L , M Double staining of the QC184 β - glucuronidase ( GUS ) marker ( light blue ) and starch granules ( dark brown ) in WT ( L ) and seu - 3 ( M ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm .

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L, M Double staining of the QC184 β-glucuronidase (GUS) marker (light blue) and starch granules (dark brown) in WT (L) and seu-3 (M) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.


N , O Expression pattern of J2341 in WT ( N ) and seu - 3 ( O ) seedlings at 5 DAG . P , Q Expression pattern of CS9227 in WT ( P ) and seu - 3 ( Q ) seedlings at 5 DAG . In white arrows indicate the CSCs . Scale bars : 20 μm .

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N, O Expression pattern of J2341 in WT (N) and seu-3 (O) seedlings at 5 DAG. P, Q Expression pattern of CS9227 in WT (P) and seu-3 (Q) seedlings at 5 DAG. In white arrows indicate the CSCs. Scale bars: 20 μm.


A - C Expression pattern of pWOX5 : : GFP in WT ( A ) , scr - 3 ( B ) and plt1 - 4 plt2 - 2 ( C ) at 5 DAG . D Quantification of GFP fluorescence in the QC of pWOX5 : : GFP transgenic seedlings , as shown in ( A - C ) . GFP signal intensity in each genotype was normalized relative to that in the WT . Data information : In ( D ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( D ) different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) .

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A-C Expression pattern of pWOX5::GFP in WT (A), scr-3 (B) and plt1-4 plt2-2 (C) at 5 DAG. D Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic seedlings, as shown in (A-C). GFP signal intensity in each genotype was normalized relative to that in the WT. Data information: In (D) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (D) different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).


E RT - qPCR analysis of the relative expression levels of WOX5 in WT , scr - 3 , and plt1 - 4 plt2 - 2 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data information : In ( E ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm .

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E RT-qPCR analysis of the relative expression levels of WOX5 in WT, scr-3, and plt1-4 plt2-2 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data information: In (E) , **P < 0.01 (Student's t-test). Scale bars: 20 µm.


F Primary root length of the indicated genotypes at 5 DAG . Data information : In ( F ) n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . In ( F ) , different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) .

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F Primary root length of the indicated genotypes at 5 DAG. Data information: In (F) n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. In (F), different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01).


G - J Representative confocal images of the indicated genotypes at 5 DAG . White arrows indicate the QC .

G-J Representative confocal images of the indicated genotypes at 5 DAG. White arrows indicate the QC.


K Quantification of GFP fluorescence in the QC of pSCR : : GFP and pSCR : : GFP - SCR transgenic seedlings , as shown GFP signal intensity of each genotype was normalized relative to that of the WT . Data information ( K ) , n denotes the total number of scored samples . Individual values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . ( K ) , * * P < 0 . 01 ( Student ' s t - test ) . Scale bars : 20 µm .

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K Quantification of GFP fluorescence in the QC of pSCR::GFP and pSCR::GFP-SCR transgenic seedlings, as shown GFP signal intensity of each genotype was normalized relative to that of the WT. Data information (K), n denotes the total number of scored samples. Individual values (black dots) are shown. Data represent mean ± SD of three independent replicates. (K), **P < 0.01 (Student's t-test). Scale bars: 20 µm.


L - Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG . White dashed lines indicate the QC region . The numbers denote total number of scored samples , with similar phenotypes showing in ( L - Q ) .

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L-Q Root stem cell niche phenotypes of the indicated genotypes at 5 DAG. White dashed lines indicate the QC region. The numbers denote total number of scored samples, with similar phenotypes showing in (L-Q).


A Yeast two - hybrid ( Y2H ) assays showing the interaction between SEU and SCR . The yeast transformants were plated on synthetic defined ( SD ) media lacking Leu and Trp ( SD / - 2 ) or lacking Ade , His , Leu , and Trp ( SD / - 4 ) to assess protein - protein interactions . AD , GAL4 activation domain ; BD , GAL4 DNA - binding domain .

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A Yeast two-hybrid (Y2H) assays showing the interaction between SEU and SCR. The yeast transformants were plated on synthetic defined (SD) media lacking Leu and Trp (SD/-2) or lacking Ade, His, Leu, and Trp (SD/-4) to assess protein-protein interactions. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain.


B In vitro pull - down assays showing that SEU directly interacts with SCR but not with SHR . SCR - FLAG was pulled down by SEU - MBP immobilized on amylose resin . Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti - FLAG antibody . The asterisk indicates the position of SEU - MBP .

