A - D . Response of HyPer7 probes targeted to indicated compartments to reoxygenation after hypoxia ( red curve data points ) or to continued hypoxia ( black data points ) . HEK293 were grown either with glucose or galactose as carbon source . Solid represents mean , points colored in lighter version of respective color are corresponding ratios measured in . E . Model . grown on galactose - containing medium exhibit increased production of H2O2 thus also increased detection of H2O2 indicating H2O2 gradients of different steepness around mitochondria . Data information : As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * * * , p ≤ 0 .

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

A-D. Response of HyPer7 probes targeted to indicated compartments to reoxygenation after hypoxia (red curve and data points) or to continued hypoxia (black line and data points). HEK293 cells were grown either with glucose or galactose as carbon source. Solid line represents mean, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. E. Model. Cells grown on galactose-containing medium exhibit increased production of H2O2 and thus also increased detection of cytosolic H2O2 indicating H2O2 gradients of different steepness around mitochondria. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs.***, p ≤ 0.001


C . Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO - expressing HEK293 with 2 mM D - Ala ( generated with Flp - In T - REX - system ) . HEK293 were grown either with glucose ( black curve data points ) or galactose ( red curve data points ) as carbon source . Solid represents average , points colored in lighter version of respective color are corresponding ratios measured in . Data information : As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * * * , p ≤ 0 .

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C. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing HEK293 cells with 2 mM D-Ala (cells generated with the Flp-In T-REX-system). HEK293 cells were grown either with glucose (black curve and data points) or galactose (red curve and data points) as carbon source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001


A . Response of HyPer7 probes targeted to indicated compartments to treatment with increasing amounts of exogenous H2O2 ( 2 , 4 8 µM H2O2 ; 20 mM DTT ) . HEK293 were grown either with glucose ( black curve data points ) or galactose ( red curve data points ) as carbon source . As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * * * , p ≤ 0 .

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

A. Response of HyPer7 probes targeted to indicated compartments to treatment with increasing amounts of exogenous H2O2 (2, 4 and 8 µM H2O2; 20 mM DTT). HEK293 cells were grown either with glucose (black curve and data points) or galactose (red curve and data points) as carbon source. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001


A . in grown on glucose galactose . lysates were analyzed by quantitative mass spectrometry ( n = 4 biological replicates of antioxidative enzymes ( especially peroxiredoxins , PRDX in blue ) are not changed except for thioredoxin reductase ( TXNRD1 ) .

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A. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by quantitative mass spectrometry (n = 4 biological replicates Levels of antioxidative enzymes (especially peroxiredoxins, PRDX in blue) are not changed except for cytosolic thioredoxin reductase (TXNRD1).


B . in grown on glucose galactose . lysates were analyzed by immunoblot ( n = 2 biological replicates ) . thioredoxin reductase ( TXNRD1 ) is increased by more than two - fold in grown on galactose compared to glucose - grown . dotted depicts 1 , meaning no change in .

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B. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by immunoblot (n = 2 biological replicates). Cytosolic thioredoxin reductase (TXNRD1) is increased by more than two-fold in cells grown on galactose compared to glucose-grown cells.The dotted line depicts 1, meaning no change in protein levels.


B . Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A ( AntA , red curve data points ) or ethanol as control ( black curve data points ) . HeLa were grown with galactose as carbon source . Solid represents average , points colored in lighter version of respective color are corresponding ratios measured in . Data information : As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * * * , p ≤ 0 .

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 O O O O 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. Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A (AntA, red curve and data points) or ethanol as control (black curve and data points). HeLa cells were grown with galactose as carbon source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001


B . Peroxiredoxin in peroxiredoxin 1 ( PRDX1 KO ) peroxiredoxin 2 ( PRDX2 KO ) knockout . quantitative proteomics ( n = 4 , technical replicates ) right subpanel of B lists identified belonging to antioxidative systems which are not altered for PRDX1 KO or PRDX2 KO , respectively . In both quantitative proteomics experiments 4403 were detected in total .

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B. Peroxiredoxin levels in peroxiredoxin 1 (PRDX1 KO) and peroxiredoxin 2 (PRDX2 KO) knockout cells. quantitative proteomics (n = 4, technical replicates) The right subpanel of B lists the identified proteins belonging to cellular antioxidative systems which are not altered for PRDX1 KO or PRDX2 KO, respectively. In both quantitative proteomics experiments 4403 proteins were detected in total.


D . Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO - expressing lines with 4 mM D - Ala ( black , wild type ; red , PRDX1 KO ; blue , PRDX2 KO ; generated with piggyBAC system ) . were grown in glucose - containing medium . Solid represents average , points colored in lighter version of respective color are corresponding ratios measured in . Data information As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * , p ≤ 0 . 05 , * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

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D. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1 KO; blue, PRDX2 KO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001


B . Peroxiredoxin in peroxiredoxin 1 2 double knockout ( PRDX1 / 2 DKO ) . quantitative proteomics ( B ) . right subpanel in ( B ) lists detected but not altered belonging to antioxidative systems .

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B. Peroxiredoxin levels in peroxiredoxin 1 and 2 double knockout cells (PRDX1/2 DKO). quantitative proteomics (B).The right subpanel in (B) lists all detected but not altered proteins belonging to cellular antioxidative systems.


E . Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO - expressing lines with 4 mM D - Ala ( black , wild type ; red , PRDX1 / 2 DKO ; generated with piggyBAC system ) . were grown in glucose - containing medium . Solid represents average , points colored in lighter version of respective color are corresponding ratios measured in . Data information : Statistical was only performed to compare PRDX1 / 2 DKO with wildtype . As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * , p ≤ 0 . 05 , * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

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E. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1/2 DKO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: Statistical analysis was only performed to compare the PRDX1/2 DKO with the wildtype. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001


( a ) ternary NDP52 - GAL8 - LC3C ( green ) , binary NDP52 - GAL8 ( blue ) NDP52 alone ( red ) by SEC - MALS . dotted represents measured MW vertical bars ( 1 2 ) indicate gap in MW . SEC - MALS result of NDP52 alone ( red ) shows that oligomeric state is ambiguous between dimer trimer . However , NDP52 - GAL8 shows a MW difference of 70 kDa compared with NDP52 alone , which confirmed that two molecules of monomeric GAL8 ( MW : ~ 33 . 5 kDa ; Supplementary Fig . S1b ) bound to homodimeric NDP52 . Furthermore , ternary NDP52 - GAL8 - LC3C showed a MW difference of ~ 30 kDa compared with binary NDP52 - GAL8 , which also confirmed that two additional molecules of monomeric LC3C bound to binary .

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(a) The ternary NDP52-GAL8-LC3C complex (green line), binary NDP52-GAL8 complex (blue line) and NDP52 complex alone (red line) by SEC-MALS. The dotted line represents measured MW and the vertical bars (1 and 2) indicate the gap in MW. The SEC-MALS result of NDP52 alone (red line) shows that the oligomeric state is ambiguous between dimer and trimer. However, the NDP52-GAL8 complex shows a MW difference of 70 kDa compared with NDP52 alone, which confirmed that two molecules of monomeric GAL8 (MW: ~33.5 kDa; Supplementary Fig. S1b) bound to homodimeric NDP52. Furthermore, the ternary NDP52-GAL8-LC3C complex showed a MW difference of ~30 kDa compared with the binary NDP52-GAL8 complex, which also confirmed that two additional molecules of monomeric LC3C bound to the binary complex.


( b ) - exclusion chromatography data of NDP52 - GAL8 - LC3C . first peak ( marked by an orange bar ) represents ternary , as shown by SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) results marked by an orange bar ( lane 1 , 2 3 ) . second merged peak ( marked by green yellow bar ) corresponding SDS - PAGE results show remaining free GAL8 ( lane 4 , 5 , 6 7 ) LC3C ( lane 5 , 6 , 7 8 ) .

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(b) Size-exclusion chromatography data of NDP52-GAL8-LC3C complex. The first peak (marked by an orange bar) represents the ternary complex, as shown by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results marked by an orange bar (lane 1, 2 and 3). The second merged peak (marked by green and yellow bar) and corresponding SDS-PAGE results show the remaining free GAL8 (lane 4, 5, 6 and 7) and LC3C (lane 5, 6, 7 and 8).


( a ) Overall structure of GAL8 with bound Galbi peptide ( cyan ) NAD ( purple ) in asymmetric unit . other pair of crystallographic dimer is coloured grey . first β - strand forming unique interaction is labelled as β1 . ( b ) A topology diagram of GAL8 . β - strands in each N - CRD C - CRD are numbered sequentially with prime ( ′ ) in C - CRD . β - strands with a solid indicate a carbohydrate - binding side in each CRD those of with a dashed indicate an opposite side . elongated purple cyan ovals indicate NAD - NDP52 - binding sites , respectively . ( c ) Overall view of Galbi peptide ( cyan ) bound to C - CRD of GAL8 ( β - strands in yellow residues in gray ) .

