( a , b ) dRagA activation increases sensitivity to starvation . Expression of dRagAQ61L using the fat - body - specific Cg - GAL4 driver significantly decreased survival of adult female flies under starvation ( b ) but not fed ( a ) conditions , relative to controls ( Cg - GAL4 alone ) . Asterisks indicate significant difference , compared with controls . Data are mean ± s . d . , * P 0 . 05 , Students two - tailed t - test ( n = 150 flies / genotype / treatment ) .

O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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-ORGANISM O O O O O O

(a, b) dRagA activation increases sensitivity to starvation. Expression of dRagAQ61L using the fat-body-specific Cg-GAL4 driver significantly decreased survival of adult female flies under starvation (b) but not fed (a) conditions, relative to controls (Cg-GAL4 alone). Asterisks indicate significant difference, compared with controls. Data are mean ± s.d., *P 0.05, Students two-tailed t-test (n = 150 flies/genotype/treatment).


( C ) Asynchronous exponentially growing cells were treated with 0 . 2M HU ( + HU ) , then HU was removed from the medium ( - HU ) . SDS - PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift ( Rad53 - P = * * ) . Red and green asterisk indicate the time where mec1 - S1991 phosphomutants show altered Rad53 recovery . A 5 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - the indicated dose of HU .

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(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.


Analysis of replication fork progression at the single - molecule level by DNA combing . ( C ) Representative images of DNA fibers . Green : ssDNA , red : BrdU . Scale bar corresponds to 20 kb . ( D ) Graph depict the distribution of BrdU track length . WT 90 min ( n = 427 ) and 180 min ( n = 483 ) , mec1 - S1991A 90 min ( n = 309 ) and 180 min ( n = 477 ) .

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Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).


( A ) tRNA level measured by RT - qPCR in asynchronous ( async . ) culture and after 90 min on HU in the indicated strains . Expression is normalized to ACT1 . The repression is expressed as a ratio of HU treated / asynchronous cells in percent .

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(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.


( A - D ) Exponentially growing cells were treated with 0 . 2M HU and total protein extracts were collected at the indicated time points ( in min ) , samples were subjected to SDS - PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies . ( B , D ) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin .

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(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin.


( E , F ) Drop assay on HU showing a 10 - fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC + / - 200mM HU . ( F ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


( A ) Preparations of chromatin - bound proteins were prepared in triplicate and subjected to SDS - PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3 - TAP Quantification of chromatin - bound Rpb1 - S2P and Rpb1 - S5P is shown on the right . Mcm2 is used as a loading control .

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(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control.


( F , G ) A 10 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - 200mM of HU . A high level of HU was used to be able to demonstrate robust suppression of the mec1 - S1991A phenotype . ( G ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


( B ) Amongst the Mec1 - S1991 - dependent phosphopeptides on HU , factors involved in transcription are highlighted .

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(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted.


( E ) Position relative to the nuclear envelope ( Zone 1 of the lacO - tagged GAL1 - GAL10 locus in the indicated strains grown either 12h on glucose , or 90 min galactose or galactose + 0 . 2M HU . The third panel shows the relative retention at the nuclear periphery ( Zone 1 ) on galactose + HU . The number of cells is > 300 for each condition .

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(E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.


Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α - factor and released into S phase at 25 ° C . Actin was used as a loading control .

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Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control.


A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization . Occasionally , additional expression is observed at the posterior tip of the embryo ( asterisk ) . Double labelling by staining for LacZ expression from the CcnoTA allele ( shown in B ) and in situ hybridization for Dand5 , marking crown cells at the circumference of the node . Ccno expression is restricted to ciliated , ventral pit cells of the node as detailed in the transverse section at the indicated plane .

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A Ccno mRNA is expressed in the embryonic node at E8 as shown by in situ hybridization. Occasionally, additional expression is observed at the posterior tip of the embryo (asterisk). Double labelling by staining for LacZ expression from the CcnoTA allele (shown in B) and in situ hybridization for Dand5, marking crown cells at the circumference of the node. Ccno expression is restricted to ciliated, ventral pit cells of the node as detailed in the transverse section at the indicated plane.


C Whole embryo X ‐ Gal staining of E16 CcnoTA / + and wild ‐ type control embryos and indicated section planes of ( D - F ' ) .

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C Whole embryo X‐Gal staining of E16 CcnoTA/+ and wild‐type control embryos and indicated section planes of (D-F').


D - F ' Histological sections of LacZ ‐ stained CcnoTA / + embryo at E16 reveal Ccno expression in the epithelium of ( D ) plexus choroideus and ependyme , ( E ) snout epithelium , and ( F , F ' ) trachea and bronchi . pc , plexus choroideus ; se , snout epithelium ; tr , trachea ; and br , bronchus .

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D-F' Histological sections of LacZ‐stained CcnoTA/+ embryo at E16 reveal Ccno expression in the epithelium of (D) plexus choroideus and ependyme, (E) snout epithelium, and (F, F') trachea and bronchi. pc, plexus choroideus; se, snout epithelium; tr, trachea; and br, bronchus.


G Co ‐ expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X ‐ Gal and immunohistochemistry using a FOXJ1 ‐ specific antibody .

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G Co‐expression of Ccno and Foxj1 in multiciliated cells of the trachea shown by double staining for X‐Gal and immunohistochemistry using a FOXJ1‐specific antibody.


A Ccno ‐ deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 ( arrow ) .

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A Ccno‐deficient mice develop severe hydrocephalus resulting in characteristic head deformation at P21 (arrow).


B , C Cranial MRI analysis ( B ) shows the drastically enlarged ventricular cavity ( marked by V ) and diminished cerebral cortex tissue , ( C ) also seen when brains are cut in coronal orientation .

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B, C Cranial MRI analysis (B) shows the drastically enlarged ventricular cavity (marked by V) and diminished cerebral cortex tissue, (C) also seen when brains are cut in coronal orientation.


D , D ' Alcian ‐ blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium ( arrows ) .

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D, D' Alcian‐blue staining of paranasal cavities reveals mucus congestion along the nasal epithelium (arrows).


F , F ' Scanning electron microscopy ( SEM ) of P21 adult trachea shows reduced numbers of cilia of Ccno ‐ deficient MCCs . Remaining cilia are found in the central regions of the cell surface , and cell margins frequently lack the ciliary decoration ( arrows ) .

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F, F' Scanning electron microscopy (SEM) of P21 adult trachea shows reduced numbers of cilia of Ccno‐deficient MCCs. Remaining cilia are found in the central regions of the cell surface, and cell margins frequently lack the ciliary decoration (arrows).


G Transmission electron microscopy ( TEM ) of wild ‐ type and CcnoRA / RA MCCs from adult trachea . Ccno ‐ deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface ( arrows ) . Ectopic electron ‐ dense material is found within the cytoplasm of Ccno ‐ deficient MCCs ( arrowheads ) .

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G Transmission electron microscopy (TEM) of wild‐type and CcnoRA/RA MCCs from adult trachea. Ccno‐deficient MCCs show reduced numbers of basal bodies and cilia that correctly docked to the apical cell surface (arrows). Ectopic electron‐dense material is found within the cytoplasm of Ccno‐deficient MCCs (arrowheads).


