F . Addition of AKT inhibitor MK2206 ( AKTi ) abolishes Rab25 - dependent glycogen storage . ( a ) p < 0 . 05 versus shRNA control ( b ) p < 0 . 01 SF + AKTi versus SF .

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F. Addition of AKT inhibitor MK2206 (AKTi) abolishes Rab25-dependent glycogen storage. (a) p < 0.05 versus shRNA control and (b) p < 0.01 SF + AKTi versus SF.


G . Inhibiting AKT pathway glucose metabolism decreases viability . were pretreated with 10 µM of PI103 or AKTi , 100 µM LND or 3BrPy for 2 h before switching to SF media in presence of inhibitors for an addition16 h before titre blue viability . * p < 0 . 001 shRNA Rab25 versus shRNA control .

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G. Inhibiting AKT pathway and glucose metabolism decreases cell viability. Cells were pretreated with 10 µM of PI103 or AKTi, 100 µM LND or 3BrPy for 2 h before switching to SF media in the presence of inhibitors for an addition16 h before cell titre blue viability assay. *p < 0.001 shRNA Rab25 versus shRNA control.


A . Rab25 over - expression increases H3 - labelled 2DG uptake . ( a ) p < 0 . 001 Rab25 versus pcDNA . Results are mean ± s . d . of a triplicate in one of representative experiment .

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A. Rab25 over-expression increases H3-labelled 2DG uptake. (a) p < 0.001 Rab25 versus pcDNA. Results are mean ± s.d. of a triplicate in one of representative experiment.


B . Down - regulation of Rab25 or AKT expression , or inhibition AKT activity by MK2206 decreases glucose uptake in HEY ( a ) p < 0 . 001 versus NT siRNA control , ( b ) p < 0 . 05 versus siRab25 ( c ) p < 0 . 05 versus siAKT .

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B. Down-regulation of Rab25 or AKT expression, or inhibition AKT activity by MK2206 decreases glucose uptake in HEY cells (a) p < 0.001 versus NT siRNA control, (b) p < 0.05 versus siRab25 and (c) p < 0.05 versus siAKT.


C . Effect of down regulation of Rab25 AKT inhibitor MK2206 ( AKTi ) on glucose uptake . were either pretreated for 2 h with AKTi before assessing glucose uptake . ( a ) p < 0 . 05 versus shRNA control ( b ) p < 0 . 05 versus shRab25 .

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C. Effect of down regulation of Rab25 and AKT inhibitor MK2206 (AKTi) on glucose uptake. Cells were either pretreated for 2 h with AKTi before assessing glucose uptake. (a) p < 0.05 versus shRNA control and (b) p < 0.05 versus shRab25.


D . Rab25 co - localization with GLUT1 in A2780 . Immunofluorescence microscopy of GLUT1 ( red ) Rab25 ( green ) . Co - localization of GLUT1 Rab25 ( i . e . yellow ) is indicated by arrows . Single scale bar , 10 µM .

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D. Rab25 co-localization with GLUT1 in A2780 cells. Immunofluorescence microscopy staining of GLUT1 (red) and Rab25 (green). Co-localization of GLUT1 and Rab25 (i.e. yellow) is indicated by arrows. Single scale bar, 10 µM.


E - G . Resistance to 2DG induced in Rab25 is mediated through PI3K / AKT pathway . Inhibition of PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . E . AKT inhibition increases sensitivity to 2DG induced . * p < 0 . 001 versus HEYpcDNA .

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E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.E. AKT inhibition increases sensitivity to 2DG induced cell death. *p < 0.001 versus HEYpcDNA cells.


E - G . Resistance to 2DG induced in Rab25 is mediated through PI3K / AKT pathway . Inhibition of PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . F . Down - regulation of AKT by siRNA specific to AKT .

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E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.F. Down-regulation of AKT by siRNA specific to AKT.


E - G . Resistance to 2DG induced in Rab25 is mediated through PI3K / AKT pathway . Inhibition of PI3K / AKT pathway was achieved by addition of 10 µM of PI103 or MK2206 . G . Or down - regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced . * p < 0 . 001 versus RNAi control .

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E-G. Resistance to 2DG induced cell death in Rab25 cells is mediated through the PI3K/AKT pathway. Inhibition of the PI3K/AKT pathway was achieved by addition of 10 µM of PI103 or MK2206.G. Or down-regulation of Rab25 by shRNA specific to Rab25 enhances 2DG induced cell death. *p < 0.001 versus RNAi control cells.


Ovarian cancer HEY ( upper panel ) were co - transfected with AKT - IFPN Rab25 - IFPC or with AKT - IFPC Rab25 - IFPN . become due to an interaction between AKT Rab25 bringing two halves of into proximity creating a stable restoring . Hela stably expressing AKT - IFPN ( lower panel ) were transfected with Rab25 - IFPC or PDK - IFPC as control . A brightfield image corresponding to image is shown transfected indicated by an arrow .

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Ovarian cancer HEY cells (upper panel) were co-transfected with AKT-IFPN and Rab25-IFPC or with AKT-IFPC and Rab25-IFPN. Cells become fluorescent due to an interaction between AKT and Rab25 bringing the two halves of the fluorescent protein into proximity creating a stable complex and restoring fluorescence. Hela cells stably expressing AKT-IFPN (lower panel) were transfected with Rab25-IFPC or PDK-IFPC as control. A brightfield image corresponding to the fluorescent image is shown and transfected cells indicated by an arrow.


Detection of Rab25 AKT interaction by immunoprecipitation . Ovarian expressing HA - tagged Rab25 were IP with anti - HA antibody . resultant was separated by gel electrophoresis detected by WB using anti - AKT antibody .

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Detection of Rab25 and AKT interaction by immunoprecipitation. Ovarian cells expressing HA-tagged Rab25 were IP with anti-HA antibody. The resultant complex was separated by gel electrophoresis and detected by WB using anti-AKT antibody.


Generation of Rab25 deletion mutants . Expression of deletion mutants was detected by immunofluorescence ( upper panel ) WB ( middle panel ) using anti - GFP antibody which interacts with YFP . Diagrammatic representation of structure of mutants is shown in lower panel .

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Generation of Rab25 deletion mutants. Expression of the deletion mutants was detected by immunofluorescence staining (upper panel) and WB (middle panel) using anti-GFP antibody which interacts with YFP. Diagrammatic representation of the structure of the mutants is shown in the lower panel.


Rab25 deletion mutants do not alter glycogen ATP . Ovarian cancer lysates were collected 24 h post transfection . ( a ) p < 0 . 05 versus empty vector transfected .

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Rab25 deletion mutants do not alter cellular glycogen and ATP levels. Ovarian cancer cell lysates were collected 24 h post transfection. (a) p < 0.05 versus empty vector transfected cells.


A . Rab25 expression correlates with glycogen in patienttumours . Total total extracts , isolated from 31 ovarian cancer specimens , were subjected to Rab25 gene expression glycogen content using qPCR glycogen , respectively .

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A. Rab25 expression correlates with glycogen levels in patienttumours. Total RNA and total cellular extracts, isolated from 31 ovarian cancer patient specimens, were subjected to Rab25 gene expression and glycogen content analysis using qPCR and glycogen assay, respectively.


B - C . Rab25 expression signature identifies with a poor prognosis . Ovarian cancer were classified as either mirroring Rab25 - associated gene expression signature ( Rab25 signature ) or not ( non - Rab25 signature ) using linear discriminant in BRB tools , in two independent published ovarian datasets . Progression free survival curvesfor with advanced disease ( Stage II to IV ) are shown . Univariate analyses were plotted using Kaplan - Meier method Coxplots used for multivariate analyses ( co - variates included stage , grade , histology residual disease ) . B . Tothill et al , 2008 . C . Dressman et al , 2007 .

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B-C. The Rab25 expression signature identifies patients with a poor prognosis. Ovarian cancer patients were classified as either mirroring the Rab25-associated gene expression signature (Rab25 signature) or not (non-Rab25 signature) using linear discriminant analysis in BRB tools, in two independent published ovarian datasets. Progression free survival curvesfor patients with advanced disease (Stage II to IV) are shown. Univariate analyses were plotted using Kaplan-Meier method and Coxplots used for multivariate analyses (co-variates included stage, grade, histology and residual disease).B. Tothill et al, 2008.C. Dressman et al, 2007.


D . Prediction of breast cancer overall survival in two independent published breast datasets ( upper panel ) Pawitan et al , 2005 ( lower panel ) Chin et al , 2006 , on Rab25 - associated gene signature . Breast classification was determined by BRB tool class prediction function to identify with Rab25 - associated gene expression signature ( Rab25 - signature ) or without ( non - Rab25 signature ) . from datasets were included in class prediction .

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D. Prediction of breast cancer overall survival in two independent published breast datasets (upper panel) Pawitan et al, 2005 and (lower panel) Chin et al, 2006, based on Rab25-associated gene signature. Breast patient classification was determined by BRB tool class prediction function to identify patient with Rab25-associated gene expression signature (Rab25-signature) or without (non-Rab25 signature). All patients from the datasets were included in class prediction.