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 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

(a) Overall structure of GAL8 with bound Galbi peptide (cyan) and NAD (purple) in the asymmetric unit. The other pair of crystallographic dimer is coloured grey. The first β-strand forming unique interaction is labelled as β1. (b) A topology diagram of GAL8. The β-strands in each N-CRD and C-CRD are numbered sequentially with prime (′) in the C-CRD. The β-strands with a solid line indicate a carbohydrate-binding side in each CRD and those of with a dashed line indicate an opposite side. The elongated purple and cyan ovals indicate NAD- and NDP52-binding sites, respectively. (c) Overall view of Galbi peptide (cyan) bound to the C-CRD of GAL8 (β-strands in yellow and residues in gray).


( a ) Magnified view showing detailed interaction around Leu374 ( p ) . ( b ) Magnified view showing detailed interaction around Tyr380 ( p ) . ( c ) Magnified view showing importance of main - chain hydrogen bonds of Galbi peptide . ( a - c ) Red dashed lines designate hydrogen bonds .

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

(a) Magnified view showing the detailed interaction around Leu374(p). (b) Magnified view showing the detailed interaction around Tyr380(p). (c) Magnified view showing the importance of main-chain hydrogen bonds of the Galbi peptide. (a-c) Red dashed lines designate hydrogen bonds.


( d ) An FP using FITC - labelled Galbi peptides , including mutants ( L374A , Y380F Y380A ) against an increasing of GAL8 . ( e ) An FP using a FITC - labelled Galbi peptide against an increasing of GAL8 mutants ( E268A , I270A , Y272A Trp317 ) . ( d , e ) KD values were calculated from at least three independent experiments . ND , not determined .

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(d) An FP assay using FITC-labelled Galbi peptides, including mutants (L374A, Y380F and Y380A) against an increasing concentration of GAL8. (e) An FP assay using a FITC-labelled Galbi peptide against an increasing concentration of GAL8 mutants (E268A, I270A, Y272A and Trp317). (d,e) The KD values were calculated from at least three independent experiments. ND, not determined.


( a ) alignment of N - CRD C - CRD of GAL8 . residues important for interaction with Galbi peptide NAD are marked by yellow magenta circles , respectively . ( b ) Detailed view of peptide - binding site of C - CRD superposed with N - CRD . residues for Galbi peptide ( cyan ) interaction are labelled side chains of corresponding N - CRD residues ( magenta ) are shown . ( c ) A detailed view of NAD - binding site of N - CRD superposed with C - CRD . residues for NAD ( purple ) interaction are labelled side chains of corresponding C - CRD residues ( yellow ) are shown .

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

(a) Sequence alignment of N-CRD and C-CRD of GAL8. The residues important for interaction with the Galbi peptide and NAD are marked by yellow and magenta circles, respectively. (b) Detailed view of the peptide-binding site of C-CRD superposed with N-CRD. The residues for the Galbi peptide (cyan) interaction are labelled and the side chains of the corresponding N-CRD residues (magenta) are shown. (c) A detailed view of the NAD-binding site of N-CRD superposed with C-CRD. The residues for NAD (purple) interaction are labelled and the side chains of corresponding C-CRD residues (yellow) are shown.


( a ) Three GAL8 structures , including both CRDs , are available from PDB ( 4FQZ - lactose bound in only N - CRD ; 3VKL - lactose bound in both N - CRD N - C - CRD ; 3VKM - sialylated lactose in N - CRD N - CRD in C - CRD ) . C - CRDs of these Gal8 structures were superposed with that of our Gal8 . For clarity , only Cα - traces of each structure ( our GAL8 - yellow ; 4FQZ - red ; 3VKM - blue ; 3VKL - cyan ) are shown . large rotation of N - CRD domain relative to GAL8 compared with other NDP52 structures is observed in GAL8 - NDP52 . A representative angle between CRD of our Gal8 that of 4FQZ structure is indicated in degrees . ( b ) A 90 ° rotation of panel ( a ) along horizontal axis showing a top view . Arg59 , which is a unique residue of Gal8CRD that provides carbohydrate specificity , is used as a standard for measuring of domain movement .

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

(a) Three GAL8 structures, including both CRDs, are available from PDB (4FQZ-lactose bound in only N-CRD; 3VKL-lactose bound in both N-CRD and N-C-CRD; 3VKM-sialylated lactose in N-CRD and N-CRD in C-CRD). The C-CRDs of these Gal8 structures were superposed with that of our Gal8. For clarity, only Cα-traces of each structure (our GAL8-yellow; 4FQZ-red; 3VKM-blue; and 3VKL-cyan) are shown. The large rotation of the N-CRD domain relative to GAL8 compared with the other NDP52 structures is observed in the GAL8-NDP52 complex. A representative angle between CRD of our Gal8 and that of the 4FQZ structure is indicated in degrees. (b) A 90° rotation of panel (a) along the horizontal axis showing a top view. Arg59, which is a unique residue of Gal8CRD that provides carbohydrate specificity, is used as a standard for measuring the distance of domain movement.


( A ) Immunofluorescent of ABCA12 ( green ) Insulin ( red ) in mouse islets showing extensive co - localisation in β - as well as expression in other islet populations ( arrowheads , scale bar = 25μm ) . ( B ) Co - expression of Abca12 ( green ) Insulin ( red ) in MIN6 ( scale bar = 10μm ) .

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(A) Immunofluorescent staining of ABCA12 (green) and Insulin (red) in mouse islets showing extensive co-localisation in β-cells as well as expression in other islet populations (arrowheads, scale bar=25μm). (B) Co-expression of Abca12 (green) and Insulin (red) in MIN6 cells (scale bar = 10μm).


( C ) LacZ ( blue ) to detect gene expression of knocked - in LacZ cassette from Abca12 locus in islets ( islet outlined , scale bar = 25μm ) .

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(C) LacZ staining (blue) to detect gene expression of the knocked-in LacZ cassette from the Abca12 locus in islets (islet outlined, scale bar = 25μm).


( D , E ) ABCA12 immunostaining development of hyperkeratotic skin disease in E18 . 5 Abca12tm1a / tm1a mouse ( brackets indicate thickened epidermis , dashed indicates epidermal basement , scale bar = 50μm ) .

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(D,E) ABCA12 immunostaining and the development of hyperkeratotic skin disease in E18.5 Abca12tm1a/tm1a mouse embryos (brackets indicate thickened epidermis, dashed line indicates epidermal basement membrane, scale bar = 50μm).


( F , G ) Immunohistochemical detection of cre expression ( green ) in pancreatic islets from conditional mouse lines showing co - expression with Insulin ( red ) ( scale bar = 50μm ) .

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(F, G) Immunohistochemical detection of cre expression (green) in pancreatic islets from conditional mouse lines showing co-expression with Insulin (red)(scale bar = 50μm).


( H ) Detection of recombination of Abca12 locus by PCR , indicating different alleles ( tm1c , wild type tm1d ) .

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(H) Detection of recombination of the Abca12 locus by PCR, indicating different alleles (tm1c, wild type and tm1d).


( I - K ) Detection of ABCA12 expression ( green ) deletion in pancreas sections from mice of indicated genotypes ( islets outlined , scale bar = 25μm ) .

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(I-K) Detection of ABCA12 expression (green) and deletion in pancreas sections from mice of indicated genotypes (islets outlined, scale bar = 25μm).


( L ) Quantitation of ABCA12 expression by densitometry of Western blots from purified pancreatic islets ( values relative to wild type , n = 3 - 8 per genotype , mean ± , * p = < 0 . 05 , Students t - ) , showing representative blot with Actin loading control ( right ) .

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(L) Quantitation of ABCA12 expression by densitometry of Western blots from purified pancreatic islets (values relative to wild type, n=3-8 animals per genotype, mean±SEM, *p=<0.05, Students t-test), showing representative blot with Actin loading control (right).


Average daily food intake of conditionally modified mice ( n = 4 - 10 / genotype , mean ± ) .

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Average daily food intake of conditionally modified mice (n=4-10 animals/genotype, mean±SEM).


( N ) ( n = 4 - 10 / genotype , mean ± ) .

(N) animal body weight (n=4-10 animals/genotype, mean±SEM).


( A - F ) Oral intraperitoneal glucose tolerance testing in Abca12tm1d mice at 6 ( A , B ) , 8 ( C , D ) 12 ( E , F ) ( mean ± , n = 4 - 9 mice per group , * p = < 0 . 05 , * * p < 0 . 01 Students t - ) .

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(A-F) Oral and intraperitoneal glucose tolerance testing in Abca12tm1d mice at 6 (A,B), 8 (C, D) and 12 weeks (E,F) (mean±SEM, n=4-9 mice per group, * p=<0.05, **p<0.01 Students t-test).


( G ) Insulin tolerance in 12 week old mice ( mean ± , n = 4 - 9 mice per group ) .

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(G) Insulin tolerance in 12 week old mice (mean±SEM, n=4-9 mice per group).


( H ) Whole blood Hb1Ac in 16 week old Abca12tm1d mice ( mean ± , n = 4 - 9 mice per group , * * p < 0 . 01 Students t - ) .

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(H) Whole blood Hb1Ac levels in 16 week old Abca12tm1d mice (mean±SEM, n=4-9 mice per group, **p<0.01 Students t-test).