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B In vitro pull-down assays showing that SEU directly interacts with SCR but not with SHR. SCR-FLAG was pulled down by SEU-MBP immobilized on amylose resin. Protein bound to the amylose resin was eluted and analyzed by immunoblotting using anti-FLAG antibody. The asterisk indicates the position of SEU-MBP.


C Verification of in vivo interactions between SCR and SEU in N . benthamiana leaves via Co - IP assays . SCR - GFP and SEU - myc were transiently coexpressed in N . benthamiana leaves . Protein samples were immunoprecipitated using anti - myc antibody .

O O O O O O O O O O O O O O O O O O O O O O O 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 Verification of in vivo interactions between SCR and SEU in N. benthamiana leaves via Co-IP assays. SCR-GFP and SEU-myc were transiently coexpressed in N. benthamiana leaves. Protein samples were immunoprecipitated using anti-myc antibody.


D Co - IP assays of SEU with SCR in Arabidopsis . Protein extracts from WT and SCR - GFP roots were isolated at 5 DAG and immunoprecipitated with anti - GFP antibody .

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D Co-IP assays of SEU with SCR in Arabidopsis. Protein extracts from WT and SCR-GFP roots were isolated at 5 DAG and immunoprecipitated with anti-GFP antibody.


F , G SCR and SEU physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP antibody . The precipitated DNA was used as a template for qPCR analysis . A , B , and I indicated the PCR amplicons ACT7 , control . Data information : In ( F ) , ( G ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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F, G SCR and SEU physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP antibody. The precipitated DNA was used as a template for qPCR analysis. A, B, and I indicated the PCR amplicons ACT7, control. Data information: In (F), (G) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter , as shown by ChIP - qPCR analysis . Chromatin was extracted from SEU - GFP and SEU - GFP scr - 3 seedlings at 5 DAG and precipitated with anti - GFP antibody . ChIP signals were quantified by qPCR as a percentage of total input DNA . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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H Mutation of the SCR gene impairs the recruitment of SEU to the WOX5 promoter, as shown by ChIP-qPCR analysis. Chromatin was extracted from SEU-GFP and SEU-GFP scr-3 seedlings at 5 DAG and precipitated with anti-GFP antibody. ChIP signals were quantified by qPCR as a percentage of total input DNA. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


I SEU stimulated SCR - mediated WOX5 promoter activation in transient expression assays in N . benthamiana leaves . The pWOX5 : : LUC reporter was cotransformed with the indicated effector constructs . The pWOX5 : : LUC activity was normalized relative to the internal control ( LUC / Renilla luciferase [ REN ] ) . The schematic diagram shows the construct used in the transient expression assays . Arrows indicate promoter regions , and boxes indicate coding sequences . Different lowercase letters indicate significant differences by one - way ANOVA followed by Tukey ' s multiple comparison test ( P < 0 . 01 ) . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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I SEU stimulated SCR-mediated WOX5 promoter activation in transient expression assays in N. benthamiana leaves. The pWOX5::LUC reporter was cotransformed with the indicated effector constructs. The pWOX5::LUC activity was normalized relative to the internal control (LUC/Renilla luciferase [REN]). The schematic diagram shows the construct used in the transient expression assays. Arrows indicate promoter regions, and boxes indicate coding sequences. Different lowercase letters indicate significant differences by one-way ANOVA followed by Tukey's multiple comparison test (P < 0.01). Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


J - M Representative confocal images of the indicated genotypes at 5 DAG . Scale bars : 20 μm . Insets show the QC region in which the solid white lines indicate the QC of WT , and the dashed white lines indicate the QC of seu - 3 . ( Insets scale bars : 5 μm ) . N Quantification of GFP signal intensity in the QC of WT and seu - 3 seedlings expressing pWOX5 : : SEU - GFP and pWOX5 : : SCR - GFP . GFP signal intensity in seu - 3 seedlings was normalized relative to the WT . Individual values ( black dots ) are shown . n denotes the total number of scored samples . Data information : In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 ( Student ' s t - test ) .

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J-M Representative confocal images of the indicated genotypes at 5 DAG. Scale bars: 20 μm. Insets show the QC region in which the solid white lines indicate the QC of WT, and the dashed white lines indicate the QC of seu-3. (Insets scale bars: 5 μm). N Quantification of GFP signal intensity in the QC of WT and seu-3 seedlings expressing pWOX5::SEU-GFP and pWOX5::SCR-GFP. GFP signal intensity in seu-3 seedlings was normalized relative to the WT. Individual values (black dots) are shown. n denotes the total number of scored samples. Data information: In (N), data represent mean ± SD of three independent replicates. **P < 0.01 (Student's t-test).