A Determination of a time window for 2a2iL treatment needed to induce a naïve - like pluripotency state in primed hPSCs . One day treatment with 2a followed by addition of 2a2iL for up to 14 days . Scale bars : 200 µm .

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A Determination of a time window for 2a2iL treatment needed to induce a naïve-like pluripotency state in primed hPSCs. One day treatment with 2a followed by the addition of 2a2iL for up to 14 days. Scale bars: 200 µm.


C Characteristics of 2a2iL - RH6 represented by dome - shaped colonies , ALP activity , a normal karyotype ( 46 , XY ) . 2a2iL - RH6 expressed common pluripotency markers OCT4 , NANOG , SOX2 , TRA - 1 - 81 , but not SSEA1 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) Propidium iodide ( PI ) . Scale bars : 200 , 100 , 50 µm in inset .

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C Characteristics of 2a2iL-RH6 cells represented by dome-shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL-RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA-1-81, but not SSEA1. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI). Scale bars: 200, 100, and 50 µm in the inset.


A Doubling time in pRH6 versus 2a2iL - RH6 . * * P < 0 . 01 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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A Doubling time in pRH6 versus 2a2iL-RH6. **P<0.01 (t-test). Data presented as mean ± SD (n=3).


B Cloning efficiency in presence ( + ) absence ( − ) of ROCK inhibitor ( ROCKi ) in pRH6 versus 2a2iL - RH6 . ROCKi ( − ) * P < 0 . 05 , ROCKi ( + ) * P < 0 . 01 ( t - ) , data presented as mean ± SD ( n = 3 ) .

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B Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL-RH6. ROCKi (−)*P<0.05, ROCKi (+) *P<0.01 (t-test), data presented as mean ± SD (n=3).


C Firefly luciferase expression of constructs that contain distal proximal enhancers of OCT4 after transfection of pRh6 2a2iL - RH6 . of firefly luciferase expression to Renilla luciferase expression ( expressed from co - transfected plasmid ) measured 48 h after transfection showed proximal enhancer activity in pRH6 distal enhancer activation in 2a2iL - RH6 . OCT4 PE - Luc * * * P < 0 . 001 , OCT4 DE - Luc * P < 0 . 05 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P<0.001, OCT4 DE-Luc *P<0.05 (t-test). Data presented as mean ± SD (n=3).


D qRT - PCR of pluripotency - related genes in pRH6 2a2iL - RH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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D qRT-PCR analysis of pluripotency-related genes in pRH6 and 2a2iL-RH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


E Immunofluorescence for several naïve specific markers in 2a2iL - RH6 . Scale bar : 100 µm .

E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm.


F Western blot of naïve - related pluripotency markers in pRH6 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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F Western blot analysis of naïve-related pluripotency markers in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Immunofluorescence against pRH5 2a2iL - RH5 ( 46 , XX ) for H3K27me3 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Reactivation of inactive X - chromosome in 2a2iL - RH5 was confirmed by lack of H3K27me3 foci . Scale bar : 50 µm .

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G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.


A Phase contrast of embryoid bodies ( EBs ) from 2a2iL - RH6 that differentiated into neurons . Scale bars : 200 100 µm in inset .

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A Phase contrast images of embryoid bodies (EBs) from 2a2iL-RH6 cells that differentiated into neurons. Scale bars: 200 and 100 µm in the inset.


B qRT - PCR of 20 day - EBs from pRH6 2a2iL - RH6 for markers of different embryonic germ layer derivatives . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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B qRT-PCR analysis of 20 day-EBs from pRH6 and 2a2iL-RH6 for markers of different embryonic germ layer derivatives. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


C Immunofluorescence of differentiated 2a2iL - RH6 that depict expression of markers for neuron - like ( TUJ - 1 ) , cardiac - muscle - like ( CTNT ) , hepatocyte - like ( SOX17 ) . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


D 2a2iL - RH6 form teratomas that consist of three embryonic germ layer - derived tissues . ( i ) Rosette structures ( ectoderm ) , ( ii ) cartilage ( mesoderm ) , ( iii ) primitive gut ( endoderm ; scale bar 50 µm ) .

D 2a2iL-RH6 cells form teratomas that consist of three embryonic germ layer-derived tissues. (i) Rosette structures (ectoderm), (ii) cartilage (mesoderm), and (iii) primitive gut (endoderm; scale bar 50 µm).


E From left to right : Phase contrast GFP representation in 2a2iL - RH6 - eGFP ; presence of injected 2a2iL - RH6 - eGFP in mouse blastocyst integration into inner mass ( ICM ) after 3 , 24 48 h incubation periods . Scale bars : 200 , 100 , 50 µm in inset .

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E From left to right: Phase contrast and GFP representation in 2a2iL-RH6-eGFP; presence of injected 2a2iL-RH6-eGFP in the mouse blastocyst and integration into the inner cell mass (ICM) after 3, 24 and 48 h incubation periods. Scale bars: 200, 100, and 50 µm in the inset.


F of tissue - sections from human - mouse chimeras in E10 . 5 showed positive for GFP ( top ) human antigen ( HNA ) ( bottom ) in different parts of , including head ( i ) , trunk ( ii ) , tail ( iii ) with higher magnification in right panels . Scale bars : 200 100 µm in inset .

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F Fluorescence images of tissue-sections from human-mouse chimeras in E10.5 showed cells positive for GFP (top) and human nuclear antigen (HNA) (bottom) in different parts of the embryos, including the head (i), trunk (ii), and tail (iii) with the higher magnification in the right panels. Scale bars: 200 and 100 µm in the inset.


G Genomic PCR for a human specific mitochondrial ( 391 bp ) in human - mouse chimeras .

G Genomic PCR analysis for a human specific mitochondrial sequence (391 bp) in human-mouse chimeras.


H Immunofluorescence against NANOG shows lack of expression in E10 . 5 human - mouse chimera tissue sections . Scale bars : 200 100 µm in inset .

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H Immunofluorescence staining against NANOG shows lack of expression in E10.5 human-mouse chimera tissue sections. Scale bars: 200 and 100 µm in the inset.


I Immunofluorescence against TUJ1 , BRACHYURY ( BRA ) , SOX17 in E10 . 5 human - mouse chimera tissue sections represent differentiation - derivatives of 2a2iL - RH6 . Scale bars : 200 100 µm in inset .

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I Immunofluorescence staining against TUJ1, BRACHYURY (BRA), and SOX17 in E10.5 human-mouse chimera tissue sections represent differentiation-derivatives of 2a2iL-RH6. Scale bars: 200 and 100 µm in the inset.


B of ICM - outgrowths by protocols 1 2 during first 4 days after seeding . nRH11 - 14 ( naïve Royan H11 , 12 , 13 , 14 ) : naïve hESC lines produced from human using 2a2iL condition . Scale bar : 100 µm .

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B Morphology of ICM-outgrowths by protocols 1 and 2 during the first 4 days after seeding. nRH11-14 (naïve Royan H11, 12, 13, and 14): naïve hESC lines produced from human embryos using 2a2iL condition. Scale bar: 100 µm.


C Comparison of efficiency of naïve - like hESCs derivation from human blastocysts by two protocols .

C Comparison of the efficiency of naïve-like hESCs derivation from human blastocysts by two protocols.


D Representative example of naïve - like hESCs ( nRH13 ) at passage 33 . Dome - shaped colonies were stained for ALP activity karyotyped . Scale bars : 200 100 µm in inset .

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D Representative example of naïve-like hESCs (nRH13) at passage 33. Dome-shaped colonies were stained for ALP activity and karyotyped. Scale bars: 200 and 100 µm in the inset.


E Expression of common pluripotency markers OCT4 , SOX2 , TRA - 1 - 81 , naïve - related pluripotency markers NANOG , KLF17 , TFCP2L1 in nRH13 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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E Expression of the common pluripotency markers OCT4, SOX2, and TRA-1-81, and naïve-related pluripotency markers NANOG, KLF17, and TFCP2L1 in nRH13. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


F qRT - PCR for pluripotency markers in nRH13 in comparison to a primed hESC , pRH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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F qRT-PCR analysis for pluripotency markers in nRH13 in comparison to a primed hESC line, pRH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Expression of lineage markers after embryoid ( EB ) formation spontaneous differentiation in nRH13 . * P < 0 . 05 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

G Expression of lineage markers after embryoid body (EB) formation and spontaneous differentiation in nRH13 cells. *P<0.05, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


A Pairwise Pearson correlation coefficient of transcriptome profiles ( - seq ) from 2a2iL - , pRH5 , pRH6 lines . Each of shows pairwise Pearson correlation coefficient of genes with largest expression variances in a pair of samples .

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A Pairwise Pearson correlation coefficient heatmap of transcriptome profiles (RNA-seq) from 2a2iL-, pRH5, and pRH6 cell lines. Each cell of the heatmap shows the pairwise Pearson correlation coefficient heatmap of genes with the largest expression variances in a pair of samples.