( I ) Acute phase insulin secretion after intraperitoneal injection of glucose ( mean ± , n = 4 - 9 per group , / grey / black = Abca12 + / + , cre / + Abca12tm1d respectively , mice at 8 of , * * p < 0 . 01 Students t - ) .

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(I) Acute phase insulin secretion after intraperitoneal injection of glucose (mean±SEM, n=4-9 animals per group, white/grey/black = Abca12+/+, cre/+ and Abca12tm1d respectively, mice at 8 weeks of age, **p<0.01 Students t-test).


( J ) Silencing of ABCA12 in MIN6 as assessed by Western blot of ABCA12 immuno - precipitate ( top panel ) or confocal microscopy in transfected with Dy547 - siRNAABCA12 ( lower panel , scale bar = 10μm ) .

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(J) Silencing of ABCA12 in MIN6 cells as assessed by Western blot of ABCA12 immuno-precipitate (top panel) or confocal microscopy in cells transfected with Dy547-siRNAABCA12 (lower panel, scale bar = 10μm).


( K ) Glucose stimulated insulin secretion ( GSIS ) from MIN6 after silencing of ABCA12 ( mean ± , n = 4 replicates from assays in quadruplicate , * * * p < 0 . 0001 , Students t - ) .

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(K) Glucose stimulated insulin secretion (GSIS) from MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate, ***p<0.0001, Students t-test).


( L ) insulin content in MIN6 after silencing of ABCA12 ( mean ± , n = 4 replicates from assays in quadruplicate ) .

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(L) Cellular insulin content in MIN6 cells after silencing of ABCA12 (mean±SEM, n=4 replicates from assays in quadruplicate).


( A - C ) ( A ) ABCA1 , ( B ) ABCG1 ( C ) LXRβ expression in islets of 8 week old mice assessed by Western blotting ( mean ± , n = 3 - 6 mice per genotype , relative to β - actin controls , * p < 0 . 05 , * * p = < 0 . 01 , Students t - ) .

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(A-C) (A) ABCA1, (B) ABCG1 and (C) LXRβ expression in islets of 8 week old mice assessed by Western blotting (mean±SEM, n=3-6 mice per genotype, relative to β-actin controls, *p<0.05, **p=<0.01, Students t-test).


( D ) Western blot of lysates of MIN6 after silencing of Abca12 , probed with antibodies against ABCA1 , ABCG1 , SR - B1 LXRβ .

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(D) Western blot of cell lysates of MIN6 cells after silencing of Abca12, probed with antibodies against ABCA1, ABCG1, SR-B1 and LXRβ.


( E ) Total cholesterol ( COH ) or cholesteryl esters ( CE ) in isolated pancreatic islets of mice at 8 of with deletion of Abca12 in β - ( mean ± n = 9 , 5 5 mice per genotype respectively ) .

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(E) Total cholesterol (COH) or cholesteryl esters (CE) levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=9, 5 and 5 mice per genotype respectively).


( F ) Ceramides in isolated pancreatic islets of mice at 8 of with deletion of Abca12 in β - ( mean ± n = 10 , 4 5 mice per genotype respectively ) .

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(F) Ceramides levels in isolated pancreatic islets of mice at 8 weeks of age with deletion of Abca12 in β-cells (mean±SEM n=10, 4 and 5 mice per genotype respectively).


( G ) Lipid in MIN6 in which ABCA12 had been depleted by siRNA treatment ( mean ± , n = 5 ( biological replicates assayed in triplicate ) , FC = free cholesterol , CE = cholesterol ester , Cer = ceramide , SM = sphingomyelin , TG = triacylglycerol , PC = phosphatidylcholine , * p < 0 . 05 , * * p = < 0 . 01 , Students t - ) .

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(G) Lipid levels in MIN6 cells in which ABCA12 had been depleted by siRNA treatment (mean±SEM, n=5 (biological replicates assayed in triplicate), FC = free cholesterol, CE = cholesterol ester, Cer = ceramide, SM = sphingomyelin, TG = triacylglycerol, PC = phosphatidylcholine, *p<0.05, **p=<0.01, Students t-test).


( H ) Cholesterol efflux to lipid - free apoA - I ( 30 μg / ml , 2 h ) from MIN6 transfected with siRNAscr or siRNAABCA12 activated or not with TO901317 ( 4 μM ) ( mean ± , n = 4 , ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - ) .

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(H) Cholesterol efflux to lipid-free apoA-I (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM)(mean±SEM, n=4, (biological replicates assayed in triplicate), *p=<0.05, Students t-test).


( I ) Cholesterol efflux to HDL ( 30 μg / ml , 2 h ) from MIN6 transfected with siRNAscr or siRNAABCA12 activated or not with TO901317 ( 4 μM ) ; ( mean ± , n = 4 ( biological replicates assayed in triplicate ) ) .

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(I) Cholesterol efflux to HDL (30 μg/ml, 2 h) from MIN6 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 (4 μM); (mean±SEM, n=4 (biological replicates assayed in triplicate)).


( J ) effect of overexpression of ABCA1 on GSIS from MIN6 with ABCA12 deficiency ( mean ± , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - ) , inset shows a Western blot of lysates probed with anti - ABCA1 antibody in which order of bands corresponds to order of bars ) .

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(J) The effect of overexpression of ABCA1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test), inset shows a Western blot of cell lysates probed with anti-ABCA1 antibody in which the order of bands corresponds to the order of bars).


( K ) effect of overexpression of ABCG1 on GSIS from MIN6 with ABCA12 deficiency ( mean ± , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - , inset shows a Western blot of lysate probed with anti - ABCG1 anitbodies in which order of bands corresponds to order of bars ) .

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(K) The effect of overexpression of ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test, inset shows a Western blot of cell lysate probed with anti-ABCG1 anitbodies in which the order of bands corresponds to the order of bars).


( L ) effect of overexpression of ABCA1 ABCG1 on GSIS from MIN6 with ABCA12 deficiency ( mean ± , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - ) .

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(L) The effect of overexpression of ABCA1 and ABCG1 on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).


( M ) effect of activation of with TO901317 ( 4 μM ) on GSIS from MIN6 with ABCA12 deficiency ( mean ± , n = 4 ( biological replicates assayed in triplicate ) , * p = < 0 . 05 , Students t - ) .

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(M) The effect of activation of cells with TO901317 (4 μM) on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates assayed in triplicate), *p=<0.05, Students t-test).


( N ) effect of overexpression of LXRβ on GSIS from MIN6 with ABCA12 deficiency ( mean ± , n = 4 ( biological replicates ) , * p = < 0 . 05 , Students t - , inset shows a Western blot of lysates probed with anti - LXRβ antibody in which order of bands corresponds to order of bars ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The effect of overexpression of LXRβ on GSIS from MIN6 cells with ABCA12 deficiency (mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test, inset shows a Western blot of cell lysates probed with anti-LXRβ antibody in which the order of bands corresponds to the order of bars).


( B ) Scatter plot heat map of differential gene expression between cre control Abca12tm1d mice at 8 of . Legend indicates Log2 fold change values .

(B) Scatter plot and heat map of differential gene expression between cre control and Abca12tm1d mice at 8 weeks of age. Legend indicates Log2 fold change values.


( C ) Circulating glucose after administration of intraperitoneal L - arginine in mice at 8 of , mean ± , n = 3 mice per genotype , * p = < 0 . 05 of cre relative to tm1d , Students t - ) .

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(C) Circulating glucose levels after administration of intraperitoneal L-arginine in mice at 8 weeks of age, mean±SEM, n =3 mice per genotype, *p=<0.05 of cre relative to tm1d, Students t-test).


( D ) Representative of insulin granule fusion events ( yellow circles ) in mice of indicated genotypes in response to administration of 15mM glucose ( scale bar = 10μm ) . of proportion of responding density of granule fusion events are presented in graphs in lower panel ( mean ± , 8 - 10 islets / mouse , > 4 mice / genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 of cre relative to tm1d , Students t - ) .

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(D) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 15mM glucose (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the lower panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01, ***p=<0.001 of cre relative to tm1d, Students t-test).


( E ) Representative of insulin granule fusion events ( yellow circles ) in mice of indicated genotypes in response to administration of 20mM KCl ( scale bar = 10μm ) . of proportion of responding density of granule fusion events are presented in graphs in right panel ( mean ± , 8 - 10 islets / mouse , > 4 mice / genotype , * * p = < 0 . 01 of cre relative to tm1d , Students t - ) .

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(E) Representative images of insulin granule fusion events (yellow circles) in mice of indicated genotypes in response to administration of 20mM KCl (scale bar = 10μm). Quantification of the proportion of responding cells and the density of granule fusion events are presented in graphs in the right panel (mean±SEM, 8-10 islets/mouse, >4 mice/genotype, **p=<0.01 of cre relative to tm1d, Students t-test).