A - H X ‐ Gal ‐ staining of ( A - C ) control and ( D - H ) CcnoTA / + embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi . Expression is extending to more distal regions , and from E16 staining is also found in bronchioli . I X ‐ Gal ‐ staining indicating LacZ expression from the CcnoTA / + allele in mTEC cultures after 5 days of differentiation ‐ onset by switching to air - liquid interface ( ALI ) conditions ( ALI d5 ) .

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A-H X‐Gal‐staining of (A-C) control and (D-H) CcnoTA/+ embryonic lungs at indicated stages showing onset of Ccno expression from E13 in the proximal trachea and in the main bronchi. Expression is extending to more distal regions, and from E16 staining is also found in bronchioli.I X‐Gal‐staining indicating LacZ expression from the CcnoTA/+ allele in mTEC cultures after 5 days of differentiation‐onset by switching to air-liquid interface (ALI) conditions (ALI d5).


J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation . mRNA levels from three independent experiments were measured by qRT - PCR and levels of expression set as 1 on day 0 of ALI cultures . Scales for Ccno and Deup1 are indicated on the left , and for Cep152 , Cep63 and Ift57 on the right side .

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J Transcript levels of Ccno and indicated genes with known functions for the generation of multiple cilia during mTEC differentiation. mRNA levels from three independent experiments were measured by qRT-PCR and levels of expression set as 1 on day 0 of ALI cultures. Scales for Ccno and Deup1 are indicated on the left, and for Cep152, Cep63 and Ift57 on the right side.


K Relative mRNA expression levels for indicated genes were measured by qRT - PCR in wild ‐ type and Ccno ‐ deficient mTEC cultures at indicated days after differentiation ‐ onset . Relative values were calculated as in ( J ) relative to day 0 of ALI cultures .

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K Relative mRNA expression levels for indicated genes were measured by qRT-PCR in wild‐type and Ccno‐deficient mTEC cultures at indicated days after differentiation‐onset. Relative values were calculated as in (J) relative to day 0 of ALI cultures.


A - B ' SEM of tracheae from ( A , A ' ) wild ‐ type and ( B , B ' ) CcnoRA / RA embryos at E17 . In comparison to wild ‐ type MCCs , Ccno ‐ deficient MCCs almost completely lack formation of multiple cilia and only single , or doublet ( arrow ) cilia are observed at E17 .

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A-B' SEM of tracheae from (A, A') wild‐type and (B, B') CcnoRA/RA embryos at E17. In comparison to wild‐type MCCs, Ccno‐deficient MCCs almost completely lack formation of multiple cilia and only single, or doublet (arrow) cilia are observed at E17.


C - E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α ‐ tubulin staining as marker for axonemes in ( D ) Ccno ‐ deficient MCCs of E17 embryonic trachea as quantified in ( E ) . FOV , field of view .

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C-E The lack of multiple cilia at E17 stages is reflected by the almost complete absence of acetylated α‐tubulin staining as marker for axonemes in (D) Ccno‐deficient MCCs of E17 embryonic trachea as quantified in (E). FOV, field of view.


F - I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in ( F , F ' , H ) Ccno ‐ proficient and ( G , G ' , I ) Ccno ‐ deficient MCCs . Asterisks in ( F , G ) indicate microvilli that extend from the apical cell surface . ( F ' , G ' ) Deuterosomes in Ccno ‐ deficient MCCs are significantly enlarged ( identical size bars in F ' and G ' ) and show irregular morphology , which is different to the annular shape of wild ‐ type deuterosomes as seen in ( F ' , H ) . Procentrioles ( arrowheads in F ' and I ) that are found at deuterosomes of ( I ) Ccno ‐ deficient MCCs were recurrently found being shorter and appeared less structured in comparison to ( F ' , H ) wild ‐ type cells .

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F-I TEM of MCCs from E17 embryonic tracheae shows deuterosomes with forming procentrioles in (F, F', H) Ccno‐proficient and (G, G', I) Ccno‐deficient MCCs. Asterisks in (F, G) indicate microvilli that extend from the apical cell surface. (F', G') Deuterosomes in Ccno‐deficient MCCs are significantly enlarged (identical size bars in F' and G') and show irregular morphology, which is different to the annular shape of wild‐type deuterosomes as seen in (F', H). Procentrioles (arrowheads in F' and I) that are found at deuterosomes of (I) Ccno‐deficient MCCs were recurrently found being shorter and appeared less structured in comparison to (F', H) wild‐type cells.


J The average length of procentrioles was quantified for wild ‐ type and Ccno ‐ deficient MCCs of E17 tracheae ( 49 procentrioles in n = 3 independent samples for wild ‐ types and 47 procentrioles in n = 3 independent samples for Ccno ‐ deficient MCCs ) . K The diameter of deuterosomes ( 41 deuterosomes in n = 3 independent samples for wild ‐ types ; 38 deuterosomes in n = 5 independent samples for Ccno ‐ deficient MCCs ) was measured in wild ‐ type and Ccno ‐ deficient MCCs of E17 tracheae .

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J The average length of procentrioles was quantified for wild‐type and Ccno‐deficient MCCs of E17 tracheae (49 procentrioles in n = 3 independent samples for wild‐types and 47 procentrioles in n = 3 independent samples for Ccno‐deficient MCCs).K The diameter of deuterosomes (41 deuterosomes in n = 3 independent samples for wild‐types; 38 deuterosomes in n = 5 independent samples for Ccno‐deficient MCCs) was measured in wild‐type and Ccno‐deficient MCCs of E17 tracheae.


Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification ( ALI day 3 ) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS ‐ 6 . Deuterosome numbers are decreased and deuterosome size enlarged in Ccno ‐ deficient MCCs as quantified in ( C ) . Deuterosome ‐ dependent centriole biogenesis is severely reduced in Ccno ‐ deficient MCCs as shown by reduced staining for the early centriole marker SAS ‐ 6 .

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Double IF analysis of mTEC cultures at early stage of MCC differentiation and centriole amplification (ALI day 3) using antibodies for the deuterosome protein DEUP1 and the early centriole marker SAS‐6. Deuterosome numbers are decreased and deuterosome size enlarged in Ccno‐deficient MCCs as quantified in (C). Deuterosome‐dependent centriole biogenesis is severely reduced in Ccno‐deficient MCCs as shown by reduced staining for the early centriole marker SAS‐6.


IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno ‐ deficient MCCs at later stages of centriole biogenesis ( ALI day 7 ) . Deuterosome number and size were quantified in ( C ) . IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7 , which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno ‐ deficient MCCs as seen in z ‐ projection .

B-EXP_ASSAY O B-GENEPROD O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O O O O

IFstaining for DEUP1 reveals a further size increase of deuterosomes of Ccno‐deficient MCCs at later stages of centriole biogenesis (ALI day 7). Deuterosome number and size were quantified in (C). IF using a Centrin antibody shows globally reduced presence of centrioles at ALI day 7, which stay localized below the level of deuterosomes and fail to dock to the apical cellmembrane in Ccno‐deficient MCCs as seen in z‐projection.