C volcano plot shows a differential expression between 2a2iL - versus primed hPSCs lines . X - axis shows log2 fold - change , y - axis shows negative log10 scaled adjusted P - value ( Benjamini - Hochberg ) . Red blue points represent up - regulated genes in 2a2iL - primed , respectively ( absolute log - 2 fold - change > 1 , adjusted P - value < 0 . 05 ) . Gray points indicate non - significant genes between these states .

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C The volcano plot shows a differential expression between 2a2iL- versus primed hPSCs lines. X-axis shows the log2 fold-change, and the y-axis shows the negative log10 scaled adjusted P-value (Benjamini-Hochberg). Red and blue points represent up-regulated genes in 2a2iL- and primed cells, respectively (absolute log-2 fold-change >1, adjusted P-value <0.05). Gray points indicate non-significant genes between these states.


A Position of TGF - β family members in volcano plot of differentially expressed genes in 2a2iL - primed hPSCs . X - axis shows log2 fold - change y - axis shows negative log10 adjusted P - value < 0 . 05 ( Adjusted P - value was obtained using Benjamini Hochberg correction to determine false discovery rate ) . Red blue points represent up - regulated genes in 2a2iL - primed hPSCs , respectively . Genes with no significant expression change are shown in gray .

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A Position of TGF-β family members in the volcano plot of differentially expressed genes in 2a2iL- and primed hPSCs. X-axis shows log2 fold-change and y-axis shows negative log10 adjusted P-value <0.05 (Adjusted P-value was obtained using the Benjamini and Hochberg correction to determine the false discovery rate). Red and blue points represent up-regulated genes in 2a2iL- and primed hPSCs, respectively. Genes with no significant expression change are shown in gray.


B qRT - PCR of several TGF - β - related genes in pRH6 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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B qRT-PCR analysis of several TGF-β-related genes in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


C Inhibition of TGF - β pathway by two small molecules , SB43 A83 . Left panel : of naïve - like three days after treatment . Right panel : of treated after passaging . Scale bar : 100 µm .

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C Inhibition of the TGF-β pathway by two small molecules, SB43 and A83. Left panel: morphology of naïve-like cells three days after treatment. Right panel: morphology of treated cells after passaging. Scale bar: 100 µm.


D shows expression of putative LRH - 1 target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in naïve versus primed hPSCs , with a range from blue for lower expression to red for higher expression . E shows expression of putative RARγ target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in naïve versus primed hPSCs , with a range from blue for lower expression to red for higher expression .

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D Heatmap shows expression of putative LRH-1 target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression. E Heatmap shows expression of putative RARγ target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression.


A qRT - PCR of ligands associated with TGF - β signaling during conversion of primed to naïve - like pluripotency by 2a2iL induction protocol at days 1 , 3 , 5 . 2iL was selected as a control group . * * * P < 0 . 0001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) .

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A qRT-PCR analysis of ligands associated with TGF-β signaling during conversion of primed to naïve-like pluripotency by 2a2iL induction protocol at days 1, 3, and 5. 2iL was selected as a control group. ***P<0.0001. One-way ANOVA. Data presented as mean ± SD (n=3).


B Inhibition of TGF - β pathway using SB43 during conversion of primed to naïve pluripotency . Scale bar : 100 µm .

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B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm.


C of TGFβ2iL - treated RH6 along with ALP activity . Scale bar : 200 µm .

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C Morphology of TGFβ2iL-treated RH6 cells along with ALP activity. Scale bar: 200 µm.


D qRT - PCR of naïve related genes in pRH6 , 2a2iL - , TGFβ2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) .

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D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3).


E Bar plot representing proliferation rates of 2a2iL - TGFβ2iL - RH6 by . * * P < 0 . 01 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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E Bar plot representing cell proliferation rates of 2a2iL- and TGFβ2iL-RH6 by cell counts. **P<0.01 (t-test). Data presented as mean ± SD (n=3).


F Bar plot of cloning efficiency of 2a2iL TGFβ2iL - RH6 on number of ALP positive colonies . * * * P < 0 . 001 ( t - ) . Data presented as mean ± SD ( n = 3 ) .

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F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL-RH6 based on the number of ALP positive colonies. ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Immunofluorescence show expression of naïve pluripotency markers in TGF - β2iL - RH6 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


( D ) Number of putative enhancer regions per . Bar depicts mean . Diamonds represent numbers in each are color - coded according to cancer

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(D) Number of putative enhancer regions per cell line. Bar depicts the mean. Diamonds represent individual numbers in each cell line and are color-coded according to cancer type


( E ) Heat maps of DNase I signal for enhancer regions ( rows ) . For intergenic enhancers ( left ) signal is aligned at upstream ( relative to + strand ) enhancer edge , shown from 4 kbp upstream to 5 kbp downstream , ordered by increasing of enhancer regions . For intragenic enhancers ( right ) signal was aligned at upstream downstream enhancer edges shown within ± 4 kbp . Exemplary data from LoVo colorectal cancer . ( F ) Heat maps as in ( E ) showing composite ChIP - seq peak coverage from 326 TFs

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(E) Heat maps of DNase I signal for enhancer regions (rows). For intergenic enhancers (left) the signal is aligned at the upstream (relative to + strand) enhancer edge, shown from 4 kbp upstream to 5 kbp downstream, and ordered by increasing length of enhancer regions. For intragenic enhancers (right) the signal was aligned at the upstream and downstream enhancer edges and shown within ± 4 kbp. Exemplary data from LoVo colorectal cancer cells. (F) Heat maps as in (E) showing composite ChIP-seq peak coverage from 326 TFs


( B ) Bidirectional promoters show higher mRNA synthesis . Boxplots showing TT - seq signal for mRNAs from unidirectional ( n = 39 , 376 ) bidirectional ( n = 83 , 363 ) promoters . Bidirectional unidirectional promoters were defined for each separately then plotted together . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Notches represent 95 % confidence intervals for median values . Outliers not shown . P - value by two - sided Mann - Whitney U - .

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(B) Bidirectional promoters show higher mRNA synthesis. Boxplots showing TT-seq signal for mRNAs from unidirectional (n=39,376) and bidirectional (n=83,363) promoters. Bidirectional and unidirectional promoters were defined for each cell line separately and then plotted together. Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( A ) UCSC genome browser view of normalized TT - seq coverage on plus minus strand at TAL1 locus ( hg38 ; chr1 : 47 , 194 , 268 - 47 , 242 , 880 ; ( Kent et al . , 2002 ) ) in leukemia lines Jurkat K562 . For better visualization , TT - seq coverage is cut at 200 ( purple lines ) . eRNAs are shown between plus minus strand . Red indicates short 12 bp insertion in Jurkat , introducing novel binding sites for transcription factor

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(A) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the TAL1 locus (hg38; chr1:47,194,268-47,242,880; (Kent et al., 2002)) in the leukemia cell lines Jurkat and K562. For better visualization, TT-seq coverage is cut at 200 (purple lines). eRNAs are shown between the plus and minus strand. Red line indicates the short 12 bp insertion in Jurkat cells, introducing novel binding sites for the transcription factor MYB


( B ) UCSC genome browser view of normalized TT - seq coverage on plus minus strand at 2 . 8 Mbp gene desert surrounding MYC ( hg38 ; chr8 : 127 , 042 , 406 - 129 , 779 , 109 ; ( Kent et al . , 2002 ) ) in fourteen cancer lines . eRNAs represent union from lines . Genome - wide association studies ( GWAS ) risk loci contain GWAS single nucleotide polymorphisms ( SNPs ) for prostate cancer ( PrCa ) , acute lymphocytic leukemia ( ) , breast cancer ( BrCa ) , colorectal cancer ( CRC ) , glioblastoma ( GBM ) including SNPs in linkage disequilibrium ( r2 ≥ 0 . 8 ) . TT - seq coverage is displayed between 8 - 250 to allow for better visualization of eRNA signal surrounding MYC . Dashed rectangle is shown at full scale on right . Note : TT - seq reads were mapped against hg38 reference genome we cannot rule out existence of genomic rearrangements ( e . g . translocations ) affecting shown region in used cancer clones .

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(B) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the 2.8 Mbp gene desert surrounding MYC (hg38; chr8:127,042,406-129,779,109; (Kent et al., 2002)) in fourteen cancer cell lines. eRNAs represent the union from all cell lines. Genome-wide association studies (GWAS) risk loci contain GWAS single nucleotide polymorphisms (SNPs) for prostate cancer (PrCa), acute lymphocytic leukemia (ALL), breast cancer (BrCa), colorectal cancer (CRC), and glioblastoma (GBM) including SNPs in linkage disequilibrium (r2 ≥ 0.8). TT-seq coverage is displayed between 8-250 to allow for better visualization of the eRNA signal surrounding MYC. Dashed rectangle is shown at full scale on the right. Note: TT-seq reads were mapped against the hg38 reference genome and we cannot rule out the existence of genomic rearrangements (e.g. translocations) affecting the shown region in the used cancer cell line clones.