A Y2H assays showing that SEU interacts with SDG4 and SDG25 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions .

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A Y2H assays showing that SEU interacts with SDG4 and SDG25. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.


C SDG4 physically bind to the WOX5 promoter , as shown by ChIP - qPCR analysis . ACT7 , control . Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( C ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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C SDG4 physically bind to the WOX5 promoter, as shown by ChIP-qPCR analysis. ACT7, control. Data information Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (C) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


D , E ChIP - qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter . ChIP signal was normalized to the WT . Individual values ( black dots ) are shown . Data information : Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , and immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . The precipitated DNA was used as a template for qPCR analysis . In ( D ) , ( E ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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D, E ChIP-qPCR analysis indicating that mutations in SDG4 and SEU genes impair H3K4me3 deposition in the WOX5 promoter. ChIP signal was normalized to the WT. Individual values (black dots) are shown. Data information: Chromatin was isolated from 5 mm root tip sections of seedlings at 5 DAG, sonicated, and immunoprecipitated using anti-GFP or anti-H3K4me3 antibodies. The precipitated DNA was used as a template for qPCR analysis. In (D), (E) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


F , G Expression pattern of pWOX5 : : GFP in the indicated genotypes at 5 DAG . Scale bars : 20 µm . H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5 : : GFP . GFP signal intensity in the WT was set to 1 . n denotes the total number of scored samples . Data information : In ( H ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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F, G Expression pattern of pWOX5::GFP in the indicated genotypes at 5 DAG. Scale bars: 20 µm. H Quantification of GFP fluorescence in the indicated genotypes expressing pWOX5::GFP. GFP signal intensity in the WT was set to 1. n denotes the total number of scored samples. Data information: In (H) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


I RT - qPCR analysis of the relative expression levels of WOX5 in WT and ashr3 - 1 roots . Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG . Individual values ( black dots ) are shown . Data information : In ( I ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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I RT-qPCR analysis of the relative expression levels of WOX5 in WT and ashr3-1 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Individual values (black dots) are shown. Data information: In (I) data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


J - M mPS - PI staining of stem cell niche in the indicated genotypes at 5 DAG . Blue arrows indicate the QC , and red arrows indiate the CSCs . The numbers denote total number of scored samples , with same phenotypes showing in ( J - M ) . Scale bars : 10 μm . N Quantification of the CSC layer in the indicated genotypes at 5 DAG . Data information In ( N ) , data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - test ) .

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J-M mPS-PI staining of stem cell niche in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indiate the CSCs. The numbers denote total number of scored samples, with same phenotypes showing in (J-M). Scale bars: 10 μm. N Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data information In (N), data represent mean ± SD of three independent replicates. **P < 0.01, *P < 0.05 (Student's t-test).


A - G Representative confocal images of the indicated genotypes at 5 DAG . The white dashed lines indicate the QC region . Scale bars : 20 μm .

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A-G Representative confocal images of the indicated genotypes at 5 DAG. The white dashed lines indicate the QC region. Scale bars: 20 μm.


H Domain mapping of SEU involved in interactions with SCR and SDG4 . The yeast transformants were plated on SD / - 2 and SD / - 4 media to assess protein - protein interactions .

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H Domain mapping of SEU involved in interactions with SCR and SDG4. The yeast transformants were plated on SD/-2 and SD/-4 media to assess protein-protein interactions.


I SEU associates with SCR and SDG4 in N . benthamiana leaves , as shown by Co - IP assays . SDG4 - myc , SCR - HA , and SEU - GFP were coinfiltrated into N . benthamiana leaves . Protein samples were immunoprecipitated with anti - GFP antibody and immunoblotted with anti - myc and anti - HA antibody . N . benthamiana leaves coinfiltrated with SDG4 - myc and SCR - HA were used as a negative control .

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I SEU associates with SCR and SDG4 in N. benthamiana leaves, as shown by Co-IP assays. SDG4-myc, SCR-HA, and SEU-GFP were coinfiltrated into N. benthamiana leaves. Protein samples were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-myc and anti-HA antibody. N. benthamiana leaves coinfiltrated with SDG4-myc and SCR-HA were used as a negative control.


J Co - IP assays of SCR with SEU and SDG4 in Arabidopsis . Proteins extracted from SDG4 - myc and SDG4 - myc SCR - GFP plants were immunoprecipitated using anti - GFP antibody , and immunoblotted using anti - myc and anti - SEU antibodies .