IFstaining for DEUP1 in ( A , B ) was analysed by Imaris 7 . 7 . 2 software to calculate average deuterosome number of wild ‐ type ( n = 17 ) and Ccno ‐ deficient ( n = 19 ) MCCs at ALI day 3 , and wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 . Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3 , and in wild ‐ type ( n = 12 ) and Ccno ‐ deficient ( n = 14 ) MCCs at ALI day 7 .

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IFstaining for DEUP1 in (A, B) was analysed by Imaris 7.7.2 software to calculate average deuterosome number of wild‐type (n = 17) and Ccno‐deficient (n = 19) MCCs at ALI day 3, and wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7. Deuterosome size was measured using the spot measurement tool in n = 10 MCCs for both genotypes at ALI day 3, and in wild‐type (n = 12) and Ccno‐deficient (n = 14) MCCs at ALI day 7.


A , B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164 , which marks maturing centrioles . At ALI day 7 , centrioles of wild ‐ type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane . The fewer centrioles of Ccno ‐ deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z ‐ projection . Lack of CEP164 indicates immature centrioles in Ccno ‐ deficient MCCs . ( B ) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7 . 7 . 2 software .

O O O B-EXP_ASSAY O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O O O O B-CELL_TYPE O O O O O O O O O O O O

A, B IF analysis of mTEC cultures at ALI day 7 using antibodies against the centriole marker Centrin and CEP164, which marks maturing centrioles. At ALI day 7, centrioles of wild‐type MCCs are decorated with CEP164 and are mostly localized to the apical cellmembrane. The fewer centrioles of Ccno‐deficient MCCs do not colocalize with CEP164 and fail to reach the apical cell surface as seen in z‐projection. Lack of CEP164 indicates immature centrioles in Ccno‐deficient MCCs. (B) Total centriole numbers per MCC were counted in n = 11 MCCs for both genotypes using Imaris 7.7.2 software.


C , D Double IFstaining using Centrin ‐ and CP110 ‐ specific antibodies shows an almost complete absence of CP110 from Centrin ‐ positive centrioles in the majority of wild ‐ type MCCs at day 7 of ALI cultures . Most Ccno ‐ deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z ‐ projection . ( D ) CP110 ‐ positive MCCs were counted in wild ‐ type and Ccno ‐ deficient MTEC cultures and the percentages of CP110 ‐ positive MCCs from total MCCs calculated .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-CELL_TYPE O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O

C, D Double IFstaining using Centrin‐ and CP110‐specific antibodies shows an almost complete absence of CP110 from Centrin‐positive centrioles in the majority of wild‐type MCCs at day 7 of ALI cultures. Most Ccno‐deficient MCCs exhibit pronounced CP110 staining that colocalizes with Centrin as seen in z‐projection. (D) CP110‐positive MCCs were counted in wild‐type and Ccno‐deficient MTEC cultures and the percentages of CP110‐positive MCCs from total MCCs calculated.


Luciferase sequestration as an assay for in vitro uptake . ( A ) Luciferase cofractionating with vacuoles is proteinase K protected . In vitro uptake reactions of three times the standard volume were performed ( 60 min , 27 ° C ) in the presence or absence of the ATP - regenerating system . The vacuoles were reisolated , washed , resuspended in 150 mM KCl in PS buffer to a concentration of 0 . 23 mg / ml , and split into aliquots . After addition of 1 % Triton X - 100 or buffer , the mixture was digested with the indicated concentrations of proteinase K ( 15 min , 0 ° C ) . Proteinase treatment was stopped by adding one volume of 0 . 5 mM PMSF in 150 mM KCl in PS buffer . Then , luciferase activity was assayed .

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Luciferase sequestration as an assay for in vitro uptake. (A) Luciferase cofractionating with vacuoles is proteinase K protected. In vitro uptake reactions of three times the standard volume were performed (60 min, 27°C) in the presence or absence of the ATP-regenerating system. The vacuoles were reisolated, washed, resuspended in 150 mM KCl in PS buffer to a concentration of 0.23 mg/ml, and split into aliquots. After addition of 1% Triton X-100 or buffer, the mixture was digested with the indicated concentrations of proteinase K (15 min, 0°C). Proteinase treatment was stopped by adding one volume of 0.5 mM PMSF in 150 mM KCl in PS buffer. Then, luciferase activity was assayed.


( B ) Luciferase can be released from the vacuolar lumen by freeze - thaw treatment . An uptake reaction was performed ( 60 min , 27 ° C ) . The vacuoles were reisolated and washed as described in the Materials and Methods section . The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots . One was frozen at − 80 ° C and thawed again slowly , whereas the other remained on ice . The samples were centrifuged ( 15 min , 145 , 000 g , 4 ° C ) . The supernatants ( Sup ) were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl . Luciferase activity in both fractions was determined . The reaction tubes for this experiment had been coated with BSA ( 0 . 5 mg / ml , 15 min at room temperature ) to reduce unspecific binding of luciferase to the surface .

O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O

(B) Luciferase can be released from the vacuolar lumen by freeze-thaw treatment. An uptake reaction was performed (60 min, 27°C). The vacuoles were reisolated and washed as described in the Materials and Methods section. The final pellet was resuspended in 90 μl PS buffer with 150 mM KCl and split into two aliquots. One was frozen at −80°C and thawed again slowly, whereas the other remained on ice. The samples were centrifuged (15 min, 145,000 g, 4°C). The supernatants (Sup)were recovered and the pellets were resuspended in an equal volume of PS with 150 mM KCl. Luciferase activity in both fractions was determined. The reaction tubes for this experiment had been coated with BSA (0.5 mg/ml, 15 min at room temperature) to reduce unspecific binding of luciferase to the surface.


time course and temperature dependence of uptake . ( A ) Standard uptake reactions were performed in the presence or absence of the ATP - regenerating system . After the indicated times at 27 ° C , the vacuoles were reisolated , washed , treated with protease , and luciferaseuptake activity was assayed .

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time course and temperature dependence of uptake. (A) Standard uptake reactions were performed in the presence or absence of the ATP-regenerating system. After the indicated times at 27°C, the vacuoles were reisolated, washed, treated with protease, and luciferaseuptake activity was assayed.


( B ) A standard uptake reaction was performed . After 70 min of incubation , the vacuoles were reisolated , washed , and treated with protease . The vacuoles were reisolated again , resuspended in reaction buffer without luciferase , and incubated for the indicated times at 27 ° C . Aliquots were withdrawn to assay the total luciferase remaining . As a control , luciferase was incubated in reaction buffer for the same periods of time and assayed for activity .

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(B) A standard uptake reaction was performed. After 70 min of incubation, the vacuoles were reisolated, washed, and treated with protease. The vacuoles were reisolated again, resuspended in reaction buffer without luciferase, and incubated for the indicated times at 27°C. Aliquots were withdrawn to assay the total luciferase remaining. As a control, luciferase was incubated in reaction buffer for the same periods of time and assayed for activity.