( A ) Med1 - defined enhancers ranked by Med1 ChIP - seq signal ( Yan et al . , 2013 ) in LoVo colorectal cancer . Super - enhancers ( black rectangles ) were determined as enhancer regions lying above inflection point of

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(A) Med1-defined enhancers ranked by Med1 ChIP-seq signal (Yan et al., 2013) in LoVo colorectal cancer cells. Super-enhancers (black rectangles) were determined as enhancer regions lying above the inflection point of the curve


( B ) Increased eRNA synthesis from super - enhancers . Boxplots comparing eRNA synthesis ( TT - seq signal ) at Med1 - defined typical enhancers ( n = 2 , 111 ) super - enhancers ( n = 130 ) in LoVo ( was restricted to intergenic enhancers Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Notches represent 95 % confidence intervals for median values . Outliers not shown . P - value by two - sided Mann - Whitney U - .

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(B) Increased eRNA synthesis from super-enhancers. Boxplots comparing eRNA synthesis (TT-seq signal) at Med1-defined typical enhancers (n=2,111) and super-enhancers (n=130) in LoVo cells (analysis was restricted to intergenic enhancers Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown. P-value by two-sided Mann-Whitney U-test.


( C ) Super - enhancers are associated with production of long eRNAs . Boxplots showing of TT - seq - defined eRNAs originating from Med1 - defined super - enhancers ( n = 552 ) or other enhancer regions ( n = 6 , 934 ) . Median , hinges , whiskers notches are shown as in ( B ) . P - value by two - sided Mann - Whitney U - .

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(C) Super-enhancers are associated with the production of long eRNAs. Boxplots showing length of TT-seq-defined eRNAs originating from Med1-defined super-enhancers (n=552) or other enhancer regions (n=6,934). Median, hinges, whiskers and notches are shown as in (B). P-value by two-sided Mann-Whitney U-test.


( D ) Barplot showing number of TT - seq enhancer regions per Med1 - defined super - enhancer region in LoVo . Super - enhancers spanning a - coding TSS ( n = 32 ) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT - seq enhancer calling .

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(D) Barplot showing the number of TT-seq enhancer regions per Med1-defined super-enhancer region in LoVo cells. Super-enhancers spanning a protein-coding TSS (n=32) are excluded from this comparison as regions TSS ± 1 kbp were excluded from TT-seq enhancer calling.


( E ) UCSC genome browser view of normalized TT - seq coverage on plus minus strand at PHLDA1 locus ( hg38 ; chr12 : 75 , 612 , 350 - 76 , 042 , 552 ; in colorectal cancer HCT 116 . TT - seq coverage is cut at 250 ( purple lines ) to allow for better visualization . eRNAs are shown between plus minus strand . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) comprise ChIP - seq binding profiles of 19 TFs in HCT 116 from CISTROME height of signal indicates number of different TFs binding at a particular region . Binding regions for selected TFs ( TCF7l2 , YY1 , SP1 ) are shown below together with p300 polymerase II ( Pol II ) . TT - seq enhancer regions are shown at bottom together with a H3K27ac - defined super - enhancer annotation , which was downloaded from SEdb 2019 ) . At date of publication no Med1 ChIP - seq data was available for HCT 116 .

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(E) UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the PHLDA1 locus (hg38; chr12:75,612,350-76,042,552; in the colorectal cancer cell line HCT 116. TT-seq coverage is cut at 250 (purple lines) to allow for better visualization. eRNAs are shown between the plus and minus strand. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) comprise ChIP-seq binding profiles of 19 TFs in HCT 116 cells from CISTROME The height of the signal indicates the number of different TFs binding at a particular region. Binding regions for selected TFs (TCF7l2, YY1, SP1) are shown below together with p300 and RNA polymerase II (Pol II). TT-seq enhancer regions are shown at the bottom together with a H3K27ac-defined super-enhancer annotation, which was downloaded from SEdb 2019). At date of publication no Med1 ChIP-seq data was available for HCT 116.


( A ) Unsupervised clustering of pairwise Spearman correlations of normalized TT - seq signal for mRNAs ( left ) eRNAs ( right ) for union of transcripts over fourteen cancer lines Grey colored bars on top indicate gender cancer type , respectively .

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(A) Unsupervised clustering of all pairwise Spearman correlations of normalized TT-seq signal for mRNAs (left) and eRNAs (right) for the union of transcripts over all fourteen cancer cell lines Grey and colored bars on top indicate gender and cancer type, respectively.


( B ) Heatmaps showing mRNAs ( left ) eRNAs ( right ) as being synthesized ( red , normalized TT - seq signal FPK ≥ 15 ) or not ( blue , normalized TT - seq signal FPK < 15 ) for fourteen cancer lines ( columns ) . Heatmaps show only transcripts ( rows ) with normalized FPK ≥ 30 in at least one of lines ( mRNAs , n = 14 , 040 ; eRNAs n = 48 , 105 ) . Heatmaps are ordered by increasing type specificity , as indicated by grey bars to right showing number of lines a transcript is observed in .

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(B) Heatmaps showing mRNAs (left) and eRNAs (right) as being synthesized (red, normalized TT-seq signal FPK ≥ 15) or not (blue, normalized TT-seq signal FPK < 15) for all fourteen cancer cell lines (columns). Heatmaps show only transcripts (rows) with normalized FPK ≥ 30 in at least one of the cell lines (mRNAs, n=14,040; eRNAs n=48,105). Heatmaps are ordered by increasing cell type specificity, as indicated by the grey bars to the right showing the number of cell lines a transcript is observed in.


( D ) Boxplots showing normalized TT - seq signal for nearest active promoters in in which enhancers are exclusively transcribed ( from left to right : n = 890 ; 500 ; 494 ; 214 ; 238 ; 239 ; 378 ; 62 ; 153 ; 879 ; 586 ; 173 ; 601 ; 232 ) compared to same promoters in other lines in which respective enhancers are not transcribed . pairwise comparisons are significantly different ( P - values < 1e - 9 , two - sided Mann - Whitney U - ) . Box limits are first third quartiles , band inside box is median . ends of whiskers extend box by 1 . 5 times interquartile range . Notches represent 95 % confidence intervals for median values . Outliers not shown .

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(D) Boxplots showing normalized TT-seq signal for nearest active promoters in the cell line in which enhancers are exclusively transcribed (from left to right: n=890; 500; 494; 214; 238; 239; 378; 62; 153; 879; 586; 173; 601; 232) compared to the same promoters in all other cell lines in which the respective enhancers are not transcribed. All pairwise comparisons are significantly different (P-values < 1e-9, two-sided Mann-Whitney U-test). Box limits are the first and third quartiles, the band inside the box is the median. The ends of the whiskers extend the box by 1.5 times the interquartile range. Notches represent 95% confidence intervals for the median values. Outliers not shown.


UCSC genome browser view of normalized TT - seq coverage on plus minus strand at BMP4 locus ( hg38 ; chr14 : 53 , 935 , 697 - 54 , 264 , 430 ; in LoVo colorectal cancer . TT - seq coverage is cut at 500 ( purple lines ) to allow for better visualization . eRNAs enhancers are shown above below minus strand , respectively . Aggregated transcription factor ChIP - seq peak regions ( TF ChIP - seq ) include ChIP - seq binding profiles of 326 TFs in LoVo height of signal indicates number of different TFs binding at a particular region . Significant promoter capture Hi - C ( CHi - C ) interactions are shown in

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UCSC genome browser view of normalized TT-seq coverage on the plus and minus strand at the BMP4 locus (hg38; chr14:53,935,697-54,264,430; in LoVo colorectal cancer cells. TT-seq coverage is cut at 500 (purple lines) to allow for better visualization. eRNAs and enhancers are shown above and below the minus strand, respectively. Aggregated transcription factor ChIP-seq peak regions (TF ChIP-seq) include ChIP-seq binding profiles of 326 TFs in LoVo cells The height of the signal indicates the number of different TFs binding at a particular region. Significant promoter capture Hi-C (CHi-C) interactions are shown in purple


B . Western blots of MDI in wild type ( wt ) mdi1 flies , showing that mdi1 is a - null mutation . Tubulin was used as loading control .

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B. Western blots of MDI protein in wild type (wt) and mdi1 flies, showing that mdi1 is a protein-null mutation. Tubulin was used as loading control.


C . Ovarioles expressing MDI - GFP stained with a mitochondrial marker , ATP synthase ( ATP - S ) . Overlapping signals ( merge ) indicate that MDI localizes to mitochondria . Note that MDI is highly expressed in germ at late germarium stages ( left panels ) in egg chambers ( right panels ) . Bars , 10 m .

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C. Ovarioles expressing MDI-GFP stained with a mitochondrial marker, ATP synthase (ATP-S). Overlapping signals (merge) indicate that MDI localizes to mitochondria. Note that MDI is highly expressed in germ cells at late germarium stages (left panels) and in egg chambers (right panels). Bars, 10 m.