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J Co-IP assays of SCR with SEU and SDG4 in Arabidopsis. Proteins extracted from SDG4-myc and SDG4-myc SCR-GFP plants were immunoprecipitated using anti-GFP antibody, and immunoblotted using anti-myc and anti-SEU antibodies.


D . RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset . Cells ( column ) are clustered by the expression of the main marker genes ( row ) .

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D. RNA expression heatmap showing the top differentially expressed gene markers for each cluster of the reference single cell atlas dataset. Cells (column) are clustered by the expression of the main marker genes (row).


E . Average expression level of the genes Fas3 ( left panel ) and zfh1 ( right panel ) used as markers to assign epithelial and myoblast cells , respectively , in the reference dataset .

E. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) used as markers to assign epithelial and myoblast cells, respectively, in the reference dataset.


F . Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti - Zfh1 ( red ) and anti - Fas3 ( green ) .

F. Confocal single plane image of third instar larval wing disc and orthogonal views of the disc stained with anti-Zfh1 (red) and anti-Fas3 (green).


G . Dot plot showing the expression levels of the marker genes identified for the myoblast , epithelial and tracheal cells across the 26 clusters of the reference cell atlas . Color intensity represents the average normalized expression level . Dot diameter represents the fraction of cells expressing each gene in each cluster . Gene expression for each cell was normalized by the total expression , and then the expression of each gene was scaled .

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G. Dot plot showing the expression levels of the marker genes identified for the myoblast, epithelial and tracheal cells across the 26 clusters of the reference cell atlas. Color intensity represents the average normalized expression level. Dot diameter represents the fraction of cells expressing each gene in each cluster. Gene expression for each cell was normalized by the total expression, and then the expression of each gene was scaled.


H . Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC > GFP ( green ) stained with anti - Ct ( red ) and 4 ´ , 6 - diamidino - 2 - phenylindole ( DAPI , blue ) . Full genotype y - , w - / w - ; UAS - GFP / + ; SPARC - GAL4 / + .

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H. Confocal single plane image of third instar larval wing disc and orthogonal view of SPARC>GFP (green) stained with anti-Ct (red) and 4´,6-diamidino-2-phenylindole (DAPI, blue). Full genotype y-, w-/w-; UAS-GFP/+; SPARC-GAL4/+.


D . Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset . Cells colored by the expression of nub , wg , zfh2 , Sox15 , grn , bnl , eyg , Ance , Idg4 and drm .

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D. Average expression level of the genes used as known markers to assign each epithelial cluster in the reference atlas dataset. Cells colored by the expression of nub, wg, zfh2, Sox15, grn, bnl, eyg, Ance, Idg4 and drm.


E . Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset .

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E. Dot plot showing the expression levels of the top marker genes identified for the epithelial cells across 17 clusters of the reference cell atlas dataset.


F . Confocal single plane image of third instar larval wing disc stained with anti - Nub , anti - Wg , anti - Svp , anti - Hairy ( red ) , reporting expression of dpp - lacZ , Dad - lacZ ( anti - β - gal , red ) and counterstained with DAPI ( cyan ) . Scale bars : 100 μm . Full genotypes : P { CaryP } attP2 ( top and bottom panels ) , P { PZ } dpp [ 10638 ] ; + , and y - w - ; + ; P { lacW } Dad [ j1E4 ] .

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F. Confocal single plane image of third instar larval wing disc stained with anti-Nub, anti-Wg, anti-Svp, anti-Hairy (red), reporting expression of dpp-lacZ, Dad-lacZ (anti-β-gal, red) and counterstained with DAPI (cyan). Scale bars: 100 μm. Full genotypes: P{CaryP}attP2 (top and bottom panels), P{PZ}dpp[10638];+, and y- w-;+; P{lacW}Dad[j1E4].


B . Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset .

B. Dot plots showing the expression levels of the top variable genes between IFM and DFM myoblasts across 7 clusters of the reference cell atlas dataset.


Confocal single plane images of third instar larval wing discs stained with : ( C ) anti - Ct ( red ) and anti - Zfh1 ( green ) , ( D ) kirre [ rp298 ] - lacZ ( anti - β - gal , green ) and anti - Zfh1 ( red ) , ( E ) Ten - a [ MI04411 ] : : GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) , ( F ) anti - Nrt ( red ) and anti - Zfh1 ( cyan ) , ( G ) Argk [ CB3789 ] : : GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) , ( H ) nkd [ MI00209 ] : : GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) , ( I ) Ama [ NP1297 ] > GFP ( green ) , anti - Ct ( red ) and anti - Zfh1 ( cyan ) and ( J ) Mef2 > CD8 - GFP ( green ) , and fluorescent in situ hybridization of Ama - RNA probe ( white ) .