Requirements for in vitro uptake . Standard uptake reactions were performed ( 60 min ) ( A ) at different temperatures , or in the presence of different concentrations of ( B ) cytosol , ( C ) ATP , or ( D ) MgCl2 . Luciferase uptake was determined as described in the legend to Fig . 3 A . In B , titration curves for four independent cytosol preparations are shown . The maximal uptake signal of each titration curve was set at 100 % . In D , the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O 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-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O

Requirements for in vitro uptake. Standard uptake reactions were performed (60 min) (A) at different temperatures, or in the presence of different concentrations of (B) cytosol, (C) ATP, or (D) MgCl2. Luciferase uptake was determined as described in the legend to Fig. 3 A. In B, titration curves for four independent cytosol preparations are shown. The maximal uptake signal of each titration curve was set at 100%. In D, the sample drawn as 0 mM MgCl2 contained 10 mM EDTA to chelate free magnesium in the reaction.


In vitro uptake is independent of components involved in homotypic vacuolar fusion . ( A ) Standard uptake reactions were performed ( 60 min , 27 ° C ) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734 - 16 ( Δpep4 ) and DBY5734 - 19 ( Δpho8 ) . Therefore , homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample . Four independent experiments were averaged . Error bars indicate SD . For averaging , uptake and fusion activities of the control sample without inhibitor were set at 100 % . Inhibitors were used at the following concentrations : anti - Sec17p , 1 μM ; anti - Sec18p , 1 μM ; Gdi1p , 2 . 5 μM ; anti - Vam3p , 1 μM ; anti - Vam7p , 1 μM ; and anti - Nyv1p , 1 μM .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O B-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O 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-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O

In vitro uptake is independent of components involved in homotypic vacuolar fusion. (A) Standard uptake reactions were performed (60 min, 27°C) in the presence of the indicated reagents using a mixture of vacuoles from strains DBY5734-16 (Δpep4) and DBY5734-19 (Δpho8). Therefore, homotypic vacuolar fusion and luciferase uptake could be assayed simultaneously from the same sample. Four independent experiments were averaged. Error bars indicate SD. For averaging, uptake and fusion activities of the control sample without inhibitor were set at 100%. Inhibitors were used at the following concentrations: anti-Sec17p, 1 μM; anti-Sec18p, 1 μM; Gdi1p, 2.5 μM; anti-Vam3p, 1 μM; anti-Vam7p, 1 μM; and anti-Nyv1p, 1 μM.


( B ) Vacuoles and cytosols were prepared from sec18 - 1 and from corresponding wild - type ( Wt ) cells grown and spheroplasted at 23 ° C . The isolated vacuoles and the cytosols were exposed to heat treatment ( 10 min , 37 ° C ) . Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations . Control reactions were performed in the absence of ATP . Five independent experiments were averaged ; the sample with vacuoles and cytosol from wild - type cells was used as the 100 % reference . Error bars indicate SD . Mut , mutant .

O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O

(B) Vacuoles and cytosols were prepared from sec18-1 and from corresponding wild-type (Wt) cells grown and spheroplasted at 23°C. The isolated vacuoles and the cytosols were exposed to heat treatment (10 min, 37°C). Standard uptake reactions were performed with vacuoles and cytosols in the indicated combinations. Control reactions were performed in the absence of ATP. Five independent experiments were averaged; the sample with vacuoles and cytosol from wild-type cells was used as the 100% reference. Error bars indicate SD. Mut, mutant.


In vitro uptake depends on GTP hydrolysis and on the V - ATPase . ( A ) Standard uptake reactions were performed in the presence of different concentrations of GTPγS . Luciferase uptake was assayed as in the legend to Fig . 3 A . Four independent experiments were averaged , using the sample without inhibitor as the 100 % reference . Error bars indicate SD . GTPγS did not influence luciferase activity by itself ( not shown ) .

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In vitro uptake depends on GTP hydrolysis and on the V-ATPase. (A) Standard uptake reactions were performed in the presence of different concentrations of GTPγS. Luciferase uptake was assayed as in the legend to Fig. 3 A. Four independent experiments were averaged, using the sample without inhibitor as the 100% reference. Error bars indicate SD. GTPγS did not influence luciferase activity by itself (not shown).


( B ) Standard uptake reactions were performed in the presence of FCCP ( CMA , 20 μM ) or concanamycin A ( 20 μM ) , or in the absence of ATP .

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(B) Standard uptake reactions were performed in the presence of FCCP (CMA, 20 μM) or concanamycin A (20 μM), or in the absence of ATP.


C ) MXE saturation analysis . Whereas increasing amounts of RNA - seq reads should lead to the confirmation of further MXE candidates , more RNA - seq reads might also result in the rejection of previously validated MXEs . The green curves show the number of validated MXEs in relation to the percentage of total RNA - seq reads used for validation . The orange curves indicate the number of initially ' validated MXEs ' that were rejected with increasing amounts of reads . Grey dashed lines indicate the point of saturation , which is defined as the point where a two - fold increase in reads leads to rejection of less than 1 % of the validated MXEs . Of note , whereas the rejection of validated MXEs saturates with 20 % of the data the amount of novel MXE validations is still rapidly increasing .

O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O

C) MXE saturation analysis. Whereas increasing amounts of RNA-seq reads should lead to the confirmation of further MXE candidates, more RNA-seq reads might also result in the rejection of previously validated MXEs. The green curves show the number of validated MXEs in relation to the percentage of total RNA-seq reads used for validation. The orange curves indicate the number of initially 'validated MXEs' that were rejected with increasing amounts of reads. Grey dashed lines indicate the point of saturation, which is defined as the point where a two-fold increase in reads leads to rejection of less than 1% of the validated MXEs. Of note, whereas the rejection of validated MXEs saturates with 20% of the data the amount of novel MXE validations is still rapidly increasing.


E ) Size and distribution of multi - cluster MXEs .

E) Size and distribution of multi-cluster MXEs.


A ) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM . Here , we used the Gini coefficient , which is a measure of the inequality among values of a frequency distribution ( Ceriani & Verme , 2012 ) and has successfully been used to determine tissue - enriched gene sets ( Zhang et al , 2017 ) , to determine highly tissue - specific MXEs ( maximum normalized Gini index of cluster ) and MXEs with a broad tissue expression distribution ( minimum Gini index ) . For each MXE cluster the percent - spliced - in ( PSI ) value of the ubiquitous MXE ( minimum Gini index ) is subtracted from the PSI value of the specific MXE ( maximum Gini index of cluster ) ( delta PSI value ) and scaled between - 1 ( broad tissue distribution ) and 1 ( highly tissue specific ) . Each column represents an MXE pair and each row represents MXE expression in a tissue , cell - type , or at a developmental time point . The bar graph summarizes counts where the specific MXE is 1 . 5 fold more spliced in than the ubiquitous MXE .

O O O O O B-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O 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-GENEPROD O O O O O B-GENEPROD 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-GENEPROD O O O O O O O O O O O B-GENEPROD O

A) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM. Here, we used the Gini coefficient, which is a measure of the inequality among values of a frequency distribution (Ceriani & Verme, 2012) and has successfully been used to determine tissue-enriched gene sets (Zhang et al, 2017), to determine highly tissue-specific MXEs (maximum normalized Gini index of cluster) and MXEs with a broad tissue expression distribution (minimum Gini index). For each MXE cluster the percent-spliced-in (PSI) value of the ubiquitous MXE (minimum Gini index) is subtracted from the PSI value of the specific MXE (maximum Gini index of cluster) (delta PSI value) and scaled between -1 (broad tissue distribution) and 1 (highly tissue specific). Each column represents an MXE pair and each row represents MXE expression in a tissue, cell-type, or at a developmental time point. The bar graph summarizes counts where the specific MXE is 1.5 fold more spliced in than the ubiquitous MXE.