A - C . mtDNA replication in wt ( A ) mdi1 ovarioles ( B , C ) as illustrated by EdU incorporation . Arrows point to mitochondrial , arrowheads to nuclei . In wt ovaries ( A ) , mtDNA replication starts at germarium stage 2B lasts into egg - chamber stage 2 ( S2 ) . In most mdi1 ovarioles ( B ) , mtDNA replication is undetectable at these stages , whereas in some ( C ) it appears delayed until germarium stage 3 . Bars , 10 m .

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A-C. mtDNA replication in wt (A) and mdi1 ovarioles (B, C) as illustrated by EdU incorporation. Arrows point to mitochondrial DNA, arrowheads to nuclei. In wt ovaries (A), mtDNA replication starts at germarium stage 2B and lasts into egg-chamber stage 2 (S2). In most mdi1 ovarioles (B), mtDNA replication is undetectable at these stages, whereas in some (C) it appears delayed until germarium stage 3. Bars, 10 m.


D . Hatching rates mtDNA content of eggs laid by of different mutant genotypes relative to eggs laid by wt controls . Each data point represents mean of 3 independent replicates . Expression of mdi or hAKAP1 in mdi1 significantly restored mtDNA level hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . relative mtDNA level was determined as average of 3 biological repeats . P values of comparing mdi1 ; nos > mdi to mdi1 : hatching rate , P = 1 . 1995E - 05 ; mtDNA , P = 0 . 0078 . P values of comparing mdi1 ; nos > hAKAP1 to mdi1 : hatching rate , P = 1 . 6998E - 05 ; mtDNA level , P = 0 . 0009 .

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D. Hatching rates and mtDNA content of eggs laid by females of different mutant genotypes relative to eggs laid by wt controls. Each data point represents the mean of 3 independent replicates. Expression of mdi or hAKAP1 in mdi1 significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was determined as the average of 3 biological repeats. P values of comparing mdi1; nos> mdi to mdi1: hatching rate, P= 1.1995E-05; mtDNA, P= 0.0078. P values of comparing mdi1; nos> hAKAP1 to mdi1: hatching rate, P= 1.6998E-05; mtDNA level, P= 0.0009.


B . Representative of S2 expressing GFP fusions ( green ) of MDI diagramed in ( A ) . with MitoTracker Red ( red ) to label mitochondria shows an overlapping signal ( yellow ) , indicating proper localization to mitochondria for fusion except MDIΔMTS .

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B. Representative images of S2 cells expressing GFP fusions (green) of the MDI proteins diagramed in (A). Staining with MitoTracker Red (red) to label mitochondria shows an overlapping signal (yellow), indicating proper localization to mitochondria for all fusion proteins except MDIΔMTS.


C . Submitochondrial localization of MDI . Mitochondria from cultured expressing an MDI - myc fusion were kept intact or subjected to swelling to disrupt their outer membranes , digested or not with Protease K ( PK ) . extracts were subjected to western blot . Tom20 , cytochrome C ( cyt . C ) SOD2 were used as markers of outer , inter - space matrix , respectively . MDI behaved like an outer - .

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C. Submitochondrial localization of MDI. Mitochondria from cultured cells expressing an MDI-myc fusion protein were kept intact or subjected to swelling to disrupt their outer membranes, and digested or not with Protease K (PK). Protein extracts were subjected to western blot analysis. Tom20, cytochrome C (cyt.C) and SOD2 were used as markers of the outer membrane, inter-membrane space and matrix, respectively. MDI behaved like an outer-membrane protein.


D . Western blots of MDI in wt mdi knockout ( mdi - ko ) confirming mdi - ko completely lacked MDI . Actin was used as loading control .

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D. Western blots of MDI protein in wt and mdi knockout (mdi-ko) cells confirming the mdi-ko cells completely lacked MDI protein. Actin was used as loading control.


E . Representative of wt or mdi - ko expressing a Larp - GFP fusion stained with MitoTracker Red to label mitochondria . majority of Larp localized to mitochondria in wt ( overlapping red green signal ) , but diffused into cytoplasm of mdi - ko .

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E. Representative images of wt or mdi-ko cells expressing a Larp-GFP fusion protein and stained with MitoTracker Red to label mitochondria. The majority of Larp localized to mitochondria in wt cells (overlapping red and green signal), but diffused into the cytoplasm of mdi-ko cells.


F . Representative of mdi - ko co - expressing Larp - GFP with MDI or deletion mutants diagramed in ( A ) fused to mCherry . Of 4 MDI deletions that localized to mitochondria , 2 ( MDIΔR MDIΔTudor ) did not allow Larp - GFP to localize to mitochondria . localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants , hatching rate mtDNA level in their eggs were determined . result was summarized in ( A ) .

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F. Representative images of mdi-ko cells co-expressing Larp-GFP with MDI or the deletion mutants diagramed in (A) fused to the mCherry. Of the 4 MDI deletions that localized to mitochondria, 2 (MDIΔR and MDIΔTudor) did not allow Larp-GFP to localize to mitochondria. The localization of Larp in mdi1 flies expressing MDI or MDI deletion mutants, and the hatching rate and mtDNA level in their eggs were determined. The result was summarized in (A).


A . Co - immunoprecipitation of Larp with MDI - myc in transfected S2 . Tubulin was used as loading control .

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A. Co-immunoprecipitation of Larp with MDI-myc in transfected S2 cells. Tubulin was used as loading control.


B . mtDNA replication , as illustrated by EdU incorporation , in wt larp mutant ( larp ) ovarioles . mtDNA replication is dramatically reduced in larp ovariole . Arrows : mtDNA ; arrowheads : nuclei .

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B. mtDNA replication, as illustrated by EdU incorporation, in wt and larp mutant (larp) ovarioles. mtDNA replication is dramatically reduced in the larp ovariole. Arrows: mtDNA; arrowheads: nuclei.


C . wt mdi1 egg chambers stained for Larp ( green ) ATP - S ( red ) to reveal mitochondria . Larp closely associates with mitochondria in wt egg chambers . Mitochondria in mdi1 flies are clumped together , completely lack Larp . Bars in ( B ) ( C ) : 10 m .

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C. wt and mdi1 egg chambers stained for Larp (green) and ATP-S (red) to reveal mitochondria. Larp closely associates with mitochondria in wt egg chambers. Mitochondria in mdi1 flies are clumped together, and completely lack Larp staining. Bars in (B) (C): 10 m.


A . Nascent synthesis revealed by HpG incorporation ( green ) in wt mdi1 egg chambers . Arrowheads point to HpG signal on ER in perinuclear region periphery . Arrows point to HpG signal associated with mitochondria . Mitochondria are marked by a Tom20 - mCherry ( red ) created by inserting mCherry at endogenous Tom20 locus .

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A. Nascent protein synthesis revealed by HpG incorporation (green) in wt and mdi1 egg chambers. Arrowheads point to the HpG signal on the ER in the perinuclear region and cell periphery. Arrows point to HpG signal associated with mitochondria. Mitochondria are marked by a Tom20-mCherry (red) created by inserting mCherry at the endogenous Tom20 locus.


B . ER location is unaltered in mdi1 egg chambers . An ER marker ( ER - GFP ) was expressed in wt mdi1 egg chambers that were co - stained with ATP - S to mark mitochondria . ER localizes to perinuclear region , cytoplasm periphery in both wt mdi1 egg chambers .

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B. ER location is unaltered in mdi1 egg chambers. An ER marker (ER-GFP) was expressed in wt and mdi1 egg chambers that were co-stained with ATP-S to mark mitochondria. ER localizes to the perinuclear region, cytoplasm and cell periphery in both wt and mdi1 egg chambers.


Detection of nascent synthesis in mitochondrial fraction of ovary . A . Nascent synthesis was monitored by AHA incorporation , detected by anti - biotin antibody . Tom20 served as a loading control . Note that synthesis of - encoded mitochondrial was decreased in mdi1 background was restored by overexpressing Tom20 - Larp ( mdi1 / TL ) .

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Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. A. Nascent protein synthesis was monitored by AHA incorporation, and detected by anti-biotin antibody. Tom20 served as a loading control. Note that the synthesis of nuclear-encoded mitochondrial proteins was decreased in the mdi1 background and was restored by overexpressing Tom20-Larp (mdi1/TL).


Detection of nascent synthesis in mitochondrial fraction of ovary . B . Western blots of mitochondrial in ovarioles of wt , mdi1 mdi1 flies expressing Tom20 - Larp ( mdi1 / TL ) fusion . Note that of Tamas , TFAM , mRpL19 COX4 were reduced in mdi1 flies , but restored in mdi1 / TL flies .

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Detection of nascent protein synthesis in the mitochondrial fraction of the ovary.B. Western blots of mitochondrial proteins in ovarioles of wt, mdi1 and mdi1 flies expressing Tom20-Larp (mdi1/TL) fusion protein. Note that levels of Tamas, TFAM, mRpL19 and COX4 were reduced in mdi1 flies, but restored in mdi1/TL flies.