( F ) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old . responses were corrected for basal fill ( F / F0 ) . ( mean ± , each data point represents average of 10 / coverslip for 13 - 16 coverslips for each of 4 wild type 5 Abca12tm1d mice )

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(F) Assessment of calcium flux measured by Fluo4 in response to either Glucose or Potassium in isolated islets from 11 week old animals. Fluorescence responses were corrected for basal fill (F/F0). (mean±SEM, each data point represents the average of 10 cells/coverslip for 13-16 coverslips for each of 4 wild type and 5 Abca12tm1d mice)


( G ) Expression of GLUT2 in isolated mouse islets at 8 of . upper panel depicts a representative Western blot lower graph quantitation of from 3 mice of each genotype , normalised to β - actin ( mean ± ) .

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(G) Expression of GLUT2 in isolated mouse islets at 8 weeks of age. The upper panel depicts a representative Western blot and the lower graph the quantitation of protein levels from 3 mice of each genotype, normalised to β-actin (mean±SEM).


( H ) Insulin content of islets of Abca12tm1d mice ( mean ± , n = 3 mice of each genotype , normalised to total content ) .

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(H) Insulin content of islets of Abca12tm1d mice (mean±SEM, n=3 mice of each genotype, normalised to total protein content).


( A - C ) Transmission electron microscopy of β - from Abca12tm1d control mice at 8 of . Panels A ' - C ' depict higher resolution from boxed regions in A - C ( scale bar = 1μm ) .

(A-C) Transmission electron microscopy of β-cells from Abca12tm1d and control mice at 8 weeks of age. Panels A'-C' depict higher resolution images from the boxed regions in A-C (scale bar = 1μm).


( D ) Quantitation of of 3000 granules from each genotype ( mean ± , n = 4 , 3 3 respectively , * p = < 0 . 05 of cre relative to tm1d , Students t - ) . ( E ) Relative frequency distribution of granule areas from ( D ) .

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(D) Quantitation of the area of 3000 granules from each genotype (mean±SEM, n=4, 3 and 3 animals respectively, *p=<0.05 of cre relative to tm1d, Students t-test). (E) Relative frequency distribution of granule areas from (D).


( F ) Assessment of total cholesterol ( COH ) by mass spectroscopy in mice fed normal chow or a high cholesterol diet ( HCD ) ( mean ± , n = 5 4 respectively , * * p = < 0 . 01 , Students t - ) .

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(F) Assessment of total cholesterol (COH) by mass spectroscopy in mice fed normal chow or a high cholesterol diet (HCD)(mean±SEM, n=5 and 4 animals respectively, **p=<0.01, Students t-test).


( G , H ) of change in mean granule in Abca12tm1d on chow diet Abca12tm1d on high cholesterol diet islets assessed by transmission electron microscopy ( n = 3000 granules / genotype , mean ± , n = 4 , 3 , 3 4 respectively , * p = < 0 . 05 , Students t - ) as an average as a relative frequency distribution .

(G, H) Quantification of change in mean granule area in Abca12tm1d on chow diet and Abca12tm1d on high cholesterol diet islets assessed by transmission electron microscopy (n=3000 granules/genotype, mean±SEM, n=4, 3, 3 and 4 animals respectively, *p=<0.05, Students t-test) as an average and as a relative frequency distribution.


( I ) Correction in abnormal granule shape following high cholesterol feeding ( bar = 1μm ) , with I ' showing magnification of boxed region indicated .

(I) Correction in abnormal granule shape following high cholesterol feeding (bar = 1μm), with I' showing magnification of the boxed region indicated.


( J ) Glucose tolerance results following intraperitoneal glucose challenge in Abca12tm1d cre control mice fed normal chow or HCD ( mean ± , n = 3 mice per genotype , * p = < 0 . 05 , for tm1d versus tm1d + HCD , Students t - ) .

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(J) Glucose tolerance test results following intraperitoneal glucose challenge in Abca12tm1d and cre control mice fed normal chow or HCD (mean±SEM, n=3 mice per genotype, *p=<0.05, for tm1d versus tm1d+HCD, Students t-test).


( K ) Serum insulin in Abca12tm1d + / - HCD mice after i . p glucose challenge ( mean ± , n = 3 mice / genotype , * * p = < 0 . 01 , Students t - ) .

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(K) Serum insulin in Abca12tm1d +/- HCD mice after i.p glucose challenge (mean±SEM, n=3 mice/genotype, **p=<0.01, Students t-test).


( A ) Detection of lipid rafts in 8 week old islets with recombinant cholera toxin subunit B ( CT - B ) conjugated to Alexa Fluor 488 ( green ) . Islets are co - stained with DAPI ( blue ) ( scale bar = 50μm . ) .

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(A) Detection of lipid rafts in 8 week old islets with recombinant cholera toxin subunit B (CT-B) conjugated to Alexa Fluor 488 (green). Islets are co-stained with DAPI (blue) (scale bar = 50μm.).


( B ) FACS profiling of dispersed islets from 12 week old mice stained with insulin CT - B .

(B) FACS profiling of dispersed islets from 12 week old mice stained with insulin and CT-B.


( C ) Confocal of rafts ( using CTB - Alexa Fluor 647 ) in MIN6 transfected with Fluorescein - 488 - labelled siRNAScr or Dy547 - labelled siRNAABCA12 ( scale bar = 10μm ) . ( D ) Quantitation by confocal microscopy of effect of ABCA12 - deficincy on CTB binding to MIN6 ( mean ± , * p = < 0 . 05 , Students t - , n = 4 biological replicates , 50 - 100 for each ) . ( E ) Quantitation by flow cytometry of effect of ABCA12 - deficiency on CTB binding to MIN6 ( mean ± , n = 3 biological replicates , * p = < 0 . 05 , Students t - ) .

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(C) Confocal images of rafts (using CTB-Alexa Fluor 647) in MIN6 cells transfected with Fluorescein-488-labelled siRNAScr or Dy547-labelled siRNAABCA12 (scale bar=10μm). (D) Quantitation by confocal microscopy of the effect of ABCA12-deficincy on CTB binding to MIN6 cells (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each). (E) Quantitation by flow cytometry of the effect of ABCA12-deficiency on CTB binding to MIN6 cells (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test).


( F ) effect 10 mM MβCD on GSIS in MIN6 depleted or not for ABCA12 ( incubation for 5 or 10 minutes as indicated , mean ± , n = 4 ( biological replicates ) , * p = < 0 . 05 , Students t - ) .

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(F) The effect 10 mM MβCD on GSIS in MIN6 cells depleted or not for ABCA12 (incubation for 5 or 10 minutes as indicated, mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test).


( G ) Western blot of abundance of CDC42 in ABCA12 deficient MIN6 .

(G) Western blot of the abundance of CDC42 in ABCA12 deficient MIN6 cells.


( H ) effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 ( BDK , 100 ng / ml , 4 min ; * p = < 0 . 05 , Students t - , significance versus ABCA12 - deficient BDK activated is indicated , n = 4 biological replicates , mean ± ) .

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(H) The effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 cells (BDK, 100 ng/ml, 4 min; *p=<0.05, Students t-test, significance versus ABCA12-deficient BDK activated cells is indicated, n=4 biological replicates, mean±SEM).


( I , J ) ( I ) Confocal of F - actin in ABCA12 - deficient MIN6 ( scale bar = 20μm ) ( J ) of abundance showing effect of ABCA12 - deficiency ( mean ± , * p = < 0 . 05 , Students t - , n = 4 biological replicates , 50 - 100 for each ) .

(I,J) (I) Confocal images of F-actin in ABCA12-deficient MIN6 cells (scale bar = 20μm) and (J) quantification of abundance showing the effect of ABCA12-deficiency (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each).


( K ) F - actin assessed by FACS sorting of labelled MIN6 transfected with siRNAABCA12 ( mean ± , n = 3 biological replicates , * p = < 0 . 05 , Students t - ) .

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(K) F-actin levels assessed by FACS sorting of labelled MIN6 cells transfected with siRNAABCA12 (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test).


( L , M ) ( L ) Representative image of F - actin using LifeAct of purified islets isolated from Abca12tm1d versus control mice ( scale bar = 10μm ) ( M ) quantitation of signals from these samples ( n = 3 biological replicates , mean ± , * * * p = < 0 . 001 , * * * * p = < 0 . 0001 Students t - ) .

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(L,M) (L) Representative image of F-actin using LifeAct staining of purified islets isolated from Abca12tm1d versus control mice (scale bar = 10μm) and (M) quantitation of fluorescent signals from these samples (n=3 biological replicates, mean±SEM, ***p=<0.001, ****p=<0.0001 Students t-test).


( N ) Colocalization between ABCA12 ( red ) CDC42 ( green ) in MIN6 ( scale bar = 10μm ) .

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(N) Colocalization between ABCA12 (red) and CDC42 (green) in MIN6 cells (scale bar = 10μm).


O ) Effects of overexpression of a constitutively active form of CDC42 on GSIS from MIN6 with ABCA12 - deficiency ( mean ± , n = 4 biological replicates , * p = < 0 . 05 , Students t - ) .

O) Effects of overexpression of a constitutively active form of CDC42 on GSIS from MIN6 cells with ABCA12-deficiency (mean±SEM, n=4 biological replicates, *p=<0.05, Students t-test).


( P ) effect of overexpression of consitutively active CDC42 on ABCA1 abundance in MIN6 with ABCA12 - deficiency . Treatments are defined in table below .