C ) Heatmap showing the the delta PSI values ( PSI value of the non - SNP - containing MXE subtracted from the PSI value of the SNP - containing MXE ) of MXE clusters containing pathogenic SNPs scaled between - 1 and 1 ( blue = high expression non - SNP - containing MXE , red = high expression SNP - containing MXE ) . Columns represent MXE clusters and rows tissues , cell types , and developmental stages . The column bar graph summarizes counts where the SNP - containing MXE is 1 . 5 fold more expressed than the non - SNP - containing MXE , whereas the row bar graph shows this for each tissue , cell type , and developmental stage .

O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O

C) Heatmap showing the the delta PSI values (PSI value of the non-SNP-containing MXE subtracted from the PSI value of the SNP-containing MXE) of MXE clusters containing pathogenic SNPs scaled between -1 and 1 (blue = high expression non-SNP-containing MXE, red = high expression SNP-containing MXE). Columns represent MXE clusters and rows tissues, cell types, and developmental stages. The column bar graph summarizes counts where the SNP-containing MXE is 1.5 fold more expressed than the non-SNP-containing MXE, whereas the row bar graph shows this for each tissue, cell type, and developmental stage.


( A ) Surface plasmon resonance ( SPR ) KAP1 - KRAB binding assay . MBP - KRAB was immobilized on the chip . WT or R311E KAP1 RBCC were flowed over the chip . Data points are shown in dark red or blue ; fits are shown as light red or blue lines .

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(A) Surface plasmon resonance (SPR) KAP1-KRAB binding assay. MBP-KRAB was immobilized on the chip. WT or R311E KAP1 RBCC were flowed over the chip. Data points are shown in dark red or blue; fits are shown as light red or blue lines.


( C ) LINE - 1 reporter repression with single point mutants and a previously described KRAB - binding deficient KAP1 variant containing four mutations in the CC domain ( CCmut ; V293S / K296A / M297A / L300S ) .

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(C) LINE-1 reporter repression with single point mutants and a previously described KRAB-binding deficient KAP1 variant containing four mutations in the CC domain (CCmut; V293S/K296A/M297A/L300S).


( D ) SVA reporter repression with the same set of mutants as in ( B ) . Data were normalized to KAP1 KO cells transfected with an empty vector ( EV ) .

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(D) SVA reporter repression with the same set of mutants as in (B). Data were normalized to KAP1 KO cells transfected with an empty vector (EV).


A - D SQST ‐ 1 : : GFP is very weakly expressed and diffusely localized in the cytoplasm in wild ‐ type larval intestine , but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae . ( A ) and ( C ) represent DIC images of the animals in ( B ) and ( D ) , respectively .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O B-TISSUE O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O

A-D SQST‐1::GFP is very weakly expressed and diffusely localized in the cytoplasm in wild‐type larval intestine, but is expressed at much higher levels and accumulates into numerous aggregates in the intestine in bp399 mutant larvae. (A) and (C) represent DIC images of the animals in (B) and (D), respectively.


E Compared to wild ‐ type , SQST ‐ 1 : : GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay . SQST ‐ 1 : : GFP levels in bp399 mutants are greatly reduced after 12 h of starvation . 200 young adult animals for each genotype were collected for analysis .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

E Compared to wild‐type, SQST‐1::GFP levels are much higher in bp399 and bp412 mutant larvae as shown by immunoblotting assay. SQST‐1::GFP levels in bp399 mutants are greatly reduced after 12 h of starvation. 200 young adult animals for each genotype were collected for analysis.


H , I SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants are separable from LAAT ‐ 1 : : Cherry ‐ labeled lysosomes under normal conditions , but are delivered to lysosomes upon starvation . Some aggregates are enclosed by lysosomes ( inserts ) . Scale bar : 20 μm .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

H, I SQST‐1::GFP aggregates in rpl‐43(bp399) mutants are separable from LAAT‐1::Cherry‐labeled lysosomes under normal conditions, but are delivered to lysosomes upon starvation. Some aggregates are enclosed by lysosomes (inserts). Scale bar: 20 μm.


J - Q RNAi inactivation of let ‐ 363 , rpn ‐ 2 , cogc ‐ 1 , or hgrs ‐ 1 suppresses the accumulation of SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) mutants . ( J ) , ( L ) , ( N ) , and ( P ) : DIC images of the animals in ( K ) , ( M ) , ( O ) , and ( Q ) . Scale bar : 20 μm .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-Q RNAi inactivation of let‐363, rpn‐2, cogc‐1, or hgrs‐1 suppresses the accumulation of SQST‐1::GFP aggregates in rpl‐43(bp399) mutants. (J), (L), (N), and (P): DIC images of the animals in (K), (M), (O), and (Q). Scale bar: 20 μm.


R SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are greatly reduced by inactivation of rpt ‐ 3 and rpn ‐ 2 . 200 young adult animals for each genotype were collected for analysis .

O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

R SQST‐1::GFP levels in rpl‐43(bp399) mutants are greatly reduced by inactivation of rpt‐3 and rpn‐2. 200 young adult animals for each genotype were collected for analysis.


A , B SQST ‐ 1 : : GFP aggregates are absent in the rpl ‐ 43 ( bp399 ) ; sma ‐ 3 ( wk20 ) mutant intestine .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O

A, B SQST‐1::GFP aggregates are absent in the rpl‐43(bp399); sma‐3(wk20) mutant intestine.


C Percentage of the indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : many SQST ‐ 1 : : GFP aggregates in all intestinal cells , as in rpl ‐ 43 ( bp399 ) mutants ; M : fewer SQST ‐ 1 : : GFP aggregates in some but not all intestinal cells ; N : no or very few SQST ‐ 1 : : GFP aggregates in intestinal cells . For each genotype , > 30 animals were examined .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-TISSUE I-TISSUE O O O O O O O O O O O

C Percentage of the indicated animals with different levels of SQST‐1::GFP aggregates. S: many SQST‐1::GFP aggregates in all intestinal cells, as in rpl‐43(bp399) mutants; M: fewer SQST‐1::GFP aggregates in some but not all intestinal cells; N: no or very few SQST‐1::GFP aggregates in intestinal cells. For each genotype, > 30 animals were examined.


D SQST ‐ 1 : : GFP levels in rpl ‐ 43 ( bp399 ) mutants are dramatically decreased in a Western blot when sma ‐ 3 , lin ‐ 35 , or daf ‐ 2 is simultaneously depleted . Loss of function of daf ‐ 16 partially restores SQST ‐ 1 levels in rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutants .

O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

D SQST‐1::GFP levels in rpl‐43(bp399) mutants are dramatically decreased in a Western blot when sma‐3, lin‐35, or daf‐2 is simultaneously depleted. Loss of function of daf‐16 partially restores SQST‐1 levels in rpl‐43(bp399); daf‐2(RNAi) mutants.


E Endogenous LGG ‐ 1 is elevated in a Western blot in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

E Endogenous LGG‐1 is elevated in a Western blot in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants.


F mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in dbl ‐ 1 ( wk70 ) , sma ‐ 2 ( e502 ) , and sma ‐ 3 ( wk20 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O 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 mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in dbl‐1(wk70), sma‐2(e502), and sma‐3(wk20) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


G , H No SQST ‐ 1 : : GFP aggregates are observed in the lin ‐ 35 ( n745 ) ; rpl ‐ 43 ( bp399 ) mutant intestine .

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G, H No SQST‐1::GFP aggregates are observed in the lin‐35(n745); rpl‐43(bp399) mutant intestine.


I , J Numerous SQST ‐ 1 : : GFP aggregates are formed in rpl ‐ 43 ( bp399 ) ; lin ‐ 15A ( n767 ) mutants .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

I, J Numerous SQST‐1::GFP aggregates are formed in rpl‐43(bp399); lin‐15A(n767) mutants.


K Endogenous LGG ‐ 1 levels are increased in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants .

O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

K Endogenous LGG‐1 levels are increased in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants.


L mRNA levels of bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 are upregulated in lin ‐ 9 ( n112 ) , lin ‐ 15B ( n744 ) , and lin ‐ 35 ( n745 ) mutants . * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

L mRNA levels of bec‐1, epg‐8, lgg‐1, and atg‐7 are upregulated in lin‐9(n112), lin‐15B(n744), and lin‐35(n745) mutants. **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


M , N rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) mutant larvae contain no intestinal SQST ‐ 1 : : GFP aggregates .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O

M, N rpl‐43(bp399); daf‐2(RNAi) mutant larvae contain no intestinal SQST‐1::GFP aggregates.


O , P SQST ‐ 1 : : GFP aggregates are partially restored in daf ‐ 16 ( mu86 ) ; rpl ‐ 43 ( bp399 ) ; daf ‐ 2 ( RNAi ) animals .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

O, P SQST‐1::GFP aggregates are partially restored in daf‐16(mu86); rpl‐43(bp399); daf‐2(RNAi) animals.


A , B hsp ‐ 4 : : GFP is very weakly expressed in wild ‐ type animals .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O

A, B hsp‐4::GFP is very weakly expressed in wild‐type animals.


C Percentage of indicated animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined for each genotype .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

C Percentage of indicated animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined for each genotype.


D , E Phsp ‐ 4 : : GFP expression is much higher in rpt ‐ 3 ( RNAi ) animals .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

D, E Phsp‐4::GFP expression is much higher in rpt‐3(RNAi) animals.


F , G Upregulation of Phsp ‐ 4 : : GFP expression in rpt ‐ 3 ( RNAi ) animals is suppressed by the xbp ‐ 1 ( zc12 ) mutation .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

F, G Upregulation of Phsp‐4::GFP expression in rpt‐3(RNAi) animals is suppressed by the xbp‐1(zc12) mutation.


H , I SQST ‐ 1 : : GFP aggregates are absent in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

H, I SQST‐1::GFP aggregates are absent in rpl‐43(bp399); rpt‐3(RNAi) animals.


J , K Loss of function of xbp ‐ 1 restores SQST ‐ 1 : : GFP aggregates in rpl ‐ 43 ( bp399 ) ; rpt ‐ 3 ( RNAi ) animals .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

J, K Loss of function of xbp‐1 restores SQST‐1::GFP aggregates in rpl‐43(bp399); rpt‐3(RNAi) animals.


M The increase in bec ‐ 1 , epg ‐ 8 , lgg ‐ 1 , and atg ‐ 7 mRNA levels in npl ‐ 4 . 1 , rpt ‐ 3 , and pep ‐ 2 RNAi animals is dependent on xbp ‐ 1 . * P 0 . 05 , * * P 0 . 01 . 500 young adult animals were collected for analysis . Error bars indicate s . d . from three experiments .

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

M The increase in bec‐1, epg‐8, lgg‐1, and atg‐7 mRNA levels in npl‐4.1, rpt‐3, and pep‐2 RNAi animals is dependent on xbp‐1. *P 0.05, **P 0.01. 500 young adult animals were collected for analysis. Error bars indicate s.d. from three experiments.


A Phsp ‐ 60 : : GFP is weakly expressed in wild ‐ type animals .

O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O

A Phsp‐60::GFP is weakly expressed in wild‐type animals.


B egl ‐ 46 ( RNAi ) causes upregulation of Phsp ‐ 60 : : GFP .

O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O

B egl‐46(RNAi) causes upregulation of Phsp‐60::GFP.


C Percentage of indicated mutant animals with different levels of SQST ‐ 1 : : GFP aggregates . S : strong . M : medium . N : none . > 30 animals were examined in each group .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

C Percentage of indicated mutant animals with different levels of SQST‐1::GFP aggregates. S: strong. M: medium. N: none. >30 animals were examined in each group.


D Levels of SQST ‐ 1 : : GFP in various genetic mutants . 200 young adult animals for each genotype were collected for analysis .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O

D Levels of SQST‐1::GFP in various genetic mutants. 200 young adult animals for each genotype were collected for analysis.


E , F Loss of function of atfs ‐ 1 suppresses the upregulation of Phsp ‐ 60 : : GFP in egl ‐ 46 ( n1126 ) mutants . ( E ) : DIC image of ( F ) .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O

E, F Loss of function of atfs‐1 suppresses the upregulation of Phsp‐60::GFP in egl‐46(n1126) mutants. (E): DIC image of (F).


G , H The dramatic decrease in the number of SQST ‐ 1 : : GFP aggregates in the intestine in rpl ‐ 43 ( bp399 ) ; egl ‐ 46 ( RNAi ) mutants ( G ) is restored by simultaneous loss of function of atfs ‐ 1 ( gk3094 ) ( H ) .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

G, H The dramatic decrease in the number of SQST‐1::GFP aggregates in the intestine in rpl‐43(bp399); egl‐46(RNAi) mutants (G) is restored by simultaneous loss of function of atfs‐1(gk3094) (H).


I , J GFP : : LGG ‐ 1 forms numerous puncta in the intestine in egl ‐ 46 ( RNAi ) ( I ) , gfi ‐ 1 ( RNAi ) ( J ) animals .

O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

I, J GFP::LGG‐1 forms numerous puncta in the intestine in egl‐46(RNAi) (I), gfi‐1(RNAi) (J) animals.