Detection of nascent synthesis in mitochondrial fraction of ovary . C . Nascent synthesis of Tamas TFAM was decreased in mdi1 ovary , whereas mtSSB was not affected . Tfamgfp , Tamasgfp mtSSBgfp were expressed in wt or mdi1 background . Nascent were labeled by AHA incorporation then GFP - tagged was immunopurified with a GFP antibody nascent synthesis was detected by anti - biotin antibody .

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Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. C. Nascent protein synthesis of Tamas and TFAM was decreased in mdi1 ovary, whereas mtSSB was not affected. Tfamgfp, Tamasgfp and mtSSBgfp were expressed in wt or mdi1 background. Nascent proteins were labeled by AHA incorporation and then the GFP-tagged protein was immunopurified with a GFP antibody and the nascent protein synthesis was detected by anti-biotin antibody.


Detection of nascent synthesis in mitochondrial fraction of ovary . D . Representative profile of 254 nm absorbance of wt , mdi1 mdi1 / TL ovary extracts .

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Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. D. Representative profile of 254 nm absorbance of wt, mdi1 and mdi1/TL ovary extracts.


Detection of nascent synthesis in mitochondrial fraction of ovary . E . Polysome mRNA profiling for tamas , tfam , mtSSB , cox4 mRPL19 in wt , mdi1 mdi1 / TL ovary . of mRNA for each gene in non - polysomal fractions ( N . P , including ribosomal subunits monosome - associated ) polysomal fractions ( poly ) was calculated plotted . fractions of tamas , tfam , cox4 mRPL19 mRNAs in polysomal fractions were significantly decreased in mdi1 compared to wt , but were restored in mdi1 / TL flies . N = 4 for samples . P values of comparing wt to mdi1 : tamas , P = 0 . 0055 ; tfam , P = 0 . 001 ; cox4 , P = 0 . 0066 ; mRPL19 , P = 0 . 0097 . P values of comparing mdi1 / TL to mdi1 : tamas , P = 0 . 0206 ; tfam , P = 0 . 0346 ; cox4 , P = 0 . 0036 ; mRPL19 , P = 0 . 0016 .

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

Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. E. Polysome mRNA profiling for tamas, tfam, mtSSB, cox4 and mRPL19 in wt, mdi1 and mdi1/TL ovary. The percentage of mRNA for each gene in non-polysomal fractions (N.P, including ribosomal subunits and monosome-associated) and polysomal fractions (poly) was calculated and plotted. The fractions of tamas, tfam, cox4 and mRPL19 mRNAs in the polysomal fractions were significantly decreased in mdi1 compared to wt, but were restored in mdi1/TL flies. N=4 for all samples. P values of comparing wt to mdi1: tamas, P= 0.0055; tfam, P= 0.001; cox4, P=0.0066; mRPL19, P=0.0097. P values of comparing mdi1/TL to mdi1: tamas, P= 0.0206; tfam, P= 0.0346; cox4, P=0.0036; mRPL19, P=0.0016.


Detection of nascent synthesis in mitochondrial fraction of ovary . F . nascent synthesis of COX4 mRPL19 were decreased in mdi1 ovary were restored mdi1 / TL flies . were immunopurified with antibodies against endogenous .

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Detection of nascent protein synthesis in the mitochondrial fraction of the ovary. F. The nascent protein synthesis of COX4 and mRPL19 were decreased in mdi1 ovary and were restored mdi1/TL flies. The proteins were immunopurified with antibodies against the endogenous proteins.


A . Mitochondrial localization of a Tom20 - LarpGFP ( Tom20 - Larp ) fusion in an mdi1 egg chamber stained with ATP - S to reveal mitochondria . Bars , 20 m .

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A. Mitochondrial localization of a Tom20-LarpGFP (Tom20-Larp) fusion protein in an mdi1 egg chamber stained with ATP-S to reveal mitochondria. Bars, 20 m.


B . Hatching rates mtDNA contents of eggs produced by flies with different genotypes relative to wt control ; each data point represents mean of 3 independent replicates . Bars , std . Expression of Tom20 - Larp in mdi1 ( mdi1 ; Tom20 - Larp ) significantly restored mtDNA level hatching rate . N = 3 x > 100 eggs / genotype for hatching rate . relative mtDNA level was average of 3 biological repeats . P values of comparing mdi1 ; Tom20 - Larp to mdi1 : for mtDNA level , P = 0 . 0209 ; for hatching rate , P = 3 . 4758E - 05 .

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B. Hatching rates and mtDNA contents of eggs produced by flies with different genotypes relative to wt control; each data point represents the mean of 3 independent replicates. Bars, std. Expression of Tom20-Larp in mdi1 (mdi1;Tom20-Larp) significantly restored the mtDNA level and hatching rate. N= 3 x >100 eggs / genotype for hatching rate. The relative mtDNA level was the average of 3 biological repeats. P values of comparing mdi1;Tom20-Larp to mdi1: for mtDNA level, P=0.0209; for hatching rate, P=3.4758E-05.


C Representative D - type motor neurons in wild - type mxl - 1 mutant 24 h after surgery . In wild - type , a severed axon has regenerated a growth cone ( arrow ) . In mutants , proximal ends of axons failed to regenerate ( arrowhead ) . Scale bar = 10 μm .

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C Representative D-type motor neurons in wild-type and mxl-1 mutant animals 24 h after laser surgery. In wild-type animals, a severed axon has regenerated a growth cone (arrow). In mutants, proximal ends of axons failed to regenerate (arrowhead). Scale bar = 10 μm.


D Percentages of axons that initiated regeneration 24 h after surgery . numbers ( n ) of examined are shown . Error bars indicate 95 % confidence intervals ( CI ) . Data information statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * * P < 0 . 001 ; NS ; not significant .

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D Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% confidence intervals (CI). Data information statistical significance was determined by Fisher's exact test; *P < 0.05; ***P <0.001; NS; not significant.


Percentages of axons that initiated regeneration 24 h after surgery . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information : In ( A ) , statistical significance was determined by Fisher ' s exact ; * * * P < 0 . 001 ; NS ; not significant .

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Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: In (A), statistical significance was determined by Fisher's exact test; ***P <0.001; NS; not significant.


B Induction of Psvh - 2 : : nls : : venus expression in D - type motor neurons by surgery . Expression of in D - type motor neurons of wild - type , mxl - 1 , tdpt - 1 mxl - 1 ; tdpt - 1 3 h after surgery are shown . Yellow arrowheads arrows indicate axon bodies , respectively , of D - type neurons . arrows indicate sites of surgery . D neurons are visualized by CFP under control of unc - 25 promoter . bodies of D - type neurons are magnified shown within red boxes . Most of intestinal in these photos is from endogenous variable background autofluorescence . A schematic representation of D - type motor neurons is shown on bottom . Scale bars = 10 μm .

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B Induction of Psvh-2::nls::venus expression in D-type motor neurons by laser surgery. Expression of fluorescent proteins in D-type motor neurons of wild-type, mxl-1, tdpt-1 and mxl-1; tdpt-1 animals 3 h after laser surgery are shown. Yellow arrowheads and white arrows indicate axon and cell bodies, respectively, of D-type neurons. White arrows indicate the sites of laser surgery. D neurons are visualized by CFP under control of the unc-25 promoter. Cell bodies of D-type neurons are magnified and shown within the red boxes. Most of the intestinal fluorescence in these photos is from endogenous and variable background autofluorescence. A schematic representation of D-type motor neurons is shown on the bottom. Scale bars = 10 μm.


Percentages of axons that initiated regeneration 24 h after surgery . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information : statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

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Percentages of axons that initiated regeneration 24 h after laser surgery. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


B Interaction of MXL - 1 with TDPT - 1 by yeast two - hybrid assays . reporter yeast L40u was co - transformed with expression vectors encoding LexA DBD - MXL - 1 GAL4 AD - TDPT - 1 as indicated . Yeasts carrying indicated plasmids were grown on a selective plate lacking histidine containing 10 mM 5 - aminotriazole for 4 days .

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B Interaction of MXL-1 with TDPT-1 by yeast two-hybrid assays. The reporter yeast strain L40u was co-transformed with expression vectors encoding LexA DBD-MXL-1 and GAL4 AD-TDPT-1 as indicated. Yeasts carrying the indicated plasmids were grown on a selective plate lacking histidine and containing 10 mM 5-aminotriazole for 4 days.


B Interaction between ETS - 4 TDPT - 1 . COS - 7 were transfected with plasmids encoding T7 - TDPT - 1 , HA - ETS - 4 ( WT ) , HA - ETS - 4 ( S73E ) ( SE ) HA - ETS - 4 ( S73A ) ( SA ) , as indicated . Whole - extracts ( WCE ) immunoprecipitated complexes obtained with anti - T7 antibody ( IP : T7 ) were analyzed by immunoblotting ( IB ) .

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B Interaction between ETS-4 and TDPT-1. COS-7 cells were transfected with plasmids encoding T7-TDPT-1, HA-ETS-4 (WT), HA-ETS-4(S73E) (SE) and HA-ETS-4(S73A) (SA), as indicated. Whole-cell extracts (WCE) and immunoprecipitated complexes obtained with anti-T7 antibody (IP: T7) were analyzed by immunoblotting (IB).