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(P) The effect of overexpression of consitutively active CDC42 on ABCA1 abundance in MIN6 cells with ABCA12-deficiency. Treatments are defined in the table below.


( Q ) Effects of Jasplakinolide on F - actin ( green ) in siRNA treated ( scale bar = 10μm ) .

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(Q) Effects of Jasplakinolide on F-actin levels (green) in siRNA treated cells (scale bar=10μm).


( R ) Effects of Abca12 knockdown Jasplakinolide treatment on MIN6 in low high glucose ( mean ± , n = 4 biological replicates , * p = < 0 . 05 , * * p = < 0 . 01 , significance relative to untreated , Students t - )

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(R) Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose (mean±SEM, n=4 biological replicates, *p=<0.05, **p=<0.01, significance relative to untreated cells, Students t-test)


( A ) Mass spectroscopy of of cholesterol cholesteryl esters in purified islets from Abca12tm1d mice versus control at 16 of ( mean ± , n = 10 , 4 5 mice per genotype respectively ; COH = cholesterol , CE = cholesteryl ester ) .

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(A) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively; COH=cholesterol, CE=cholesteryl ester).


( B ) Mass spectroscopy of lipids families in purified islets from Abca12tm1d mice versus control at 16 of ( mean ± , n = 10 , 4 5 mice per genotype respectively , * * p = < 0 . 01 Students t - ) ; CE = cholesteryl ester , Cer = ceramide , DHC ( Hex2Cer ) = dihexosylceramide , MHC ( HexCer ) = monohexosylceramide , SM = sphingomyelin , GM3 = GM3 ganglioside , dhCer = dihydroceramide , DG = diacylglyerol , TG = triacylglycerol ) .

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(B) Mass spectroscopy analysis of lipids families in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, **p=<0.01 Students t-test); CE=cholesteryl ester, Cer=ceramide, DHC (Hex2Cer)=dihexosylceramide, MHC (HexCer)=monohexosylceramide, SM=sphingomyelin, GM3=GM3 ganglioside, dhCer=dihydroceramide, DG=diacylglyerol, TG=triacylglycerol).


( C ) Mass spectroscopy of triglyceride species in purified islets from Abca12tm1d mice versus control at 16 of ( mean ± , n = 10 , 4 5 mice per genotype respectively , * p = < 0 . 05 , * * p = < 0 . 01 , Students t - )

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(C) Mass spectroscopy analysis of triglyceride species in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, *p=<0.05, **p=<0.01, Students t-test)


( D ) Immunohistochemical of IL - 1β expression in sections of pancreas from Abca12tm1d mice versus control at 24 of ( bar = 25μm , islets outlined ) .

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(D) Immunohistochemical staining of levels IL-1β expression in sections of pancreas from Abca12tm1d mice versus control animals at 24 weeks of age (bar = 25μm, islets outlined).


( E ) of islet specific IL1β from sections of Abca12tm1d , Cre wild type mice ( mean ± , n = 3 , 3 4 mice of each genotype respectively ; * p = < 0 . 05 , Students t - ) .

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(E) Quantification of islet specific IL1β staining from sections of Abca12tm1d, Cre and wild type mice (mean±SEM, n=3, 3 and 4 mice of each genotype respectively; *p=<0.05, Students t-test).


of Abca12tm1d pancreata from mice at 24 of compared to control mice assessing β - mass ( F ) ( I ) ( mean ± , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - ) .

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Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing β-cell mass (F) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


of Abca12tm1d pancreata from mice at 24 of compared to control mice assessing caspase 3 ( G ) ( I ) ( mean ± , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students 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

Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing cleaved caspase 3 (G) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


of Abca12tm1d pancreata from mice at 24 of compared to control mice assessing glucagon ( H ) ( I ) ( mean ± , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - ) .

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

Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing glucagon (H) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


of Abca12tm1d pancreata from mice at 24 of compared to control mice assessing F480 + macrophage populations ( I ) ( mean ± , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - ) .

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Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing F480+ macrophage populations (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).


( J ) Fasting blood glucose coincident with emergence of inflammatory ( mean ± , n = 3 , 4 5 mice per genotype respectively , * p = < 0 . 05 , Students t - ) .

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(J) Fasting blood glucose levels coincident with the emergence of inflammatory phenotypes (mean±SEM, n=3, 4 and 5 mice per genotype respectively, *p=<0.05, Students t-test).


B ) Proliferation of H3122 parental isogenic drug - resistant , treated with indicated concentrations of crizotinib for 72h . P < 0 . 0001 was calculated for IC50 shift as indicated in methods .

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B) Proliferation assay of H3122 parental and isogenic drug-resistant cells, treated with the indicated concentrations of crizotinib for 72h. P < 0.0001 was calculated for IC50 shift as indicated in the methods.


C ) Gene - set enrichment after - seq of H3122 sensitive vs CrizR1 crizotinib resistant . D ) Epithelial to mesenchymal transition enrichment plot from - seq of C ) , using HALLMARK gene collection .

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C) Gene-set enrichment analysis after RNA-seq of H3122 sensitive vs CrizR1 crizotinib resistant cells. D) Epithelial to mesenchymal transition enrichment plot from the RNA-seq of C), using the HALLMARK gene collection.


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E) H3122 parental and drug-resistant isogenic cell lines were RNA-extracted and gene expression was analysed by RT-qPCR. On the right hand side is a western blot analysis of the AXL and EML4-ALK protein levels from the same cell lines.AXL: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P=0.03, CeritR vs H3122 P=0.2; LOX: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P=0.008; SNAI2: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P =0.003, CeritR vs H3122 P =0.003; VIM: CrizR1 vs H3122 P <0.001, CrizR4 vs H3122 P <0.001, CrizR5 vs H3122 P <0.001, CeritR vs H3122 P <0.001.


F ) Gene expression by RT - qPCR of CrizR1 treated with indicated concentrations of bemcentinib for 48h . LOX : P = 0 . 03 ; SNAI2 : P = 0 . 02 ; VIM : P = 0 . 03 .

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F) Gene expression analysis by RT-qPCR of CrizR1 cells treated with the indicated concentrations of bemcentinib for 48h. LOX: P=0.03; SNAI2: P =0.02; VIM: P =0.03.


G ) Proliferation of CrizR1 CrizR4 treated with indicated concentrations of bemcentinib ± 1uM crizotinib for 72h . * * * = P < 0 . 001 for IC50 shift , as indicated in methods ( n = 4 ) .

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G) Proliferation assay of CrizR1 and CrizR4 cells treated with the indicated concentrations of bemcentinib ± 1uM crizotinib for 72h. *** = P < 0.001 for IC50 shift, as indicated in the methods (n=4).


H ) Annexin V + apoptotic in CrizR1 treated with 1uM crizotinib , 2 . 5uM bemcentinib , or combination . STE - 1 parental were used as crizotinib drug control . crizotinib : P = 0 . 2 ; bemcentinib : P = 0 . 4 ; combination : P = 0 . 3 .

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H) Annexin V+ apoptotic assay in CrizR1 cells treated with 1uM crizotinib, 2.5uM bemcentinib, or combination. STE-1 parental cells were used as crizotinib drug control. crizotinib: P =0.2; bemcentinib: P =0.4; combination: P=0.3.


A ) GSEA enrichment using KEGG gene set identifiers . Shown are significantly dysregulated pathways ( P < 0 . 05 ) . B ) cycle enrichment plot from A ) .

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

A) GSEA enrichment analysis using the KEGG gene set identifiers. Shown are the significantly dysregulated pathways (P < 0.05). B) Cell cycle enrichment plot from A).


C ) Western blot of H3122 parental isogenic drug - resistant lines for indicated .

C) Western blot analysis of H3122 parental and isogenic drug-resistant cell lines for the indicated proteins.


D ) Western blot of Ste - 1 parental isogenic Alectinib resistant lines for indicated .

D) Western blot analysis of Ste-1 parental and isogenic Alectinib resistant cell lines for the indicated proteins.


E ) Proliferation of CrizR1 treated with indicated concentrations of alvocidib ± 1uM crizotinib for 72h . P = 0 . 2 ( n = 4 ) .

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E) Proliferation assay of CrizR1 cells treated with the indicated concentrations of alvocidib ± 1uM crizotinib for 72h. P=0.2 (n=4).


F ) As above , in a proliferation , H3122 CrizR1 were treated in parallel with DMSO or 1uM crizotinib as drug control . H3122 vs DMSO P < 0 . 0001 , CrizR1 vs DMSO P = 0 . 1 ( n = 4 ) .

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F) As above, in a proliferation assay, H3122 and CrizR1 cells were treated in parallel with DMSO or 1uM crizotinib as drug control. H3122 vs DMSO P <0.0001, CrizR1 vs DMSO P=0.1 (n=4).


Proliferation of CrizR1 , CrizR4 , CrizR5 , CeritR isogenic drug - resistant lines , treated with indicated concentrations of alvocidib ( G ) for 72h . P > 0 . 05 was calculated for IC50 shift , as indicated in methods .

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Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR isogenic drug-resistant cell lines, treated with the indicated concentrations of alvocidib(G) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.