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

Confocal single plane images of third instar larval wing discs stained with: (C) anti-Ct (red) and anti-Zfh1 (green), (D) kirre[rp298]-lacZ (anti-β-gal, green) and anti-Zfh1 (red), (E) Ten-a[MI04411]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (F) anti-Nrt (red) and anti-Zfh1 (cyan), (G) Argk[CB3789]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (H) nkd[MI00209]::GFP (green), anti-Ct (red) and anti-Zfh1 (cyan), (I) Ama[NP1297]>GFP (green), anti-Ct (red) and anti-Zfh1 (cyan) and (J) Mef2>CD8-GFP (green), and fluorescent in situ hybridization of Ama-RNA probe (white).


Confocal single plane images of third instar larval wing discs stained with : K . Ama [ NP1297 ] > gTRACE showing the lineage of Ama - GAL4 ( green ) and the active GAL4 ( red ) , and fluorescent in situ hybridization of Ama - RNA probe ( white ) . L . Confocal single plane images of adult DFM of Ama [ NP1297 ] > gTRACE showing the lineage of Ama - GAL4 ( green ) and the active GAL4 ( red ) stained with DAPI ( white ) dorsal right , anterior up . DFM are numbered in white following ( Lawrence , 1982 ) .

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

Confocal single plane images of third instar larval wing discs stained with: K. Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red), and fluorescent in situ hybridization of Ama-RNA probe (white). L. Confocal single plane images of adult DFM of Ama[NP1297]>gTRACE showing the lineage of Ama-GAL4 (green) and the active GAL4 (red) stained with DAPI (white) dorsal right, anterior up. DFM are numbered in white following (Lawrence, 1982).


A . Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset .

O 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. Dot plot showing the expression levels of the top variable genes between myoblasts across 7 clusters of the reference cell atlas dataset.


Confocal single plane images of third instar larval wing discs stained with anti - Ct ( red ) and ( B ) E ( spl ) m6 - BFM - gapGFP ( green ) , ( C ) E ( spl ) m7 - HLH [ VK00037 ] - GFP ( green )

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

Confocal single plane images of third instar larval wing discs stained with anti-Ct (red) and (B) E(spl)m6-BFM-gapGFP (green), (C) E(spl)m7-HLH[VK00037]-GFP (green)


Confocal single plane images of third instar larval wing discs stained with ( D ) E ( spl ) m3 - HLH [ GMR10E12 ] > GFP ( green ) .

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

Confocal single plane images of third instar larval wing discs stained with (D) E(spl)m3-HLH[GMR10E12]>GFP (green).


H - I . Confocal single plane images of third instar larval wing discs stained with anti - Ct ( cyan ) showing the lineage of GAL4 ( green ) and the active GAL4 ( red ) in ( H ) E ( spl ) m3 - HLH [ GMR10E12 ] > gTRACE and ( I ) E ( spl ) m6 - BFM - GAL4 > gTRACE .

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

H-I. Confocal single plane images of third instar larval wing discs stained with anti-Ct (cyan) showing the lineage of GAL4 (green) and the active GAL4 (red) in (H) E(spl)m3-HLH[GMR10E12]>gTRACE and (I) E(spl)m6-BFM-GAL4>gTRACE.


A - E Confocal single plane images of third instar larval wing discs stained with ( A ) anti - LamC ( green ) and anti - Zfh1 ( red ) , ( B ) edl [ k12907 ] - lacZ ( green ) and anti - Zfh1 ( red ) , ( C ) anti - H ( green ) and anti - Zfh1 ( red ) , ( D ) Cg25C [ k00405 ] - lacZ ( green ) and anti - Zfh1 ( red ) , or ( E ) vgk [ G00454 ] - GFP ( green ) and anti - Ct ( red ) .

A-E Confocal single plane images of third instar larval wing discs stained with (A) anti-LamC (green) and anti-Zfh1 (red), (B) edl[k12907]-lacZ (green) and anti-Zfh1 (red), (C) anti-H (green) and anti-Zfh1 (red), (D) Cg25C[k00405]-lacZ (green) and anti-Zfh1 (red), or (E) vgk[G00454]-GFP (green) and anti-Ct (red).


