CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , or HIF1α cultivated under hypoxia . Whole lysates were used for immunoblot assays for phospho - AURKA AURKA , resulting data were compared to those of normoxic hypoxic control . were normalized against β - ACTIN in same blots . NOR , normoxia ; HYP , hypoxia .

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The CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, or HIF1α and cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for phospho-AURKA and AURKA, and the resulting data were compared to those of normoxic and hypoxic control cells. Protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


CEP41 - deficient HUVECs were transfected with expression vectors encoding nothing ( MOCK ) , CEP41 , HIF1α , or HIF1α with CEP41 exposed to hypoxia . VEGFA ( E ) VEGFR2 ( F ) mRNA were analyzed by qRT - PCR using cDNA from transfected . Data are shown as mean ± SD of three independent experiments . Statistical significance was determined using one - way ANOVA followed by Tukey ' s post hoc ( * * * P < 0 . 001 , ns : non - significant ) . NOR , normoxia ; HYP , hypoxia .

B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 CEP41-deficient HUVECs were transfected with expression vectors encoding nothing (MOCK), CEP41, HIF1α, or HIF1α with CEP41 and exposed to hypoxia. VEGFA (E) and VEGFR2 (F) mRNA levels were analyzed by qRT-PCR using cDNA from transfected cells. Data are shown as mean ± SD of three independent experiments. Statistical significance was determined using the one-way ANOVA followed by Tukey's post hoc test (***P < 0.001, ns: non-significant). NOR, normoxia; HYP, hypoxia.


Control or CEP41 - depleted HUVECs were transfected with expression vectors encoding nothing ( MOCK ) or HIF1α treated with MG132 or not , then were cultivated under hypoxia . Whole lysates were used for immunoblot assays for HIF1α , phospho - AURKA , AURKA , were normalized against β - ACTIN in same blots . NOR , normoxia ; HYP , hypoxia .

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

Control or CEP41-depleted HUVECs were transfected with expression vectors encoding nothing (MOCK) or HIF1α and treated with MG132 or not, and then the cells were cultivated under hypoxia. Whole cell lysates were used for immunoblot assays for HIF1α, phospho-AURKA, and AURKA, and the protein levels were normalized against β-ACTIN in the same blots. NOR, normoxia; HYP, hypoxia.


of tube node number in ( H ) tube in ( I ) from in vitro angiogenesis assays with HIF1α # 1 or # 2 siRNAs - transfected HUVECs overexpressing nothing ( MOCK ) or CEP41 under hypoxia . Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition .

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Quantification of tube node number in (H) and tube length in (I) from in vitro angiogenesis assays with HIF1α #1 or #2 siRNAs-transfected HUVECs overexpressing nothing (MOCK) or CEP41 under hypoxia. Data are shown as mean ± SD of five independent experiments with ≥ 5 tubulogenesis regions per condition.


VEGFA ( J ) VEGFR2 ( K ) mRNA were analyzed by qRT - PCR using cDNA from hypoxic HIF1α - depleted HUVECs transfected with control or CEP41 expression vectors . results were compared to those of control normoxic HIF1α - depleted . Data are shown as mean ± SD of three independent experiments .

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

VEGFA (J) and VEGFR2 (K) mRNA levels were analyzed by qRT-PCR using cDNA from hypoxic HIF1α-depleted HUVECs transfected with control or CEP41 expression vectors. The results were compared to those of control and normoxic HIF1α-depleted cells. Data are shown as mean ± SD of three independent experiments.


A Expression patterns of pSEU : : SEU - GFP pWOX5 : : GFP in of indicated genotypes at dermatogen , early globular , heart stages . arrows indicate QC precursor , dashed lines indicate . Scale bars : 10 µm .

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A Expression patterns of pSEU::SEU-GFP and pWOX5::GFP in the embryos of the indicated genotypes at dermatogen, early globular, and heart stages. White arrows indicate the QC precursor cell, and white dashed lines indicate embryos. Scale bars: 10 µm.


