( C ) Co - labelling of puncta with mC - ATG5 and GFP - ATG12 co - expressed in promastigotes incubated in PBS for 2 h at 26 ° C . ( D ) In nutrient - rich medium , GFP - ATG8 punctum without mC - ATG5 staining is arrowed . ( E ) Incubated in PBS for 30 min at 26 ° C . ( F ) In nutrient - rich medium at 26 ° C . ( G ) Incubated in PBS for 30 min at 26 ° C , the small panels are enlargements of the merged panels . ( H - I ) Promastigotes in nutrient - rich medium . ( J - K ) Promastigotes at late log phase in nutrient - rich conditions . ( L ) Promastigotes at stationary phase in nutrient - rich conditions . Scale bar throughout , 10 µm .

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(C) Co-labelling of puncta with mC-ATG5 and GFP-ATG12 co-expressed in promastigotes incubated in PBS for 2 h at 26°C. (D) In nutrient-rich medium, GFP-ATG8 punctum without mC-ATG5 staining is arrowed. (E) Incubated in PBS for 30 min at 26°C. (F) In nutrient-rich medium at 26°C. (G) Incubated in PBS for 30 min at 26°C, the small panels are enlargements of the merged panels. (H-I) Promastigotes in nutrient-rich medium. (J-K) Promastigotes at late log phase in nutrient-rich conditions. (L) Promastigotes at stationary phase in nutrient-rich conditions. Scale bar throughout, 10 µm.


( A ) Growth curve of Leishmania promastigotes in HOMEM medium at 26 ° C . * , Δatg5 differed significantly from WT ( p < 0 . 05 ) .

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(A) Growth curve of Leishmania promastigotes in HOMEM medium at 26°C. *, Δatg5 differed significantly from WT (p<0.05).


( B ) The occurrence of GFP - ATG8 puncta in promastigotes after incubation in nutrient - deprived ( PBS , ND ) and nutrient - rich ( HOMEM medium , NR ) conditions for 2 h at 26 ° C . Scale bar , 10

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(B) The occurrence of GFP-ATG8 puncta in promastigotes after incubation in nutrient-deprived (PBS, ND) and nutrient-rich (HOMEM medium, NR) conditions for 2 h at 26°C. Scale bar, 10 µm


( C ) Occurrence of GFP - ATG8 puncta in promastigotes when incubated in the conditions detailed in ( B ) . Means ± SD from four independent experiments . * and * * , occurrence of GFP - ATG8 puncta in Δatg5 were significantly different from in WT in nutrient - deprived and nutrient - rich conditions ( p < 0 . 05 ) .

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(C) Occurrence of GFP-ATG8 puncta in promastigotes when incubated in the conditions detailed in (B). Means ± SD from four independent experiments. * and **, occurrence of GFP-ATG8 puncta in Δatg5 were significantly different from in WT in nutrient-deprived and nutrient-rich conditions (p<0.05).


( D ) Western blot analysis of extracts of promastigotes expressing GFP - ATG8 at logarithmic growth under standard conditions and probed with α - GFP antibody . The faster migrating , lipidated band is labelled GFP - ATG8 - II while the un - lipidated band migrating more slowly is labelled GFP - ATG8 - I .

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(D) Western blot analysis of extracts of promastigotes expressing GFP-ATG8 at logarithmic growth under standard conditions and probed with α-GFP antibody. The faster migrating, lipidated band is labelled GFP-ATG8-II while the un-lipidated band migrating more slowly is labelled GFP-ATG8-I.


( A ) Enlarged ( EM ) and swollen ( WM ) mitochondria seen by transmission electron microscopy ( TEM ) in Δatg5 promastigotes under standard growth conditions . WT is shown at bottom right . Scale bar , 500 nm .

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(A) Enlarged (EM) and swollen (WM) mitochondria seen by transmission electron microscopy (TEM) in Δatg5 promastigotes under standard growth conditions. WT is shown at bottom right. Scale bar, 500 nm.


( B ) Fluorescent intensity from MitoTracker Red ( MTR , 0 . 1 µM ) and MitoTracker Green ( MTG , 0 . 2 µM ) in 2 × 106promastigotes after 30 min incubation at 26 ° C . Values shown are the means ± SD from three independent experiments . * and * * , fluorescence was significantly different between WT and Δatg5 ( p < 0 . 05 ) .

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(B) Fluorescent intensity from MitoTracker Red (MTR, 0.1 µM) and MitoTracker Green (MTG, 0.2 µM) in 2×106promastigotes after 30 min incubation at 26°C. Values shown are the means ± SD from three independent experiments. * and **, fluorescence was significantly different between WT and Δatg5 (p<0.05).


( C ) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP - GFP . Scale bar , 10 µm .

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(C) Types of mitochondrial morphology observed by fluorescence microscopy of Δatg5 promastigotes expressing the mitochondrial marker protein MUP-GFP. Scale bar, 10 µm.


( D ) Differential staining of promastigotes with both MTR ( 0 . 1 µM ) and MTG ( 0 . 2 µM ) . Scale bar , 10 µm .

(D) Differential staining of promastigotes with both MTR (0.1 µM) and MTG (0.2 µM). Scale bar, 10 µm.


( E ) Viability , as measured by Alamar Blue reduction , of promastigotes . All data are means ± SD from three independent experiments . * , Alamar blue reduction was significantly different ( p < 0 . 05 ) .

(E) Viability, as measured by Alamar Blue reduction, of promastigotes. All data are means ± SD from three independent experiments. *, Alamar blue reduction was significantly different (p<0.05).