The muscle - specific ( A - B ) Mef2 - GAL4 drivers were crossed to UAS - RNAi lines to knockdown the expression of the gene markers for the myoblast clusters . ( A , Viability test quantified by scoring the percentage of viable animals at each developmental stage , including early pupa ( black ) , pharate ( grey ) and adult ( white ) , relative to the total number of animals . Data are expressed as stacked bars showing the median with interquartile range ( n ≥ 3 independent biological replicates ) , unless noted as viable ( NQ ) and lethal ( NQ ) , which stands for animals that were viable and lethal , respectively , but no quantitative analysis were performed . ( B , Flight ability scored by quantifying the percentage of flies landing on each section of the column ( top , middle and bottom ) as indicated on the diagram on the right . Data are expressed as stacked bars . The total number of flies used in each assay was pooled together and displayed next to each bar ( N ≥ 6 ) . n / a stands for non - applicable since animals are not viable .

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

The muscle-specific (A-B) Mef2-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. (A, Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. (B, Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.


The muscle - specific 151 - GAL4 drivers were crossed to UAS - RNAi lines to knockdown the expression of the gene markers for the myoblast clusters . C ) . Viability test quantified by scoring the percentage of viable animals at each developmental stage , including early pupa ( black ) , pharate ( grey ) and adult ( white ) , relative to the total number of animals . Data are expressed as stacked bars showing the median with interquartile range ( n ≥ 3 independent biological replicates ) , unless noted as viable ( NQ ) and lethal ( NQ ) , which stands for animals that were viable and lethal , respectively , but no quantitative analysis were performed . D ) . Flight ability scored by quantifying the percentage of flies landing on each section of the column ( top , middle and bottom ) as indicated on the diagram on the right . Data are expressed as stacked bars . The total number of flies used in each assay was pooled together and displayed next to each bar ( N ≥ 6 ) . n / a stands for non - applicable since animals are not viable .

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

The muscle-specific 151-GAL4 drivers were crossed to UAS-RNAi lines to knockdown the expression of the gene markers for the myoblast clusters. C). Viability test quantified by scoring the percentage of viable animals at each developmental stage, including early pupa (black), pharate (grey) and adult (white), relative to the total number of animals. Data are expressed as stacked bars showing the median with interquartile range (n≥ 3 independent biological replicates), unless noted as viable (NQ) and lethal (NQ), which stands for animals that were viable and lethal, respectively, but no quantitative analysis were performed. D). Flight ability scored by quantifying the percentage of flies landing on each section of the column (top, middle and bottom) as indicated on the diagram on the right. Data are expressed as stacked bars. The total number of flies used in each assay was pooled together and displayed next to each bar (N≥6). n/a stands for non-applicable since animals are not viable.


E . Confocal single plane images of third instar larval wing discs from Mef2 > mCherry - RNAi , ( left panel ) and Mef2 > chinmo [ HM04048 ] - RNAi ( right panel ) stained with anti - Zfh1 ( red ) and DAPI ( cyan ) . Scale bars : 50 μm . F . Quantification of phenotype displayed in ( E ) . Stacked bars showing the percentage of wing discs scored as either normal or as abnormal . Data are expressed as mean ± sem , N = 32 discs per genotype , n = 3 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 O O O O O O O

E. Confocal single plane images of third instar larval wing discs from Mef2>mCherry-RNAi, (left panel) and Mef2>chinmo[HM04048]-RNAi (right panel) stained with anti-Zfh1 (red) and DAPI (cyan). Scale bars: 50 μm. F. Quantification of phenotype displayed in (E). Stacked bars showing the percentage of wing discs scored as either normal or as abnormal. Data are expressed as mean ± sem, N = 32 discs per genotype, n = 3 independent experiments.


G . Quantification of viability test . Since the UAS - Ama - RNAi [ GD12733 ] transgene is inserted on Chromosome X the genotype of females is Mef2 > Ama - RNAi whereas males are Mef2 > + . Box plot showing the ratio of flies with genotype of interest ( females ) over control flies ( males ) . The whiskers show 5 - 95 percentile , and the middle line the median . * p = 0 . 014 ( Mann - Whitney test , N ≥ 6 independent biological replicates ) . Full genotypes are w , UAS - Dicer2 ; + ; mCherry - RNAi / Mef2 - GAL4 and w , UAS - Dicer2 , UAS - Ama - RNAi [ GD12733 ] ; + ; Mef2 - GAL4 .