B of pWOX5 : : GFP GFP in wild - type ( WT ) seu - 3 mutant . at early globular stage of WT was set to 1 . Data Information : In ( B ) data represent mean ± SD of three independent replicates . n denotes total number of scored samples . values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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B Quantification of pWOX5::GFP GFP fluorescence in wild-type (WT) and seu-3 mutant embryos. Fluorescence intensity at the early globular stage of WT embryos was set to 1. Data Information: In (B) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


C , D Expression pattern of pWOX5 : : GFP in WT ( C ) seu - 3 ( D ) at 5 days after germination ( DAG ) . Scale bars : 20 µm . E of GFP in QC of pWOX5 : : GFP transgenic , as shown in ( C ) ( D ) . was normalized to WT . Data Information : ( E ) , data represent mean ± SD of three independent replicates . n denotes total number of scored samples . values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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

C, D Expression pattern of pWOX5::GFP in WT (C) and seu-3 (D) embryos at 5 days after germination (DAG). Scale bars: 20 µm. E Quantification of GFP fluorescence in the QC of pWOX5::GFP transgenic plants, as shown in (C) and (D). Fluorescence intensity was normalized to the WT. Data Information: (E), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


F RT - qPCR of relative expression of WOX5 in WT seu - 3 roots . Total was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data Information : ( F ) data represent mean ± SD of three independent replicates . n denotes total number of scored samples . values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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

F RT-qPCR analysis of the relative expression levels of WOX5 in WT and seu-3 roots. Total RNA was extracted from 5 mm root tip sections of seedlings at 5 DAG. Data Information: (F) data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


G - J Modified pseudo - Schiff propidium iodide ( mPS - PI ) of niche areas in indicated genotypes at 5 DAG . Blue arrows indicate QC , red arrows indicate columella ( CSCs ) . numbers denote total number of scored samples , with similar showing in ( G - J ) . Scale bars : 20 μm . K of CSC layer in indicated genotypes at 5 DAG . Data Information ( K ) , data represent mean ± SD of three independent replicates . n denotes total number of scored samples . values ( black dots ) are shown . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-J Modified pseudo-Schiff propidium iodide (mPS-PI) staining of stem cell niche areas in the indicated genotypes at 5 DAG. Blue arrows indicate the QC, and red arrows indicate the columella stem cells (CSCs). The numbers denote total number of scored samples, with similar phenotypes showing in (G-J). Scale bars: 20 μm. K Quantification of the CSC layer in the indicated genotypes at 5 DAG. Data Information (K), data represent mean ± SD of three independent replicates. n denotes the total number of scored samples. Individual values (black dots) are shown. **P < 0.01, *P < 0.05 (Student's t-test).


L , M Double of QC184 β - glucuronidase ( GUS ) marker ( blue ) starch granules ( brown ) in WT ( L ) seu - 3 ( M ) seedlings at 5 DAG . In arrows indicate 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 ) seu - 3 ( O ) seedlings at 5 DAG . P , Q Expression pattern of CS9227 in WT ( P ) seu - 3 ( Q ) seedlings at 5 DAG . In arrows indicate 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 ) plt1 - 4 plt2 - 2 ( C ) at 5 DAG . D of GFP in QC of pWOX5 : : GFP transgenic seedlings , as shown in ( A - C ) . GFP signal in each genotype was normalized relative to that in WT . Data information : In ( D ) n denotes total number of scored samples . 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 ( P < 0 . 01 ) .

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

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 of relative expression of WOX5 in WT , scr - 3 , plt1 - 4 plt2 - 2 roots . Total was extracted from 5 mm root tip sections of seedlings at 5 DAG . Data information : In ( E ) , * * P < 0 . 01 ( Student ' s t - ) . 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 root of indicated genotypes at 5 DAG . Data information : In ( F ) n denotes total number of scored samples . 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 ( 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 of indicated genotypes at 5 DAG . arrows indicate QC .

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


K of GFP in QC of pSCR : : GFP pSCR : : GFP - SCR transgenic seedlings , as shown GFP signal of each genotype was normalized relative to that of WT . Data information ( K ) , n denotes total number of scored samples . values ( black dots ) are shown . Data represent mean ± SD of three independent replicates . ( K ) , * * P < 0 . 01 ( Student ' s t - ) . 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 niche of indicated genotypes at 5 DAG . dashed lines indicate QC region . numbers denote total number of scored samples , with similar 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 interaction between SEU SCR . yeast transformants were plated on synthetic defined ( SD ) media lacking Leu Trp ( SD / - 2 ) or lacking Ade , His , Leu , Trp ( SD / - 4 ) to assess - interactions . AD , GAL4 activation domain ; BD , GAL4 - 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 . bound to amylose resin was eluted analyzed by immunoblotting using anti - FLAG antibody . asterisk indicates 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 SEU in N . benthamiana leaves via Co - IP assays . SCR - GFP SEU - myc were transiently coexpressed in N . benthamiana leaves . samples were immunoprecipitated using anti - myc antibody .