C In vitro SUMOylation of ETS - 4 . COS - 7 were transfected with plasmids encoding HA - ETS - 4 ( WT ) HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , as indicated . lysates were immunoprecipitated with anti - HA antibody . immunoprecipitates were subjected to in vitro SUMOylation assays . Reaction mixtures were analyzed by immunoblotting ( IB ) with anti - SUMO - 2 / 3 anti - HA antibodies . Arrows mark SUMOylated - ETS - 4 bands .

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C In vitro SUMOylation of ETS-4. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro SUMOylation assays. Reaction mixtures were analyzed by immunoblotting (IB) with anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands.


E SUMOylation of ETS - 4 in mammalian . HEK293 were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , T7 - TDPT - 1 , His - SUMO - 1 , His - SUMO - 2 Myc - MXL - 1 , as indicated . SUMO - conjugated were isolated by cobalt affinity chromatography . ETS - 4 was detected by immunoblotting with anti - HA antibody . Whole - extracts ( WCE ) were analyzed by immunoblotting ( IB ) .

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

E SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), T7-TDPT-1, His-SUMO-1, His-SUMO-2 and Myc-MXL-1, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).


A Effect of phosphorylation of ETS - 4 on SUMOylation of ETS - 4 in mammalian . HEK293 were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , HA - ETS - 4 ( S73A ) ( SA ) , HA - ETS - 4 ( S73E ) ( SE ) , T7 - TDPT - 1 His - SUMO - 2 , as indicated . SUMO - conjugated were isolated by cobalt affinity chromatography . ETS - 4 was detected by immunoblotting with anti - HA antibody . Whole - extracts ( WCE ) were analyzed by immunoblotting ( IB ) .

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

A Effect of phosphorylation of ETS-4 on SUMOylation of ETS-4 in mammalian cells. HEK293 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(K32A; K83A) (2KA), HA-ETS-4(S73A) (SA), HA-ETS-4(S73E) (SE), T7-TDPT-1 and His-SUMO-2, as indicated. SUMO-conjugated proteins were isolated by cobalt affinity chromatography. ETS-4 was detected by immunoblotting with anti-HA antibody. Whole-cell extracts (WCE) were analyzed by immunoblotting (IB).


B Effect of SUMOylation of ETS - 4 on PKA phosphorylation of ETS - 4 in vitro . COS - 7 were transfected with plasmids encoding HA - ETS - 4 ( WT ) , HA - ETS - 4 ( S73A ) ( SA ) HA - ETS - 4 ( K32A ; K83A ) ( 2KA ) , as indicated . lysates were immunoprecipitated with anti - HA antibody . immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation then in vitro PKA kinase assays , using recombinant GST - fused active PKA , as indicated . Reaction mixtures were analyzed by immunoblotting ( IB ) with anti - phospho - PKA substrate , anti - SUMO - 2 / 3 anti - HA antibodies . Arrows mark SUMOylated - ETS - 4 bands . Asterisk indicates non - SUMOylated - phosphorylated - ETS - 4 bands .

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

B Effect of SUMOylation of ETS-4 on PKA phosphorylation of ETS-4 in vitro. COS-7 cells were transfected with plasmids encoding HA-ETS-4 (WT), HA-ETS-4(S73A) (SA) and HA-ETS-4(K32A; K83A) (2KA), as indicated. Cell lysates were immunoprecipitated with anti-HA antibody. The immunoprecipitates were subjected to in vitro PKA kinase assays or in vitro SUMOylation and then in vitro PKA kinase assays, using recombinant GST-fused active PKA, as indicated. Reaction mixtures were analyzed by immunoblotting (IB) with anti-phospho-PKA substrate, anti-SUMO-2/3 and anti-HA antibodies. Arrows mark the SUMOylated-ETS-4 bands. Asterisk indicates the non-SUMOylated- and phosphorylated-ETS-4 bands.


Percentages of axons that initiated regeneration 24 h after surgery are shown . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

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

Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


Percentages of axons that initiated regeneration 24 h after surgery are shown . numbers ( n ) of examined are shown . Error bars indicate 95 % CI . Data information : statistical significance was determined by Fisher ' s exact ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; NS ; not significant .

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

Percentages of axons that initiated regeneration 24 h after laser surgery are shown. The numbers (n) of animals examined are shown. Error bars indicate 95% CI. Data information: statistical significance was determined by Fisher's exact test; *P < 0.05; **P < 0.01; ***P < 0.001; NS; not significant.


( B ) Autophagy ‐ negative phenotype of apg16Δ . autophagic ability of wild ‐ type ( TN125 ) , apg12Δ ( YNM107 ) apg16Δ ( YNM114 ) was measured by alkaline phosphatase before ( black bars ) after ( bars ) nitrogen starvation for 4 h . Error bars indicate standard deviation of three independent experiments .

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(B) Autophagy‐negative phenotype of the apg16Δ strain. The autophagic ability of wild‐type (TN125), apg12Δ (YNM107) and apg16Δ cells (YNM114) was measured by the alkaline phosphatase assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate the standard deviation of three independent experiments.


( C ) Loss of viability during starvation . Wild ‐ type ( KA311B , ○ ) , apg12Δ ( YNM101 , ● ) apg16Δ ( YNM124 , ▴ ) were cultured in nitrogen starvation medium , their viability was determined by phloxine B .

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(C) Loss of viability during starvation. Wild‐type (KA311B, ○), apg12Δ (YNM101, ●) and apg16Δ cells (YNM124, ▴) were cultured in nitrogen starvation medium, and their viability was determined by phloxine B staining.


( D ) Defect in API maturation in apg16Δ . Transport of pro ‐ API to vacuole was examined by immunoblotting with anti ‐ API antiserum . positions of precursor mature API are indicated .

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(D) Defect in API maturation in apg16Δ cells. Transport of pro‐API to the vacuole was examined by immunoblotting with anti‐API antiserum. The positions of precursor and mature API are indicated.


( E ) Apg12p - Apg5p conjugation in apg16Δ . Total lysates were prepared by NaOH / 2 ‐ mercaptoethanol extraction method from apg12Δ ( YNM107 ) apg12Δapg16Δ ( YNM115 ) , both carrying pHA ‐ APG12 ( CEN ) . Western blot was performed using anti ‐ HA antibody .

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(E) The Apg12p-Apg5p conjugation in apg16Δ cells. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115), both carrying pHA‐APG12 (CEN). Western blot analysis was performed using anti‐HA antibody.


Apg16p interacts with Apg12p - Apg5p conjugate but not with Apg12p monomer . apg12Δapg16Δ ( YNM115 ) were transformed with high copy ( 2μ ) plasmids encoding MycApg12p / or HAApg16p as indicated . Total lysates were immunoprecipitated with anti ‐ Myc ( lanes 1 - 4 ) or anti ‐ HA antibody ( lanes 5 - 8 ) detected by immunoblotting using anti ‐ Myc or anti ‐ HA antibody . positions of IgG heavy chain κ chain are indicated .

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

Apg16p interacts with the Apg12p-Apg5p conjugate but not with the Apg12p monomer. The apg12Δapg16Δ cells (YNM115) were transformed with high copy (2μ) plasmids encoding MycApg12p and/or HAApg16p as indicated. Total lysates were immunoprecipitated with anti‐Myc (lanes 1-4) or anti‐HA antibody (lanes 5-8) and detected by immunoblotting using anti‐Myc or anti‐HA antibody. The positions of the IgG heavy chain and κ light chain are indicated.


Apg16p interacts preferentially with Apg12p ‐ modified Apg5p . Total lysates were prepared from apg16Δ ( YNM124 ) or apg12Δapg16Δ ( YNM115 ) harboring both pHA ‐ APG5 pMyc ‐ APG16 CEN plasmids . lysates were immunoprecipitated analyzed as described in Figure 3 .

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

Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δ cells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.


Apg16p interacts preferentially with Apg12p ‐ modified Apg5p . Total lysates were prepared from apg16Δcells ( YNM124 ) or apg12Δapg16Δ ( YNM115 ) harboring both pHA ‐ APG5 pMyc ‐ APG16 CEN plasmids . lysates were immunoprecipitated analyzed as described in Figure 3 .

Apg16p interacts preferentially with Apg12p‐modified Apg5p. Total lysates were prepared from apg16Δcells (YNM124) or apg12Δapg16Δ cells (YNM115) harboring both pHA‐APG5 and pMyc‐APG16 CEN plasmids. The lysates were immunoprecipitated and analyzed as described in Figure 3.


( A ) PJ69 ‐ 4A were co ‐ transformed with each pGBD pGAD plasmid as indicated . Transformants were selected on Trp − Leu − plates , then two ‐ hybrid interaction ( + or − ) was assessed for growth on Ade − Trp − Leu − plates .

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(A) PJ69‐4A cells were co‐transformed with each pGBD and pGAD plasmid as indicated. Transformants were selected on Trp− Leu− plates, and then two‐hybrid interaction (+ or −) was assessed for growth on Ade− Trp− Leu− plates.


