP‐WIPI‐1, incubated for 4 h in starvation media, fixed and immunostained with anti‐Atg16 antibody (red). GFP‐WIPI1‐transfected cell (A), endogenous Atg16 immunostaining (B), colocalization of GFP‐WIPI‐1 and Atg16 is indicated in yellow in merge image (C). Boxed areas are shown at higher magnification as inset. Scale bars, 10 and 5 μm in insets.


( D - I ) C2C12 were transfected for 48 h with control ( scramble ) ( D , E ) or Jumpy siRNA ( F - H ) , followed by a second transfection overnight with corresponding siRNA GFP ‐ WIPI ‐ 1 . were incubated for 4 h in full ( D , G ) or starvation media ( E , F , H ) in presence ( F ) or absence ( E - H ) of 100 nM wortmannin , fixed analysed by confocal microscopy . Quantitation of WIPI ‐ 1 per ( I ) . Bars , s . e . m . ( n = 3 independent experiments , with an average of 30 per experiment ) . * P0 . 05 , * * P0 . 01 ( t ‐ ) .

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

(D-I) C2C12 cells were transfected for 48 h with control (scramble) (D, E) or Jumpy siRNA (F-H), followed by a second transfection overnight with corresponding siRNA and GFP‐WIPI‐1. Cells were incubated for 4 h in full (D, G) or starvation media (E, F, H) in presence (F) or absence (E-H) of 100 nM wortmannin, fixed and analysed by confocal fluorescence microscopy. Quantitation of WIPI‐1 puncta per cell (I). Bars, s.e.m. (n=3 independent experiments, with an average of 30 cells per experiment). *P0.05, **P0.01 (t‐test).


( A - T ) C2C12 were transfected for 24 h with GFP , GFP ‐ Jumpy WT or GFP ‐ Jumpy C330S ( Jumpy CS ) Tomato ‐ LC3 ( LC3 ) HA ‐ Atg9 , fixed immunostained with anti ‐ HA . GFP ( I ) , Jumpy WT ( M ) , Jumpy CS ( green ) ( A , E , Q ) , LC3 ( red ) ( C , G , J , N , R ) Atg9 ( in B , F , K , O , S or blue in D , H , I , P , T ) . Boxed areas ( A - D ) are shown at higher magnification in corresponding panel below ( E - H ) . arrows indicate colocalization between Jumpy CS , LC3 Atg9 , blue arrow indicates colocalization between Jumpy CS Atg9 but not LC3 yellow arrow indicates colocalization between Jumpy CS LC3 but not Atg9 . boxes ( I - T ) show LC3 location . Scale bars , 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-T) C2C12 cells were transfected for 24 h with GFP, GFP‐Jumpy WT or GFP‐Jumpy C330S (Jumpy CS) and Tomato‐LC3 (LC3) and HA‐Atg9, fixed and immunostained with anti‐HA. GFP (I), Jumpy WT (M), Jumpy CS (green) (A, E, Q), LC3 (red) (C, G, J, N, R) and Atg9 (white in B, F, K, O, S or blue in D, H, I, P, T). Boxed areas (A-D) are shown at higher magnification in the corresponding panel below (E-H). White arrows indicate colocalization between Jumpy CS, LC3 and Atg9, blue arrow indicates colocalization between Jumpy CS and Atg9 but not LC3 and yellow arrow indicates colocalization between Jumpy CS and LC3 but not Atg9. White boxes (I-T) show LC3 puncta location. Scale bars, 5 μm.


( U ) of LC3 colocalizing with Atg9 . n , number of counted .

(U) Percentage of LC3 puncta colocalizing with Atg9. n, number of puncta counted.


( A - K ) C2C12 were transfected for 48 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) , YFP ‐ Jumpy C330S ( Jumpy CS ) , YFP ‐ Jumpy Y462C ( Jumpy YC ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , fixed immunostained with anti ‐ p62 antibody ( red ) . p62 were quantitated by confocal microscopy . Representative confocal of YFP ( A ) , YFP ‐ Jumpy WT ( B ) , YFP ‐ Jumpy C330S ( C ) , YFP ‐ Jumpy Y462C ( D ) YFP ‐ Jumpy R336Q ( E ) transfected , immunostained for p62 ( F ) , ( G ) , ( H ) , ( I ) ( J ) , respectively . Scale bars , 5 μm . Quantitation of p62 per ( K ) . Bars , s . e . m . ( n = 3 independent experiments , 30 per experiments ) . * P0 . 05 ( t ‐ ) , ns : nonsignificant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-K) C2C12 cells were transfected for 48 h with YFP, YFP‐Jumpy WT (Jumpy WT), YFP‐Jumpy C330S (Jumpy CS), YFP‐Jumpy Y462C (Jumpy YC) or YFP‐Jumpy R336Q (Jumpy RQ), fixed and immunostained with anti‐p62 antibody (red). p62 puncta were quantitated by confocal fluorescence microscopy. Representative confocal images of YFP (A), YFP‐Jumpy WT (B), YFP‐Jumpy C330S (C), YFP‐Jumpy Y462C (D) and YFP‐Jumpy R336Q (E) transfected cells, immunostained for p62 (F), (G), (H), (I) and (J), respectively. Scale bars, 5 μm. Quantitation of p62 puncta per cell (K). Bars, s.e.m. (n=3 independent experiments, 30 cells per experiments). *P0.05 (t‐test), ns: nonsignificant.