Representative immunofluorescence images ( left ) of GAPDH ( red ) in HeLa cells untreated ( Utr ) , or treated with IR , etoposide ( ETO ) , adriamycin ( ADR ) , and camptothecin ( CPT ) as indicated . DNA was stained with DAPI ( blue ) . Quantitative analysis ( right ) of nuclear GAPDH . Scale bars , 10 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

O B-EXP_ASSAY O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative immunofluorescence images (left) of GAPDH (red) in HeLa cells untreated (Utr), or treated with IR, etoposide (ETO), adriamycin (ADR), and camptothecin (CPT) as indicated. DNA was stained with DAPI (blue). Quantitative analysis (right) of nuclear GAPDH. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


Immunoblot ( left ) and quantification ( right ) of endogenous GAPDH in subcellular fractions of HeLa cells treated 4 Gy IR and recovered for 4 h . β - Tubulin and Lamin A / C served as indicators for the cytoplasmic and nuclear fractions . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-CELL_LINE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of endogenous GAPDH in subcellular fractions of HeLa cells treated 4 Gy IR and recovered for 4 h. β-Tubulin and Lamin A/C served as indicators for the cytoplasmic and nuclear fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


Immunoblot ( upper ) and quantification ( lower ) of phospho - Src - Y529 in HeLa cells collected at the indicated time points after 4 Gy IR treatment . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (upper) and quantification (lower) of phospho-Src-Y529 in HeLa cells collected at the indicated time points after 4 Gy IR treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


HeLa cells were pretreated with or without 10 μM PP2 for 24 h and exposed to 4 Gy IR . After 4 h of recovery , nuclear and cytoplasmic fractions were separated and subjected to immunoblot with indicated antibodies ( left ) . Quantification of nuclear GAPDH is in the right panel . β - Tubulin and Lamin B1 served as indicators for the cytoplasmic and nuclear fractions . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * * P < 0 .

B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were pretreated with or without 10 μM PP2 for 24 h and exposed to 4 Gy IR. After 4 h of recovery, nuclear and cytoplasmic fractions were separated and subjected to immunoblot with indicated antibodies (left). Quantification of nuclear GAPDH is in the right panel. β-Tubulin and Lamin B1 served as indicators for the cytoplasmic and nuclear fractions. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; ***P<0.001


E - F . Immunoblot of anti - GAPDH immunoprecipitates from HEK293T treated with 4 Gy IR and recovered for 4 h ( E ) , or with 5 μM ADR for 2 h ( F ) .

O O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

E-F. Immunoblot of anti-GAPDH immunoprecipitates from HEK293T treated with 4 Gy IR and recovered for 4 h (E), or with 5 μM ADR for 2 h (F).


Cell viability analysis of HeLa cells stably expressing the sh - NC and sh - GAPDH were treated with the indicated concentration of ADR for 48 h . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell viability analysis of HeLa cells stably expressing the sh-NC and sh-GAPDH were treated with the indicated concentration of ADR for 48 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Left , representative immunofluorescence images of γH2AX ( C , green ) in HeLa cells transfected with scramble siRNA or si - GAPDH after 4 Gy IR exposure and recovered for 4 h . DNA was stained with DAPI ( blue ) . Right quantification of γH2AX foci / cell ( n = 50 ) . Mann - Whitney U - test . Scale bars , 10 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left, representative immunofluorescence images of γH2AX (C, green) in HeLa cells transfected with scramble siRNA or si-GAPDH after 4 Gy IR exposure and recovered for 4 h. DNA was stained with DAPI (blue). Right quantification of γH2AX foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Left , representative immunofluorescence images of 53BP1 ( D , red ) in HeLa cells transfected with scramble siRNA or si - GAPDH after 4 Gy IR exposure and recovered for 4 h . DNA was stained with DAPI ( blue ) . Right , quantification of 53BP1 foci / cell ( n = 50 ) . Mann - Whitney U - test . Scale bars , 10 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

O O O B-EXP_ASSAY O O B-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left, representative immunofluorescence images of 53BP1 (D, red) in HeLa cells transfected with scramble siRNA or si-GAPDH after 4 Gy IR exposure and recovered for 4 h. DNA was stained with DAPI (blue). Right, quantification of 53BP1 foci/cell (n=50). Mann-Whitney U-test. Scale bars, 10 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of γH2AX in HeLa cells pre - transfected with scrambled siRNA or si - GAPDH , recovered at indicated points after 4 Gy IR exposure . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of γH2AX in HeLa cells pre-transfected with scrambled siRNA or si-GAPDH, recovered at indicated points after 4 Gy IR exposure. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of HR proteins in HeLa cells transfected with scrambled siRNA and si - GAPDH . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of HR proteins in HeLa cells transfected with scrambled siRNA and si-GAPDH. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Quantification of RAD51 ( left ) or γH2AX ( right ) foci / cell in HeLa cells pre - transfected with scrambled siRNA or si - GAPDH , recovered at the indicated time points after 4 Gy IR exposure ( n = 50 ) . Mann - Whitney U - test . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of RAD51 (left) or γH2AX (right) foci/cell in HeLa cells pre-transfected with scrambled siRNA or si-GAPDH, recovered at the indicated time points after 4 Gy IR exposure (n=50). Mann-Whitney U-test. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of RAD51 in HeLa cells with or without GAPDH knockdown treated with 50 μg / mL CHX at the indicated time points . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of RAD51 in HeLa cells with or without GAPDH knockdown treated with 50 μg/mL CHX at the indicated time points. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of RAD51 in GAPDH knockdown HeLa cells and control cells treated with or without MG132 ( 10 μM ) for 10 h . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of RAD51 in GAPDH knockdown HeLa cells and control cells treated with or without MG132 (10 μM) for 10 h. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( left ) and quantification ( right ) of RAD51 in HeLa cells at different durations of 50 μg / mL CHX administration with or without TSA ( 10 μM , 12 h ) treatment . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of RAD51 in HeLa cells at different durations of 50 μg/mL CHX administration with or without TSA (10 μM, 12 h) treatment. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot of cell lysates or anti - RAD51 immunoprecipitates from HeLa cells with scramble siRNA or si - GAPDH transfection treated with MG132 ( 10 μM ) for 2 h .

B-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O

Immunoblot of cell lysates or anti-RAD51 immunoprecipitates from HeLa cells with scramble siRNA or si-GAPDH transfection treated with MG132 (10 μM) for 2 h.


Immunoblot ( left ) and quantification ( right ) of Flag - RAD51 in cell lysates from HEK293T cells transfected with indicated Flag - RAD51 mutations . Endogenous RAD51 and Flag - RAD51 are denoted respectively . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (left) and quantification (right) of Flag-RAD51 in cell lysates from HEK293T cells transfected with indicated Flag-RAD51 mutations. Endogenous RAD51 and Flag-RAD51 are denoted respectively. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot of cell lysates or anti - Flag immunoprecipitates from HEK293T cells transfected with indicated Flag - RAD51 mutations and then exposed to 10 μM MG132 . Immunoblot of cell lysates or anti - Flag immunoprecipitates from HEK293T cells transfected with HA - Ubiquitin and indicated Flag - RAD51 mutations and then exposed to 10 μM MG132 .

B-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O B-GENEPROD B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O

Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with indicated Flag-RAD51 mutations and then exposed to 10 μM MG132. Immunoblot of cell lysates or anti-Flag immunoprecipitates from HEK293T cells transfected with HA-Ubiquitin and indicated Flag-RAD51 mutations and then exposed to 10 μM MG132.


HeLa cells were transfected with or without Flag - GAPDH and HA - HDAC1 , then subjected to immunoprecipitation and immunoblots as indicated . Recombinant HDAC1 protein and recombinant GAPDH protein were co - incubated at 4 ℃ for 2 h . In vitro immunoprecipitation was performed using anti - HDAC1 and subjected to immunoblots as indicated .

B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O

HeLa cells were transfected with or without Flag-GAPDH and HA-HDAC1, then subjected to immunoprecipitation and immunoblots as indicated. Recombinant HDAC1 protein and recombinant GAPDH protein were co-incubated at 4℃ for 2 h. In vitro immunoprecipitation was performed using anti-HDAC1 and subjected to immunoblots as indicated.