( B ) Self ‐ multimerization of Apg16p in vivo . Lysates of apg16Δ ( YNM124 ) transformed with pMyc ‐ APG16 ( 2μ ) / or pHA ‐ APG16 ( 2μ ) were immunoprecipitated with anti ‐ Myc antibody detected with anti ‐ HA antibody .

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(B) Self‐multimerization of Apg16p in vivo. Lysates of apg16Δ cells (YNM124) transformed with pMyc‐APG16 (2μ) and/or pHA‐APG16 (2μ) were immunoprecipitated with anti‐Myc antibody and detected with anti‐HA antibody.


Apg16p mediates Apg5p assembly . Immunoprecipitation was carried out as described in Figure 3 , using apg12Δ ( YNM107 ) apg12Δapg16Δ ( YNM115 ) carrying pMyc ‐ APG12 ( CEN ) pHA ‐ APG12 ( CEN ) ( A ) , or pMyc ‐ APG12 ( CEN ) pHA ‐ APG5 ( CEN ) ( B ) . Following SDS - PAGE , immunoprecipitates were subjected to immunoblot using anti ‐ HA or anti ‐ Myc antibody as indicated . A model of Apg12p - Apg5p - Apg16p is illustrated in each panel . Apg16p forms a homo ‐ oligomer through its C ‐ terminal coiled ‐ coil region . It is not clear whether Apg16p forms a dimer or a larger multimer . N ‐ terminal region of each Apg16p interacts with Apg5p ; most of these complexes are conjugated to Apg12p ( A ) but a small population are unmodified ( B ) . In absence of Apg16p , separate Apg5ps do not assemble .

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

Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG12 (CEN) (A), or pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.


Apg16p mediates Apg5p assembly . Immunoprecipitation was carried out as described in Figure 3 , using apg12Δ ( YNM107 ) apg12Δapg16Δ ( YNM115 ) carrying pMyc ‐ APG12 ( CEN ) pHA ‐ APG5 ( CEN ) ( B ) . Following SDS - PAGE , immunoprecipitates were subjected to immunoblot using anti ‐ HA or anti ‐ Myc antibody as indicated . A model of Apg12p - Apg5p - Apg16p is illustrated in each panel . Apg16p forms a homo ‐ oligomer through its C ‐ terminal coiled ‐ coil region . It is not clear whether Apg16p forms a dimer or a larger multimer . N ‐ terminal region of each Apg16p interacts with Apg5p ; most of these complexes are conjugated to Apg12p ( A ) but a small population are unmodified ( B ) . In absence of Apg16p , separate Apg5ps do not assemble .

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 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 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 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 O O

Apg16p mediates Apg5p assembly. Immunoprecipitation was carried out as described in Figure 3, using apg12Δ (YNM107) and apg12Δapg16Δ cells (YNM115) carrying pMyc‐APG12 (CEN) and pHA‐APG5 (CEN) (B). Following SDS-PAGE, immunoprecipitates were subjected to immunoblot analysis using anti‐HA or anti‐Myc antibody as indicated. A model of the Apg12p-Apg5p-Apg16p complex is illustrated in each panel. Apg16p forms a homo‐oligomer through its C‐terminal coiled‐coil region. It is not clear whether Apg16p forms a dimer or a larger multimer. The N‐terminal region of each Apg16p interacts with Apg5p; most of these complexes are conjugated to Apg12p (A) but a small population are unmodified (B). In the absence of Apg16p, separate Apg5ps do not assemble.


Expression of Apg16p are dependent on Apg12p - Apg5p conjugate . Total lysates were prepared by NaOH / 2 ‐ mercaptoethanol extraction method from apg16Δ ( YNM124 ) , apg5Δapg16Δ ( YNM126 ) apg12Δapg16Δ ( YNM115 ) carrying pHA ‐ APG16 ( CEN ) . Western blot was performed using anti ‐ HA antibody .

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Expression levels of Apg16p are dependent on the Apg12p-Apg5p conjugate. Total lysates were prepared by the NaOH/2‐mercaptoethanol extraction method from apg16Δ (YNM124), apg5Δapg16Δ (YNM126) and apg12Δapg16Δ cells (YNM115) carrying pHA‐APG16 (CEN). Western blot analysis was performed using anti‐HA antibody.


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Apg16p exists in a pellet fraction in an Apg5p ‐ dependent manner . Spheroplasts were generated from harboring pHA ‐ APG5 ( A ) , pHA ‐ APG16 ( B ) or pHA ‐ APG16 ( 2μ ) with or without pHA ‐ APG5 ( 2μ ) ( C ) . They were lysed by Dounce homogenization . After unbroken spheroplasts were removed by low ‐ speed centrifugation , supernatant ( T ) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant ( S ) pellet ( P ) fraction . Each fraction was subjected to immunoblot using anti ‐ HA antibody .

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Apg16p exists in a pellet fraction in an Apg5p‐dependent manner. Spheroplasts were generated from cells harboring pHA‐APG5 (A), pHA‐APG16 (B) or pHA‐APG16 (2μ) with or without pHA‐APG5 (2μ) (C). They were lysed by Dounce homogenization. After unbroken spheroplasts were removed by low‐speed centrifugation, the supernatant (T) was fractionated by 100 000 g centrifugation for 60 min to generate a supernatant (S) and pellet (P) fraction. Each fraction was subjected to immunoblot analysis using anti‐HA antibody.


Pregnant dams were injected with 4 - OHT at days E10 . 5 E11 . 5 for at day E12 . 5 or E10 . 5 , E11 . 5 , E12 . 5 of development for later times of Developmental loss of VE - cadherin leads to severe edema . Fetuses of genotypes listed in top row were analyzed at developmental stages in first column . Numbers in brackets denote with signs of edema . Last column lists of viable fetuses in VE - cadherin deleted cohort Fetuses were explanted at day E14 . 5 . Arrowheads indicate prominent edema along back . are representative for 42 ( Cdh5lox / lox ) 25 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Developmental loss of VE-cadherin leads to severe edema. Fetuses of the genotypes listed in the top row were analyzed at the developmental stages in the first column. Numbers in brackets denote embryos with signs of edema. Last column lists the percentage of viable fetuses in the VE-cadherin deleted cohort Fetuses were explanted at day E14.5. Arrowheads indicate prominent edema along the back. The images are representative for 42 (Cdh5lox/lox) and 25 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals


Pregnant dams were injected with 4 - OHT at days E10 . 5 E11 . 5 for at day E12 . 5 or E10 . 5 , E11 . 5 , E12 . 5 of development for later times of Skin wholemount preparations stained for PROX1 , PECAM1 VEGFR - 3 . Maximum projections ( MIPs ) of tiled confocal stacks ( Cdh5lox / lox 2075µm x 2075µm , z = 50µm ; Cdh5lox / lox ; Prox1 - CreERT2 3765µm x 3765 µm , z = 61µm ) . Scale bars = 100 μm . data are representative for 6 ( Cdh5lox / lox ) 6 ( Cdh5lox / lox ; Prox1 - CreERT2 ) analyzed from 3

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Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Skin wholemount preparations stained for PROX1, PECAM1 and VEGFR-3. Maximum intensity projections (MIPs) of tiled confocal stacks (Cdh5lox/lox 2075µm x 2075µm, z=50µm; Cdh5lox/lox; Prox1-CreERT2 3765µm x 3765 µm, z=61µm). Scale bars = 100 μm. The data are representative for 6 (Cdh5lox/lox) and 6 (Cdh5lox/lox; Prox1-CreERT2) analyzed animals from 3 litters


Pregnant dams were injected with 4 - OHT at days E10 . 5 E11 . 5 for at day E12 . 5 or E10 . 5 , E11 . 5 , E12 . 5 of development for later times of Enumeration of PROX1 - expressing ( PROX1 + ) nuclei in three different VE - cadherin deleted wholemount preparations analogous to Fig . 1B . Nuclei were counted in a MIP corresponding to an of 1 . 5 mm2 normalized to number of nuclei in Cdh5lox / lox control preparations covered by lymphatic vessels was determined in samples evaluated in ( D ) is depicted as relative compared to Cdh5lox / lox controls . calculations measurements were obtained from three three confocal stacks per biological group . PROX1 - positive nuclei were counted using particle tool of

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

Pregnant dams were injected with 4-OHT at days E10.5 and E11.5 for analysis at day E12.5 or E10.5, E11.5, and E12.5 of development for later times of analysis Enumeration of the PROX1-expressing (PROX1+) nuclei in three different VE-cadherin deleted wholemount preparations analogous to Fig. 1B. Nuclei were counted in a MIP corresponding to an area of 1.5 mm2 and normalized to the number of nuclei in Cdh5lox/lox control preparations The area covered by lymphatic vessels was determined in the samples evaluated in (D) and is depicted as relative area compared to Cdh5lox/lox controls. The calculations and measurements were obtained from three animals and three confocal stacks per biological group. PROX1-positive nuclei were counted using the particle analysis tool of Fiji