H ) Proliferation of AlecR isogenic drug - resistant lines , treated with indicated concentrations of dinaciclib ( H ) for 72h . P > 0 . 05 was calculated for IC50 shift , as indicated in methods .

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H) Proliferation assay of AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of dinaciclib (H) for 72h. P >0.05 was calculated for IC50 shift, as indicated in the methods.


A ) Proliferation of CrizR1 , CrizR4 , CrizR5 , CeritR , AlecR isogenic drug - resistant lines , treated with indicated concentrations of THZ1 for 72h . P < 0 . 05 was calculated for IC50 shift , as indicated in methods .

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A) Proliferation assay of CrizR1, CrizR4, CrizR5, CeritR, and AlecR isogenic drug-resistant cell lines, treated with the indicated concentrations of THZ1 for 72h. P <0.05 was calculated for IC50 shift, as indicated in the methods.


B ) Crystal violet of EML4 / ALK parental drug - resistant . were treated with indicated drugs until vehicle control reached confluence , then fixed stained .

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B) Crystal violet staining of EML4/ALK parental and drug-resistant cells. Cells were treated with the indicated drugs until the vehicle control reached confluence, then fixed and stained.


C ) CrizR1 were treated with indicated drugs for 72h , then were stained with Annexin V / PI . Flow cytometry was then used to quantify Annexin V + . Alvocidib : early apoptosis P = 0 . 002 , late apoptosis P = 0 . 006 , alive P = 0 . 0008 ; Dinaciclib : early apoptosis P = 0 . 005 , late apoptosis P = 0 . 02 , alive P = 0 . 002 .

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C) CrizR1 cells were treated with the indicated drugs for 72h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells. Alvocidib: early apoptosis P=0.002, late apoptosis P=0.006, alive P=0.0008; Dinaciclib: early apoptosis P=0.005, late apoptosis P=0.02, alive P=0.002.


A ) Indicated lines were treated with DMSO , 200nM alvocidib or 25nM dinaciclib for 48h , then were stained with Annexin V / PI . Flow cytometry was then used to quantify Annexin V + ( n = 2 ) . Alvocidib : CrizR1 P = 0 . 005 , AlecR P = 0 . 001 , PC9 P = 0 . 05 , A549 P = 0 . 002 , H1299 P = 0 . 03 , H460 P = 0 . 07 ; P dinaciclib : CrizR1 P = 0 . 02 , AlecR P = 0 . 005 , PC9 P = 0 . 06 , A549 P = 0 . 08 , H1299 P = 0 . 09 , H460 P = 0 . 01 .

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A) Indicated cell lines were treated with DMSO, 200nM alvocidib or 25nM dinaciclib for 48h, then cells were stained with Annexin V/PI. Flow cytometry was then used to quantify Annexin V+ cells (n=2). Alvocidib: CrizR1 P=0.005, AlecR P =0.001, PC9 P =0.05, A549 P =0.002, H1299 P =0.03, H460 P =0.07; P dinaciclib: CrizR1 P=0.02, AlecR P =0.005, PC9 P =0.06, A549 P =0.08, H1299 P =0.09, H460 P =0.01.


( B ) CrizR1 were treated with DMSO or 200nM alvocidib for 24h extracts were hybridized to a 43 - antibody array analysed by immunoblotting . Graphs depict significant changes from two independent experiments .

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(B) CrizR1 cells were treated with DMSO or 200nM alvocidib for 24h and cell extracts were hybridized to a 43-antibody array and analysed by immunoblotting. Graphs depict the significant changes from two independent experiments.


C ) CrizR1 / CeritR / AlecR were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h extracts were analysed by western blotting .

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C) CrizR1/CeritR/AlecR cells were treated with DMSO or 200nM alvocidib or 50nM dinaciclib for 6h and cell extracts were analysed by western blotting.


D ) ( Top ) Western blot of CrizR1 treated with siScrambled or siRNA for CDK7 or CDK9 for 72h . ( Bottom ) CrizR1 were treated as above , or with DMSO / alvocidib / THZ1 were stained with Annexin V / PI analysed by flow cytometry for Annexin V + 72h post - transfection ( n = 2 ) . Annexin siCDK7 P = 0 . 2 , siCDK9 P = 0 . 015 , alvocidib P = 0 . 0001 , THZ1 P = 0 . 004 .

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D) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or siRNA for CDK7 or CDK9 for 72h. (Bottom) CrizR1 were treated as above, or with DMSO/alvocidib/THZ1 and cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection (n=2). Annexin siCDK7 P=0.2, siCDK9 P=0.015, alvocidib P=0.0001, THZ1 P =0.004.


E ) H3122 parental CrizR1 were treated with DMSO , 200nM alvocidib or 250ng / ml actinomycin D for 6 hours . was extracted mRNA of indicated genes were quantified by RT - qPCR . CDK1 : DMSO vs H3122 DMSO P = 0 . 002 , Actinomycin vs DMSO P = 0 . 0003 , Alvocidib vs DMSO P = 0 . 0005 ; CDK2 : DMSO vs H3122 DMSO P = 0 . 0001 , Actinomycin vs DMSO P < 0 . 0001 , Alvocidib vs DMSO P = 0 . 0005 ; CDK6 : DMSO vs H3122 DMSO P = 0 . 0005 , Actinomycin vs DMSO P = 0 . 0004 , Alvocidib vs DMSO P = 0 . 0004 ; CDK9 : DMSO vs H3122 DMSO P = 0 . 0003 , Actinomycin vs DMSO P = 0 . 0002 , Alvocidib vs DMSO P = 0 . 0003 ; CCNB1 : DMSO vs H3122 DMSO P = 0 . 02 , Actinomycin vs DMSO P = 0 . 0002 , Alvocidib vs DMSO P = 0 . 0003 ; CCNE1 : DMSO vs H3122 DMSO P = 0 . 0004 , Actinomycin vs DMSO P = 0 . 0004 , Alvocidib vs

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E) H3122 parental and CrizR1 cells were treated with DMSO, 200nM alvocidib or 250ng/ml actinomycin D for 6 hours. RNA was extracted and the mRNA levels of the indicated genes were quantified by RT-qPCR. CDK1: DMSO vs H3122 DMSO P=0.002, Actinomycin vs DMSO P=0.0003, Alvocidib vs DMSO P=0.0005; CDK2: DMSO vs H3122 DMSO P =0.0001, Actinomycin vs DMSO P<0.0001, Alvocidib vs DMSO P=0.0005; CDK6: DMSO vs H3122 DMSO P =0.0005, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO P =0.0004; CDK9: DMSO vs H3122 DMSO P=0.0003, Actinomycin vs DMSO P =0.0002, Alvocidib vs DMSO P =0.0003; CCNB1: DMSO vs H3122 DMSO P=0.02, Actinomycin vs DMSO P=0.0002, Alvocidib vs DMSO P=0.0003; CCNE1: DMSO vs H3122 DMSO P =0.0004, Actinomycin vs DMSO P =0.0004, Alvocidib vs DMSO


F ) RT - qPCR of MCL - 1 Survivin expression after treatment of CrizR1 with 100nM alvocidib , 25nM dinaciclib 50 nM THZ1 . was extracted after 24h of treatment . MCL - 1 alvocidib vs control P = 0 . 0001 , dinaciclib vs control P = 0 . 0001 , THZ1 vs control P = 0 . 0001 ; Survivin : alvocidib vs control P = 0 . 01 , dinaciclib vs control P = 0 . 01 , THZ1 vs control P = 0 . 0001 .

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F) RT-qPCR analysis of MCL-1 and Survivin expression after treatment of CrizR1 cells with 100nM alvocidib, 25nM dinaciclib and 50 nM THZ1. RNA was extracted after 24h of treatment. MCL-1 alvocidib vs control P=0.0001, dinaciclib vs control P=0.0001, THZ1 vs control P =0.0001; Survivin: alvocidib vs control P =0.01, dinaciclib vs control P=0.01, THZ1 vs control P =0.0001.


G ) ( Top ) Western blot of CrizR1 treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin ( siBIRC5 ) . ( Bottom ) were stained with Annexin V / PI analysed by flow cytometry for Annexin V + 72h post - transfection . Annexin : early apoptosis P = 0 . 0003 , late apoptosis P = 0 . 5 , alive P < 0 . 0001 .

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G) (Top) Western blot analysis of CrizR1 cells treated with siScrambled or with a pool of 4 different siRNAs targeting Survivin (siBIRC5). (Bottom) Cells were stained with Annexin V/PI and analysed by flow cytometry for Annexin V+ cells 72h post-transfection. Annexin: early apoptosis P =0.0003, late apoptosis P =0.5, alive P <0.0001.


H ) CCLE - seq dataset was used RPKM values were plotted for Cyclin D1 Survivn genes , indicating high expression in H3122 compared with other LUAD .

H) The CCLE RNA-seq dataset was used and RPKM values were plotted for the Cyclin D1 and Survivn genes, indicating high expression in H3122 cells compared with other LUAD cells.