HeLa cells were treated with or without 4 Gy IR . The indicated cells were lysed for immunoprecipitation with anti - HDAC1 and immunoblotting analysis ( left ) for anti - Maspin ( I ) Quantitative analysis ( right ) of indicated protein levels . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 .

B-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were treated with or without 4 Gy IR. The indicated cells were lysed for immunoprecipitation with anti-HDAC1 and immunoblotting analysis (left) for anti-Maspin (I) Quantitative analysis (right) of indicated protein levels. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001


HeLa cells were treated with or without 4 Gy IR . The indicated cells were lysed for immunoprecipitation with anti - HDAC1 and immunoblotting analysis ( left ) for anti - GAPDH ( J ) . Quantitative analysis ( right ) of indicated protein levels . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 .

B-CELL_LINE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells were treated with or without 4 Gy IR. The indicated cells were lysed for immunoprecipitation with anti-HDAC1 and immunoblotting analysis (left) for anti-GAPDH (J). Quantitative analysis (right) of indicated protein levels. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001


Co - immunoprecipitation was performed to determine the interaction between endogenous GAPDH and Maspin , full - length Flag - HDAC1 , and truncated forms of Flag - HDAC1 in HeLa cells , untreated ( L ) or treated with 4 Gy IR ( M ) . Asterisks indicate nonspecific bands .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O

Co-immunoprecipitation was performed to determine the interaction between endogenous GAPDH and Maspin, full-length Flag-HDAC1, and truncated forms of Flag-HDAC1 in HeLa cells, untreated (L) or treated with 4 Gy IR (M). Asterisks indicate nonspecific bands.


Immunoblot ( upper ) and quantification ( lower ) of RAD51 in HEK293T cells transfected with empty vector , Flag - HDAC1 , Flag - HDAC1 - S3 ( ∆ 306 - 353 ) , and Flag - HDAC1 - H141A . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (upper) and quantification (lower) of RAD51 in HEK293T cells transfected with empty vector, Flag-HDAC1, Flag-HDAC1-S3 (∆306-353), and Flag-HDAC1-H141A. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


HeLa cells were transfected with empty vector , Flag - HDAC1 , and Flag - HDAC1 - H141A , then subjected to immunoprecipitation and immunoblots as indicated .

B-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O

HeLa cells were transfected with empty vector, Flag-HDAC1, and Flag-HDAC1-H141A, then subjected to immunoprecipitation and immunoblots as indicated.


Left , GAPDH knockdown HeLa cells transfected with Flag - HDAC1 were treated with 4 Gy IR , fixed after 4 h , and immunostained with anti - RAD51 ( red ) . DNA was stained with DAPI ( blue ) . The number of RAD51 foci per cell is shown ( right , n = 50 ) . Mann - Whitney U - test . Scale bars , 5 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Left, GAPDH knockdown HeLa cells transfected with Flag-HDAC1 were treated with 4 Gy IR, fixed after 4 h, and immunostained with anti-RAD51 (red). DNA was stained with DAPI (blue). The number of RAD51 foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Immunoblot ( upper ) and quantification ( lower ) of γH2AX in GAPDH knockdown HeLa cells transfected with or without Flag - HDAC1 at the different time points after 4 Gy IR exposure . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Immunoblot (upper) and quantification (lower) of γH2AX in GAPDH knockdown HeLa cells transfected with or without Flag-HDAC1 at the different time points after 4 Gy IR exposure. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


Left , GAPDH knockdown HeLa cells transfected with Flag - HDAC1 were treated with 4 Gy IR , fixed after 4 h , and immunostained with anti - γH2AX ( green ) . DNA was stained with DAPI ( blue ) . The number of γH2AX foci per cell is shown ( right , n = 50 ) . Mann - Whitney U - test . Scale bars , 5 μm . Data information : Data represented as mean ± s . d . of at least three independent experiments . P values are from Student ' s t - tests . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : not

Left, GAPDH knockdown HeLa cells transfected with Flag-HDAC1 were treated with 4 Gy IR, fixed after 4 h, and immunostained with anti- γH2AX (green). DNA was stained with DAPI (blue). The number of γH2AX foci per cell is shown (right, n=50). Mann-Whitney U-test. Scale bars, 5 μm. Data information: Data represented as mean ± s.d. of at least three independent experiments. P values are from Student's t-tests. *P<0.05; **P<0.01; ***P<0.001; ns: not significant


b ) The affinity of the UVRAG - C2 domain for phospholipids was assessed using a protein - lipid overlay assay . The left panel indicates the identity of lipid species on PtdIns strips . Bacterial - purified GST fusion of UVRAG - C2 ( wild type , WT ) , but not GST , binds PtdIns ( 3 ) P , PtdIns ( 4 ) P and PtdIns ( 5 ) P . The K78A / R82A mutant of C2 is defective for phospholipid - binding , whereas the K87A / N88A mutant is partially impaired . Binding of the FYVE and PH domains of Hrs and FAPP1 , respectively , to PtdIns ( 3 ) P and PtdIns ( 4 ) P served as quality controls ( right ) . PE , phosphatidylethanolamine ; PC , phosphatidylcholine ; LPA , lysophosphatidic acid ; LPC , lysophosphatidylcholine ; S1P , sphingosine 1 - phosphate ; PA , phosphatidic acid ; PS , phosphatidylserine .

O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O 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-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O

b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.


( c ) The relative affinity of the GST - fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments ( n = 5 ) . Error bars represent standard deviation ( s . d . ) . A . U . , arbitrary units .

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(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.


( d ) Binding of full - length UVRAG ( untagged ) to lipid blots . The same PtdIns strips ( in b ) were used and immunoblotted with antibodies against IgG ( right ) and UVRAG ( left ) .

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(d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left).


( g ) Pulldown of UVRAG - C2 domain GST - fusion proteins by liposomes bearing the indicated phosphoinositides . GST - Hrs - FYVE and GST - FAPP1 - PH are also shown .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O

(g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.


( a - k ) Confocal analyses of the subcellular co - localization of endogenous UVRAG with ER markers , including endogenous calnexin ( red , a ) , endogenous PDI ( red , b ) , and overexpressed DSred - ER ( red , c ) ; with Golgi markers , including endogenous GM130 and p115 ( red , d , e , cis - Golgi ) , overexpressed GFP - Man II ( green , f , medial - Golgi ) , endogenous TGN46 ( red , g , trans - Golgi ) , and overexpressed PtdIns ( 4 ) P probe , GFP - FAPP1 - PH ( green , h ) ; with endogenous coatomer proteins , including Sec31 ( red , i , COPII - related ) and β ′ - COP ( red , j , COPI - related ) ; and with the endosome marker GFP - p40phox ( PX ) ( green , k ) in HeLa cells . The insets show a high magnification of the selected areas . Scale bars , 10 μm . ( l ) Confocal microscopic quantification of co - localization of UVRAG with the indicated markers ( data are mean ± s . d . , n = 150 cells obtained by gathering data from three independent experiments ) ; * * P0 . 01 ; * * * P0 . 001 .

O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(a-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4)P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.