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

G. Quantification of viability test. Since the UAS-Ama-RNAi[GD12733] transgene is inserted on Chromosome X the genotype of females is Mef2>Ama-RNAi whereas males are Mef2>+. Box plot showing the ratio of flies with genotype of interest (females) over control flies (males). The whiskers show 5-95 percentile, and the middle line the median. * p=0.014 (Mann-Whitney test, N≥6 independent biological replicates). Full genotypes are w, UAS-Dicer2;+; mCherry-RNAi/Mef2-GAL4 and w, UAS-Dicer2, UAS-Ama-RNAi[GD12733];+; Mef2-GAL4.


A . Lethality of 1151 > Ama - RNAi [ HMS00297 ] animals at pharate stage was partially rescued by overexpressing the transgene Ama - cDNA . Representative images of adults and pharate pupa are shown . Full genotypes : 1151 - GAL4 ; UAS - myrRFP ; + , 1151 - GAL4 ; UAS - myrRFP ; UAS - Ama [ HMS00297 ] - RNAi , 1151 - GAL4 ; UAS - AmaOE / CyO ; UAS - Ama [ HMS00297 ] - RNAi and 1151 - GAL4 ; UAS - AmaOE ; + .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Lethality of 1151>Ama-RNAi[HMS00297] animals at pharate stage was partially rescued by overexpressing the transgene Ama-cDNA. Representative images of adults and pharate pupa are shown. Full genotypes: 1151-GAL4;UAS-myrRFP;+ , 1151-GAL4;UAS-myrRFP;UAS-Ama[HMS00297]-RNAi , 1151-GAL4;UAS-AmaOE/CyO;UAS-Ama[HMS00297]-RNAi and 1151-GAL4;UAS-AmaOE;+.


C . Average expression level of the genes Fas3 ( left panel ) and zfh1 ( right panel ) in the 1151 > Ama - RNAi dataset .

C. Average expression level of the genes Fas3 (left panel) and zfh1 (right panel) in the 1151>Ama-RNAi dataset.


D . Confocal single plane images of 1151 > mCherry - RNAi and 1151 > Ama - RNAi wing discs stained with anti - Ct ( red ) and anti - Zfh1 ( green ) .

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

D. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Ct (red) and anti-Zfh1 (green).


E . Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151 > Ama - RNAi and 7 myoblast clusters in control dataset .

O O O O O O O O O O O O O O O O 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. Dot plots showing the expression levels of the top markers for myoblast clusters identified in the reference cell atlas across the cluster of myoblasts in 1151>Ama-RNAi and 7 myoblast clusters in control dataset.


F . The myoblast cells in 1151 > Ama - RNAi dataset projected into the reference single cell atlas by transferring cell type labels using Seurat . The bar graph represents the total number of cells in each cluster normalized to the total number of epithelial cells per genotype .

F. The myoblast cells in 1151>Ama-RNAi dataset projected into the reference single cell atlas by transferring cell type labels using Seurat. The bar graph represents the total number of cells in each cluster normalized to the total number of epithelial cells per genotype.


G - H . Confocal single plane images of 1151 > mCherry - RNAi and 1151 > Ama - RNAi wing discs stained with anti - Zfh1 ( green ) and ( G ) anti - Nrt ( white ) at wandering third instar larval stage ( 110 - 135 h AEL ) or ( H ) DAPI ( red ) at early third instar larval wing discs ( 72 - 110 h AEL ) .

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

G-H. Confocal single plane images of 1151>mCherry-RNAi and 1151>Ama-RNAi wing discs stained with anti-Zfh1 (green) and (G) anti-Nrt (white) at wandering third instar larval stage (110 - 135 h AEL) or (H) DAPI (red) at early third instar larval wing discs (72 - 110 h AEL).


Confocal single plane images of 1151 > Ama - RNAi animals and 1151 > mCherry - RNAi . A . Forming DFM at 40 h APF stained with anti - Zfh1 ( green ) , anti - Futsch ( 22c10 , red ) and anti - Kettin ( white ) . White arrows point to the wing hinge , wings pointing left , anterior up . B . Forming IFM ( DLM ) at 16 h APF , stained with anti - Futsch ( 22c10 , red ) , anti - Zfh1 ( green ) and DAPI ( cyan ) . Yellow - dashed box indicates magnified area ( bottom panel ) . Anterior up .