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

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

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

O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

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

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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 SDG25 . yeast transformants were plated on SD / - 2 SD / - 4 media to assess - 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 WOX5 promoter , as shown by ChIP - qPCR . ACT7 , control . Data information was isolated from 5 mm root tip sections of seedlings at 5 DAG , sonicated , immunoprecipitated using anti - GFP or anti - H3K4me3 antibodies . precipitated was used as a template for qPCR . In ( C ) data represent mean ± SD of three independent replicates . * * P < 0 . 01 , * P < 0 . 05 ( Student ' s t - ) .

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

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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 indicated genotypes at 5 DAG . Scale bars : 20 µm . H of GFP in indicated genotypes expressing pWOX5 : : GFP . GFP signal in WT was set to 1 . n denotes 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 - ) .

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 of relative expression of WOX5 in WT ashr3 - 1 roots . Total was extracted from 5 mm root tip sections of seedlings at 5 DAG . 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 - ) .

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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 of niche in indicated genotypes at 5 DAG . Blue arrows indicate QC , red arrows indiate CSCs . numbers denote total number of scored samples , with same showing in ( J - M ) . Scale bars : 10 μm . N of CSC layer in 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 - ) .

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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 of indicated genotypes at 5 DAG . dashed lines indicate 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 SDG4 . yeast transformants were plated on SD / - 2 SD / - 4 media to assess - 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 SDG4 in N . benthamiana leaves , as shown by Co - IP assays . SDG4 - myc , SCR - HA , SEU - GFP were coinfiltrated into N . benthamiana leaves . samples were immunoprecipitated with anti - GFP antibody immunoblotted with anti - myc anti - HA antibody . N . benthamiana leaves coinfiltrated with SDG4 - myc 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 SDG4 in Arabidopsis . extracted from SDG4 - myc SDG4 - myc SCR - GFP were immunoprecipitated using anti - GFP antibody , immunoblotted using anti - myc 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 . expression showing top differentially expressed gene markers for each cluster of reference single atlas dataset . ( column ) are clustered by expression of 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 genes Fas3 ( left panel ) zfh1 ( right panel ) used as markers to assign epithelial myoblast , respectively , in reference dataset .

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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 orthogonal views of disc stained with anti - Zfh1 ( red ) anti - Fas3 ( green ) .

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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 expression of marker genes identified for myoblast , epithelial tracheal across 26 clusters of reference atlas . Color represents average normalized expression level . Dot diameter represents fraction of expressing each gene in each cluster . Gene expression for each was normalized by total expression , then 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 orthogonal view of SPARC > GFP ( green ) stained with anti - Ct ( red ) 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 genes used as known markers to assign each epithelial cluster in reference atlas dataset . colored by expression of nub , wg , zfh2 , Sox15 , grn , bnl , eyg , Ance , Idg4 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 expression of top marker genes identified for epithelial across 17 clusters of reference 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 ) counterstained with DAPI ( cyan ) . Scale bars : 100 μm . Full genotypes : P { CaryP } attP2 ( top bottom panels ) , P { PZ } dpp [ 10638 ] ; + , 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 expression of top variable genes between IFM DFM myoblasts across 7 clusters of reference atlas dataset .

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

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

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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 expression of top variable genes between myoblasts across 7 clusters of reference atlas dataset .

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

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

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

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

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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).


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

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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.


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

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

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

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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 ] at pharate stage was partially rescued by overexpressing transgene Ama - cDNA . Representative of adults 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 1151 - GAL4 ; UAS - AmaOE ; + .

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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 genes Fas3 ( left panel ) zfh1 ( right panel ) in 1151 > Ama - RNAi dataset .

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


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

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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 expression of top markers for myoblast clusters identified in reference atlas across cluster of myoblasts in 1151 > Ama - RNAi 7 myoblast clusters in control dataset .

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

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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 of 1151 > mCherry - RNAi 1151 > Ama - RNAi wing discs stained with anti - Zfh1 ( green ) ( G ) anti - Nrt ( ) 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 ) .

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

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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR 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 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 III inhibitor , red curve data points ) or ethanol as control ( black data points ) . HEK293 were grown either with glucose or galactose as carbon source as indicated . Solid represents mean , points colored in lighter version of respective color are corresponding ratios measured in . Data information : As most of data were not normal distributed , instead of a t - , a Wilcoxon / Mann - Whitney - U - was performed samples were compared in pairs . * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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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).