A ) CrizR1 were treated with vehicle control , 200nM alvocidib or 100nM THZ1 for 6 hrs , then was precipitated with an anti - polymerase II antibody sequenced . Plotted is average number of peaks per condition .

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A) CrizR1 cells were treated with vehicle control, 200nM alvocidib or 100nM THZ1 for 6 hrs, then chromatin was precipitated with an anti-RNA polymerase II antibody and sequenced. Plotted is the average number of peaks per condition.


GSEA using HALLMARK gene collection for differentially enriched peaks around TSS ( ± 1kb ) with ( B ) alvocidib Shown on right is plot of " MYC targets " - enriched signature .

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GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (B) alvocidib Shown on the right is the plot of the "MYC targets"-enriched signature.


GSEA using HALLMARK gene collection for differentially enriched peaks around TSS ( ± 1kb ) with ( C ) THZ1 treatment . Shown on right is plot of " MYC targets " - enriched signature .

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GSEA analysis using the HALLMARK gene collection for the differentially enriched peaks around the TSS (±1kb) with (C) THZ1 treatment. Shown on the right is the plot of the "MYC targets"-enriched signature.


D ) Gene tracks of polymerase II occupancy at MCL1 , BIRC5 ( Survivin ) , CCND1 MYC genes .

D) Gene tracks of RNA polymerase II occupancy at the MCL1, BIRC5 (Survivin), CCND1 and MYC genes.


E ) qPCR of MYC CCND1 upon treatment of CrizR1 with Alvocidib or THZ1 for 6h . graph represents mean fold change ± SD . MYC : alvocidib vs DMSO P = 0 . 03 , THZ1 vs DMSO P < 0 . 0001 ; CCND1 : alvocidib vs DMSO P = 0 . 001 , THZ1 vs DMSO P = 0 . 003 .

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E) qPCR of MYC and CCND1 upon treatment of CrizR1 cells with Alvocidib or THZ1 for 6h. The graph represents the mean fold change ± SD. MYC: alvocidib vs DMSO P =0.03, THZ1 vs DMSO P <0.0001; CCND1: alvocidib vs DMSO P =0.001, THZ1 vs DMSO P =0.003.


F ) BIM is upregulated upon MYC silencing in CrizR1 .

F) BIM is upregulated upon MYC silencing in CrizR1 cells.


G ) Annexin V showing of apoptotic CrizR1 upon MYC downregulation using siRNA . Early apoptosis P = 0 . 002 , late apoptosis P = 0 . 04 , alive P = 0 . 0008 .

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G) Annexin V assay showing the percentage of apoptotic CrizR1 cells upon MYC downregulation using siRNA. Early apoptosis P =0.002, late apoptosis P=0.04, alive P =0.0008.


H ) Annexin V showing of apoptotic CrizR1 after treatment with trametinib , rapamycin or both . Trametinib : early apoptosis P = 0 . 002 , late apoptosis P = 0 . 05 , alive P = 0 . 009 ; rapamycin : early apoptosis P = 0 . 04 , late apoptosis P = 0 . 8 , alive P = 0 . 003 ; tram + rapa : early apoptosis P = 0 . 0001 , late apoptosis P = 0 . 04 , alive P = 0 . 01 .

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H) Annexin V assay showing the percentage of apoptotic CrizR1 cells after treatment with trametinib, rapamycin or both. Trametinib: early apoptosis P =0.002, late apoptosis P =0.05, alive P =0.009; rapamycin: early apoptosis P =0.04, late apoptosis P =0.8, alive P =0.003; tram+rapa: early apoptosis P =0.0001, late apoptosis P =0.04, alive P =0.01.


Tumour growth of in vivo xenografts of H3122 CrizR1 lines in response to crizotinib correspondent tumour weights ( P . O . control n = 5 ; I . P . control n = 4 ; crizotinib n = 8 Tumour weights , crizotinib P = 0 .

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Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to crizotinib and correspondent tumour weights (P.O. control n=5; I.P. control n=4; crizotinib n=8 Tumour weights, crizotinib P= 0.44


Tumour growth of in vivo xenografts of H3122 CrizR1 lines in response to alvocidib correspondent tumour weights ( P . O . control n = 5 ; I . P . control n = 4 alvocidib n = 6 ) . Tumour weights , , alvocidib P = 0 . 03 .

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Tumour growth of in vivo xenografts of H3122 CrizR1 cell lines in response to alvocidib and correspondent tumour weights (P.O. control n=5; I.P. control n=4 and alvocidib n=6). Tumour weights, , alvocidib P=0.03.


C ) Tumour growth of in vivo xenografts of Ste - 1 AlectR lines in response to alvocidib correspondent weights ( I . P . control n = 6 ; alvocidib n = 4 ) . Tumour weights alvocidib P = 0 . 004 .

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C) Tumour growth of in vivo xenografts of Ste-1 AlectR cell lines in response to alvocidib and correspondent tumor weights (I.P. control n=6; alvocidib n=4). Tumour weights alvocidib P=0.004.


D ) Number of caspase - 3 positive nuclei in alvocidib treated tumours compared to controls .

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D) Number of cleaved caspase-3 positive nuclei in alvocidib treated tumours compared to controls.


E ) Ki67 p21 of CrizR1 xenograft tumours . Scale bar = 200μm . Quantitative of is presented on right . Ki67 P = 0 . 0004 ; p21 P = 0 . 02 .

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E) Ki67 and p21 staining of CrizR1 xenograft tumours. Scale bar = 200μm. Quantitative analysis of IHC staining is presented on the right. Ki67 P=0.0004; p21 P=0.02.


F ) qPCR of Survivin ( P = 0 . 0009 ) , MCL - 1 ( P = 0 . 04 ) , MYC ( P = 0 . 007 ) BIM ( P = 0 . 03 ) in Ste - 1 AlectR xenografts treated with alvocidib ( Control n = 6 ; alvocidib n = 4 ) .

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F) qPCR of Survivin (P=0.0009), MCL-1 (P=0.04), MYC (P=0.007) and BIM (P=0.03) in Ste-1 AlectR cells xenografts treated with alvocidib (Control n=6; alvocidib n=4).


G ) Proliferation of H3122 parental isogenic drug - resistant lines , treated with DMSO , 100nM lorlatinib , 200nM alvocidib or 50nM dinaciclib for 72h ( n = 4 ) .

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G) Proliferation assay of H3122 parental and isogenic drug-resistant cell lines, treated with DMSO, 100nM lorlatinib, 200nM alvocidib or 50nM dinaciclib for 72h (n=4).


H ) Growth curve of CrizR1 xenograft tumors in response to lorlatinib ( Control n = 7 ; lorlatinib n = 7 ) .

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H) Growth curve of CrizR1 xenograft tumors in response to lorlatinib (Control n=7; lorlatinib n=7).


Immunoblot of indicated lines , starved for 6 hours ( HBSS ) as indicated , treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for indicated times .

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Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times.


In C endogenous CALCOCO1 is analysed bars represent mean ± sd of band intensities relative to actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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In C endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot of indicated lines , starved for 6 hours ( HBSS ) as indicated , treated with 25 μM MG132 or 200 ng / ml of bafilomycin A1 ( Baf A1 ) for indicated times . In E , endogenous CALCOCO1 is analysed bars represent mean ± sd of band intensities relative to actin loading control , as quantified using ImageJ of three independent experiments . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines, starved for 6 hours (HBSS) as indicated, and treated with 25 μM MG132 or 200 ng/ml of bafilomycin A1 (Baf A1) for the indicated times. In E, endogenous CALCOCO1 is analysed and the bars represent the mean±sd of band intensities relative to the actin loading control, as quantified using ImageJ of three independent experiments. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


F A representative micrograph using widefield deconvolution microscopy of HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 immunostained for endogenous GM130 . Scale bars are 5 μm 2 μm ( zoomed inset ) .

F A representative micrograph using widefield and deconvolution microscopy of HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 and immunostained for endogenous GM130. Scale bars are 5 μm and 2 μm (zoomed inset).


G Same as in E were left untreated or treated with Baf A1 for 6 hours , then immunostained for endogenous p62 LC3B . Scale bars are 5 μm for confocal microscopy 2 μm for airyscans .

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G Same cells as in E were left untreated or treated with Baf A1 for 6 hours, and then immunostained for endogenous p62 and LC3B. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


H CALCOCO1 , p62 LC3B in in indicated conditions , counted using an automated system . error bars represent mean ± of per of three independent experiments per condition 250 per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison . Significance is displayed as * * * p < 0 . 001 .

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H CALCOCO1, p62 and LC3B puncta in in the indicated conditions, counted using an automated system. The error bars represent mean±SEM of puncta per cell of three independent experiments per condition and 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.


I of co - localization of CALCOCO1 with p62 / or LC3B in treated as indicated . error bars represent mean ± of three independent experiments per condition over 250 per experiment . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison . Significance is displayed as * * * p < 0 . 001 .

I Percentage of co-localization of CALCOCO1 puncta with p62 and/or LC3B in cells treated as indicated. The error bars represent mean±SEM of three independent experiments per condition and over 250 cells per experiment. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test. Significance is displayed as ***p<0.001.


J HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 were treated with Baf A1 for 6 hours immunostained for endogenous LAMP1 . Scale bars are 5 μm for confocal microscopy 2 μm for airyscans .

J HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were treated with Baf A1 for 6 hours and immunostained for endogenous LAMP1. Scale bars are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


A GST pulldown binding of in vitro transcribed / translated 35S - Myc - CALCOCO1 with recombinant GST - tagged ATG8 family . GST GST fusion were visualized by coomassie brilliant blue ( bottom panel ) , co - precipitated Myc - CALCOCO1 was detected by autoradiography ( upper panel ) . numbers below AR represent % binding in shown AR .

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A GST pulldown binding assay of in vitro transcribed/translated 35S-Myc-CALCOCO1 with recombinant GST-tagged ATG8 family proteins. GST and GST fusion proteins were visualized by coomassie brilliant blue staining (bottom panel), and the co-precipitated Myc-CALCOCO1 was detected by autoradiography (upper panel). The numbers below the AR represent % binding in the shown AR .


B GST pulldown of transiently transfected Myc - CALCOCO1 from HEK293 extracts with recombinant GST - tagged ATG8 family . GST GST fusions were visualized by Ponceau S ( bottom panel ) , co - precipitated Myc - CALCOCO1 detected by immunoblotting with anti - Myc antibody ( upper panel ) .

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B GST pulldown assay of transiently transfected Myc-CALCOCO1 from HEK293 cell extracts with recombinant GST-tagged ATG8 family proteins. GST and GST fusions were visualized by Ponceau S staining (bottom panel), and co-precipitated Myc-CALCOCO1 detected by immunoblotting with anti-Myc antibody (upper panel).


GST pulldown assays of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family . Precipitated GST GST fusions co - precipitated Myc - CALCOCO1 constructs were analyzed as in A .

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GST pulldown assays of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins. Precipitated GST and GST fusions and co-precipitated Myc-CALCOCO1 constructs were analyzed as in A.


I GST pulldown of endogenous GABARAP from HEK293 extracts with recombinant GST - tagged CALCOCO1 constructs . GST GST fusions were visualized as in A , co - precipitated GABARAP with anti - GABARAP antibody ( upper panel ) .

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I GST pulldown assay of endogenous GABARAP from HEK293 cell extracts with recombinant GST-tagged CALCOCO1 constructs. GST and GST fusions were visualized as in A, and co-precipitated GABARAP with anti-GABARAP antibody (upper panel).


GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family ( left ) . Cartoon of CALCOCO1 with domain organization indicated location of LIR UIR motifs . presence of two well separated binding surfaces on ATG8 binding to LIR ( LDS ) UIR ( UDS ) is indicated ( right ) .

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GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins (left). Cartoon of CALCOCO1 with domain organization indicated and the location of LIR and UIR motifs. The presence of two well separated binding surfaces on ATG8 proteins binding to LIR (LDS) and UIR (UDS) is indicated (right).


B GST pulldowns testing binding of indicated in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - tagged ATG8 family

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B GST pulldowns testing binding of indicated in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-tagged ATG8 family proteins


C GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 35S - Myc - p62 with recombinant GST - GABARAPL2 ( WT indicated mutants ) .

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C GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 and 35S-Myc-p62 with recombinant GST-GABARAPL2 (WT and indicated mutants).


D GST pulldown assays of in vitro transcribed / translated 35S - Myc - TAX1BP1 ( WT indicated mutants ) with recombinant GST - GABARAP ( WT indicated mutants ) .

D GST pulldown assays of in vitro transcribed/translated 35S-Myc-TAX1BP1 (WT and indicated mutants) with recombinant GST-GABARAP (WT and indicated mutants).


Immunoblot of HeLa CALCOCO1 KO lines stably transfected with WT EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 . were induced with tetracycline for 24 hours then starved or treated with MG132 or Baf A1 as indicated . blot panels are from more than one western blot experiment but for clarity , only a single actin / GAPDH loading control is shown . In F , bars represent mean ± sd of band intensities relative to actin or GAPDH loading controls of three independent experiments quantified using ImageJ . Statistical comparison was analyzed by one - way ANOVA followed by Tukey multiple comparison significance displayed as * * * p ˂ 0 . 001 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of HeLa CALCOCO1 KO cell lines stably transfected with WT EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691. Cells were induced with tetracycline for 24 hours and then starved or treated with MG132 or Baf A1 as indicated. The blot panels are from more than one western blot experiment but for clarity, only a single actin/GAPDH loading control is shown. In F, the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading controls of three independent experiments quantified using ImageJ. Statistical comparison was analyzed by one-way ANOVA followed by Tukey multiple comparison test and significance displayed as ***p ˂ 0.001, *p ˂ 0.01; ns is not significant.


G HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 or EGFP - CALCOCO1 mLIR + ∆ 623 - 691 grown in full medium treated with Baf A1 as indicated were immunostained for endogenous p62 LC3B . Scale bars , 5 μm .

G HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 or EGFP-CALCOCO1 mLIR+∆623-691 grown in full medium and treated with Baf A1 as indicated were immunostained for endogenous p62 and LC3B. Scale bars, 5 μm.


HeLa CALCOCO1 KO stably expressing EGFP - CALCOCO1 were starved for 4 hours then immunostained with anti - ATG13 anti - WIPI2 antibodies . Scale bars in A are 5 μm for confocal microscopy 2 μm for airyscans .

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HeLa CALCOCO1 KO cells stably expressing EGFP-CALCOCO1 were starved for 4 hours and then immunostained with anti-ATG13 and anti-WIPI2 antibodies. Scale bars in A are 5 μm for the confocal microscopy images and 2 μm for the airyscans.


In B , error bars represent mean ± sd of per from three independent experiments and100 - 200 per each experiment .

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In B, the error bars represent mean±sd of puncta per cell from three independent experiments and100-200 cells per each experiment.


Immunoblot of indicated lines treated as indicated . Numbers below blots represent relative of bands in shown blots normalized against loading control ( GAPDH In D bars represent mean ± sd of band intensities relative to GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In D the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot of indicated lines treated as indicated . Numbers below blots represent relative of bands in shown blots normalized against loading control ( GAPDH In , bars represent mean ± sd of band intensities relative to GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH In , the bars represent the mean±sd of band intensities relative to the GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot of indicated lines treated as indicated . Numbers below blots represent relative of bands in shown blots normalized against loading control actin ) . In , bars represent mean ± sd of band intensities relative to actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control actin). In , the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


, I bars represent mean ± sd of band intensities relative to actin loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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, I the bars represent the mean±sd of band intensities relative to the actin loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


Immunoblot of indicated lines treated as indicated . Numbers below blots represent relative of bands in shown blots normalized against loading control (

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH


Immunoblot of indicated lines treated as indicated . Numbers below blots represent relative of bands in shown blots normalized against loading control ( GAPDH asterisk in K indicates that endogenous CALCOCO1 is detected in WT KO extracts EGFP - CALCOCO1 in extracts from rescued . In bars represent mean ± sd of band intensities relative to actin or GAPDH loading control as quantified using ImageJ , n = 5 in I , n = 3 in others . Statistical comparison was analyzed by one - way ANOVA significance displayed as * * * p ˂ 0 . 001 , * * p ˂ 0 . 005 , * p ˂ 0 . 01 ; ns is not significant .

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Immunoblot analysis of indicated cell lines treated as indicated. Numbers below the blots represent relative intensity of the bands in the shown blots normalized against the loading control (GAPDH The asterisk in K indicates that endogenous CALCOCO1 is detected in WT and KO cell extracts and EGFP-CALCOCO1 in cells extracts from the rescued cells. In the bars represent the mean±sd of band intensities relative to the actin or GAPDH loading control as quantified using ImageJ, n=5 in I, n=3 in others. Statistical comparison was analyzed by one-way ANOVA and significance displayed as ***p ˂ 0.001, **p ˂ 0.005, *p ˂ 0.01; ns is not significant.


B Co - IP of Myc - VAPA or Myc - VAPB with EGFP - CALCOCO1 from transiently transfected HEK293 . asterisks ( * ) indicate Myc - VAP previously detected on same

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B Co- IP of Myc-VAPA or Myc-VAPB with EGFP-CALCOCO1 from transiently transfected HEK293 cells. The asterisks (*) indicate Myc-VAP previously detected on the same membrane


C , D GST pulldown assays of in vitro transcribed / translated 35S - Myc - CALCOCO1 constructs with indicated recombinant GST - VAPA or GST - VAPB constructs . scheme is a representation of CALCOCO1 domains motifs .

C, D GST pulldown assays of in vitro transcribed/translated 35S-Myc-CALCOCO1 constructs with indicated recombinant GST-VAPA or GST-VAPB constructs.The scheme is a representation of CALCOCO1 domains and motifs.


E HeLa transiently co - transfected with EGFP - CALCOCO1 Myc - VAPA or - VAPB were immunostained with anti - Myc antibody . Scale bars , 5 μm .

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E HeLa cells transiently co-transfected with EGFP-CALCOCO1 and Myc-VAPA or -VAPB were immunostained with anti-Myc antibody. Scale bars, 5 μm.