( L ) HeLa were transfected for 24 h with YFP , YFP ‐ Jumpy WT ( Jumpy WT ) or YFP ‐ Jumpy R336Q ( Jumpy RQ ) , incubated with or without 50 ng / μl rapamycin ( BafA1 ( 100 nM ) was present in both control rapamycin treated ) for 2 h , lysed analysed for LC3 , YFP actin by immunoblotting . Densitometric LC3 ‐ II / actin ratios are shown underneath blot .

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

(L) HeLa cells were transfected for 24 h with YFP, YFP‐Jumpy WT (Jumpy WT) or YFP‐Jumpy R336Q (Jumpy RQ), incubated with or without 50 ng/μl rapamycin (BafA1 (100 nM) was present in both control and rapamycin treated cells) for 2 h, lysed and analysed for LC3, YFP and actin by immunoblotting. Densitometric LC3‐II/actin ratios are shown underneath the blot.


( C ) Example of yeast for each of 21 subcellular endocytic identified in this study . relevant markers are listed to left of . Scale bar : 5 µm . ( D ) Pie charts showing proportion of specific phenotype mutants ( SPMs ) that have one or more distinct aberrant , non - phenotype - specific mutants for each of compartments screened .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study. The relevant markers are listed to the left of the micrographs. Scale bar: 5 µm. (D) Pie charts showing the proportion of specific phenotype mutants (SPMs) that have one or more distinct aberrant phenotypes, and non-phenotype-specific mutants for each of the compartments screened.


Representative of wild - type mutant organized by marker phenotype ( labels on left of each panel ) . For each phenotype , three labelled for training set ( labelled single ) identified by 2NN classifier ( identified single ) are shown . table to right of shows ( from left to right ) : [ 1 ] occurrence of each phenotype in a wild - type population ( % in WT ) ; [ 2 ] number of specific phenotype mutants ( ) stringent specific phenotype mutants ( str ) for each of 17 mutant morphologies ; [ 3 ] most significantly enriched GO Slim biological process ; [ 4 ] most significantly enriched . # denotes term below statistical significance .

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

Representative images of wild-type and mutant cells organized by marker and phenotype (labels on the left of each panel). For each phenotype, three cells labelled for the training set (labelled single cells) and cells identified by the 2NN classifier (identified single cells) are shown. The table to the right of the images shows (from left to right): [1] the occurrence of each phenotype in a wild-type population (% in WT); [2] the number of specific phenotype mutants (all) and stringent specific phenotype mutants (str) for each of the 17 mutant morphologies; [3] the most significantly enriched GO Slim biological process and; [4] the most significantly enriched protein complex. # denotes term below statistical significance.


Endocytic patch formation dynamics in ydl176wΔ ( ipf1Δ ) . Patch dynamics were examined using time - lapse microscopy of wild - type ( WT ) ipf1Δ deletion strains carrying reporters for coat ( Sla1 - GFP ; green ) , actin ( Sac6 - tdTomato ; red ) modules . Left : Representative kymographs for WT ipf1Δ strains . Right : Box plot illustrating distribution of lifetimes of Sla1 - GFP Sac6 - tdTomato patches . Significance was determined using unpaired t - . Scale bar : 10 seconds .

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

Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds.


Penetrance as a function of replicative . Top : Bar graph showing fraction of outliers in populations of increasing replicative ( # of divisions ) for wild - type ( WT ) , 5 mutant strains ( rrd2Δ , rpl20bΔ , cka2Δ , vac8Δ vac17Δ ) . Data are presented as mean of 3 biological replicates + / - SD . Bottom : Box plot with distribution of sizes for same populations of . Whiskers extend to 5th 95th percentile .

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

Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile.


of young ( top row of ) older ( bottom row of ) wild - type ( WT ) expressing Vph1 - EGFP ( green vacuole ) stained with CF640R WGA ( magenta bud scars ) . Scale bar : 5 µm .

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

Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.


Combined effect of replicative a vacuole inheritance defect on penetrance . of wild - type vac17Δ expressing Vph1 - EGFP ( green vacuole ) Hta2 - mCherry ( red nucleus ) , stained with CF640R WGA ( magenta bud scars ) . with increasing bud - scar ( replicative ) are shown from left to right . Scale bar : 5 µm .