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

Confocal single plane images of 1151>Ama-RNAi animals and 1151>mCherry-RNAi. A. Forming DFM at 40 h APF stained with anti-Zfh1 (green), anti-Futsch (22c10, red) and anti-Kettin (white). White arrows point to the wing hinge, wings pointing left, anterior up. B. Forming IFM (DLM) at 16 h APF, stained with anti-Futsch (22c10, red), anti-Zfh1 (green) and DAPI (cyan). Yellow-dashed box indicates magnified area (bottom panel). Anterior up.


C . DFM at 96 h APF stained with Phalloidin ( red ) , anti - Kettin ( green ) and DAPI ( blue ) . Anterior up , dorsal right . DFM are numbered in white as in ( Lawrence , 1982 ) . D . IFM at 96 h APF , transverse section ( top ) stained with anti - PS - integrin ( green ) , Phalloidin ( red ) and DAPI ( blue ) , dorsal up ; sagittal section ( bottom ) stained with anti - Kettin ( green ) , Phalloidin ( red ) and DAPI ( blue ) , anterior up , dorsal right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. DFM at 96 h APF stained with Phalloidin (red), anti-Kettin (green) and DAPI (blue). Anterior up, dorsal right. DFM are numbered in white as in (Lawrence, 1982). D. IFM at 96 h APF, transverse section (top) stained with anti-PS-integrin (green), Phalloidin (red) and DAPI (blue), dorsal up; sagittal section (bottom) stained with anti-Kettin (green), Phalloidin (red) and DAPI (blue), anterior up, dorsal right.


C . Confirmation of sensor localization to different cellular compartments . Green , HyPer7 ; blue , DAPI

C. Confirmation of sensor localization to different cellular compartments. Green, HyPer7; blue, DAPI stain


D . - H . Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A ( a complex III inhibitor , red curve and data points ) or ethanol as control ( black line and data points ) . HEK293 cells were grown either with glucose or galactose as carbon source as indicated . Solid line represents mean , 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 . 01 , * * * , p ≤ 0 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.-H. Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A (a complex III inhibitor, red curve and data points) or ethanol as control (black line and data points). HEK293 cells were grown either with glucose or galactose as carbon source as indicated. Solid line represents mean, 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 ≤ 0.01, ***, p ≤ 0.001


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 .

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

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 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 .

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

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 ) .

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

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

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 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 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 .

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

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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 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 .

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

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

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

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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 ) 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 ) .

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 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

(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 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 .

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 B-MEASURED_VAR O 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 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 .

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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 ) 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 .

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

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

(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 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 ) .

(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 staining ( blue ) to detect gene expression of the knocked - in LacZ cassette from the 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 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 ) .

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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 the Abca12 locus by PCR , indicating different alleles ( tm1c , wild type and 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 ) and 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 animals per genotype , mean ± SEM , * p = < 0 . 05 , Students t - test ) , 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 animals / genotype , mean ± SEM ) .

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


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

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


( 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 ) .

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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 ± SEM , n = 4 - 9 mice per group ) .

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


( 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 ) .

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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 ± 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 ) .

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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 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 ) .

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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 cells after silencing of ABCA12 ( mean ± SEM , n = 4 replicates from assays in quadruplicate , * * * p < 0 . 0001 , Students t - test ) .

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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 ) Cellular insulin content in MIN6 cells after silencing of ABCA12 ( mean ± SEM , 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 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 ) .

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

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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 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 ) .

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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 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 ) .

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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 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 ) .

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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 cells transfected with siRNAscr or siRNAABCA12 and activated or not with TO901317 ( 4 μM ) ; ( mean ± SEM , 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 ) 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 ) .

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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 ) 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 ) .

(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 ) 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 ) .

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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 ) 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 ) .

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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 ) 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 ) .

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

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(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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 )

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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 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 ) .

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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 ± SEM , n = 3 mice of each genotype , normalised to total protein content ) .

(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 - 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 ) .

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(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 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 ) .

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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 ± SEM , n = 5 and 4 animals respectively , * * p = < 0 . 01 , Students t - test ) .

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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 ) 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 .

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(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 the boxed region indicated .

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


( 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 ) .

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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 ± SEM , n = 3 mice / genotype , * * p = < 0 . 01 , Students t - test ) .

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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 and CT - B .

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


( 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 ) .

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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 ) 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 ) .

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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 the abundance of CDC42 in ABCA12 deficient MIN6 cells .

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


( 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 ) .

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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 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 ) .

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

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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 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 ) .

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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 ) and CDC42 ( green ) in MIN6 cells ( 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 cells with ABCA12 - deficiency ( mean ± SEM , n = 4 biological replicates , * p = < 0 . 05 , Students t - test ) .

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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).