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

Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.


Relationship between stress response penetrance . Single - UPRE - RFP were measured in ~ 60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole . were binned into equal - sized bins , from low to high stress response , assessed as outlier or inlier , clustered on their penetrance profile ( composed from fraction of outliers in each stress - response bin ) . Each plot represents a penetrance profile . * denotes cluster with a profile most similar to wild - type . Insert pie charts show proportion of mutant strains in each cluster .

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

Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.


( C ) Blood brain tissue of CNP520 in rat after a 30 µmol / kg ( 15 . 4 mg / kg ) oral dose . Shown are unbound fraction ( main graph ) total ( insert ) of CNP520 in blood brain , using values for rat plasma binding of 97 . 8 % unspecific binding to brain homogenate of 99 % . Values are mean ± SD , with n = 5 per time point .

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

(C) Blood and brain tissue levels of CNP520 in the rat after a 30 µmol/kg (15.4 mg/kg) oral dose. Shown are unbound fraction (main graph) and total levels (insert) of CNP520 in the blood and brain, using values for rat plasma protein binding of 97.8% and unspecific binding to brain homogenate of 99%. Values are mean ± SD, with n = 5 per time point.


( A ) Subjective assessment of hair depigmentation ( score 0 - 5 , with 0 = completely black 5 = completely ) at 2 doses of either CNP520 or BACE - 1 / - 2 inhibitor NB - 360 ( mean ± , n = 8 / group ) , statistical comparisons were made for every scoring day , versus vehicle using Kruskal - Wallis with Dunn ' s post - hoc .

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

(A) Subjective assessment of hair depigmentation (score 0-5, with 0 = completely black and 5 = completely white) at 2 doses of either CNP520 or the BACE-1/-2 inhibitor NB-360 (mean ± SEM, n=8/group), statistical comparisons were made for every scoring day, versus vehicle using Kruskal-Wallis with Dunn's post-hoc test.


( B ) Summary of doses , steady - state effects on Aβ40 in brain ( % compared to vehicle - treated mice ) ratios between total skin concentrations of CNP520 NB - 360 their respective IC50 values for inhibition of BACE - 2 in vitro .

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

(B) Summary of doses, steady-state effects on Aβ40 in brain (% compared to vehicle-treated mice) and the ratios between total skin concentrations of CNP520 and NB-360 and their respective IC50 values for inhibition of BACE-2 in vitro.


( A ) Doses from 1 . 5 mg / kg ( 3 µmol / kg ) to 51 . 3 mg / kg ( 100 µmol / kg ) were given by oral gavage to rats ( n = 5 per dose ) ; were killed 4 hours later for determination of Aβ40 in brain tissue . Values are mean ± as determined by ANOVA ( GraphPad Prism ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Doses from 1.5 mg/kg (3 µmol/kg) to 51.3 mg/kg (100 µmol/kg) were given by oral gavage to rats (n = 5 per dose); animals were killed 4 hours later for determination of Aβ40 in brain tissue. Values are mean ± SEM as determined by ANOVA (GraphPad Prism).


( B ) Time - dependent reduction of Aβ40 in rat brain CSF after a single 15 . 4 mg / kg ( 30 µmol / kg ) oral dose of CNP520 .

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

(B) Time-dependent reduction of Aβ40 in rat brain and CSF after a single 15.4 mg/kg (30 µmol/kg) oral dose of CNP520.


( C ) CNP520 in dog blood CSF , following a 3 . 1 mg / kg ( 6 µmol / kg ) oral dose ( mean ± SD = 4 ) .

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

(C) CNP520 levels in dog blood and CSF, following a 3.1 mg/kg (6 µmol/kg) oral dose (mean ± SD = 4).


( D ) Time - dependent reduction of Aβ40 42 in dog CSF , after a 3 . 1 mg / kg oral CNP520 dose ( mean ± s . d . ) .

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

(D) Time-dependent reduction of Aβ40 and 42 in dog CSF, after a 3.1 mg/kg oral CNP520 dose (mean ± s.d.).


APP23 mice ( at baseline : 12 - 14 months old ) were dosed with CNP520 in food pellets for 6 months : Low dose 0 . 03 g CNP520 / kg food , corresponding to a daily oral dose of 4 mg / kg ; high dose 0 . 3 g / kg food , corresponding to a daily oral dose of 40 mg / kg . ( A ) CNP520 in blood cerebellum .

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

Male APP23 mice (age at baseline: 12-14 months old) were dosed with CNP520 in food pellets for 6 months: Low dose 0.03 g CNP520/kg food, corresponding to a daily oral dose of 4 mg/kg; high dose 0.3 g/kg food, corresponding to a daily oral dose of 40 mg/kg. (A) CNP520 levels in blood and cerebellum.