( F ) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2 × 106 / ml with H2DCFDA at 0 . 1 mM for 2 h at 26 ° C . Values shown are the means ± SD from three independent experiments . * , DCF fluorescence was significantly different ( p < 0 . 05 ) .

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(F) Spectrometric analyses of the DCF fluorescence intensity resulting from incubating promastigotesat 2×106/ml with H2DCFDA at 0.1 mM for 2 h at 26°C. Values shown are the means ± SD from three independent experiments. *, DCF fluorescence was significantly different (p<0.05).


Phospholipid accumulation in Δatg5 promastigotes . Negative ion survey scans ( 650 - 900 m / z ) of WT ( A ) and Δatg5 ( B ) promastigotes extracted for lipids and analysed by ES - MS , as described in Materials and Methods . a = ( alkylacyl ) .

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Phospholipid accumulation in Δatg5 promastigotes.Negative ion survey scans (650-900 m/z) of WT (A) and Δatg5 (B) promastigotes extracted for lipids and analysed by ES-MS, as described in Materials and Methods. a = (alkylacyl).


( A ) Proportion of metacyclic promastigotes in stationary phase cultures , assessed using the PNA assay . Values shown are the means ± SD from three independent experiments . * , differed significantly ( p < 0 . 05 ) .

(A) Proportion of metacyclic promastigotes in stationary phase cultures, assessed using the PNA assay. Values shown are the means ± SD from three independent experiments. *, differed significantly (p<0.05).


( B ) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α - HASPB . α - Cysteine Synthase was used as a loading control [ 56 ] .

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(B) Western blot analysis of extracts of 107promastigotes at stationary phase of growth probed with α-HASPB. α-Cysteine Synthase was used as a loading control [56].


( C ) Infectivity and survival of promastigotes in peritoneal macrophages in vitro , infected at a ratio 5 ∶ 1 , with the infection rates being assessed after 1 and 5 days . * , differed significantly ( p < 0 . 01 ) .

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(C) Infectivity and survival of promastigotes in peritoneal macrophages in vitro, infected at a ratio 5∶1, with the infection rates being assessed after 1 and 5 days. *, differed significantly (p<0.01).


( D ) Lesion progression in BALB / c mice inoculated with 5 × 105 stationary phase promastigotes . Values shown are the means ± SD from 5 mice . * , infection level of Δatg5 differed significantly from WT ( p < 0 . 01 ) and Δatg5 : : ATG5 ( p < 0 . 05 ) .

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(D) Lesion progression in BALB/c mice inoculated with 5×105 stationary phase promastigotes. Values shown are the means ± SD from 5 mice. *, infection level of Δatg5 differed significantly from WT (p<0.01) and Δatg5::ATG5 (p<0.05).


( E ) Morphologies of cells isolated from mouse lesions and analysed by SEM . Scale bar : 2 µm .

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(E) Morphologies of cells isolated from mouse lesions and analysed by SEM. Scale bar: 2 µm.


A ) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient - rich medium . Shown are ovoid and amastigote - like form ( left ) ; spindled - shaped form without an external flagellum ( centre left panel ) and with an external flagellum of varying lengths ( centre right and right panels ) . Scale bar , 10 µm .

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A) SEM analysis of promastigote culture initiated with Δatg5 isolated from a mouse lesion and cultured in nutrient-rich medium. Shown are ovoid and amastigote-like form (left); spindled-shaped form without an external flagellum (centre left panel) and with an external flagellum of varying lengths (centre right and right panels). Scale bar, 10 µm.


( B ) Distribution of flagella lengths and body lengths of stationary phase promastigotes . Data represent measurements from ∼ 200 cells from each promastigote population .

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(B) Distribution of flagella lengths and body lengths of stationary phase promastigotes. Data represent measurements from ∼200 cells from each promastigote population.


( a ) Identification of biomarkers modulated upon VCP silencing . HeLa , HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h . Protein cell extracts were resolved by SDS - PAGE , and filters were probed with the indicated antibodies . The extent of biomarker modulation correlated with the efficiency of silencing ( oligo 4 versus pool ) . VCP silencing determined stabilization of cyclin E ; induction of the endoplasmic reticulum chaperone GRP78 , CHOP transcription factor and its downstream target GADD - 34 ; conversion of the autophagy regulator ATG8 / light chain 3 ( LC3B ) from the free to the lipidated form ; activation of the effector caspase - 3 ; and cleavage of the caspase target PARP - 1 . A graphical representation of the cell cycle distribution ( percentage of cells ) obtained from the cell cycle analysis ( Supplementary Fig . 3b ) is reported at the bottom of each sample . C , control ; NT , nontargeting oligo ; P , oligonucleotide pool .

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(a) Identification of biomarkers modulated upon VCP silencing. HeLa, HCT116 and U2OS cells were treated with the indicated siRNA oligonucleotides for 72 h. Protein cell extracts were resolved by SDS-PAGE, and filters were probed with the indicated antibodies. The extent of biomarker modulation correlated with the efficiency of silencing (oligo 4 versus pool). VCP silencing determined stabilization of cyclin E; induction of the endoplasmic reticulum chaperone GRP78, CHOP transcription factor and its downstream target GADD-34; conversion of the autophagy regulator ATG8/light chain 3 (LC3B) from the free to the lipidated form; activation of the effector caspase-3; and cleavage of the caspase target PARP-1. A graphical representation of the cell cycle distribution (percentage of cells) obtained from the cell cycle analysis (Supplementary Fig. 3b) is reported at the bottom of each sample. C, control; NT, nontargeting oligo; P, oligonucleotide pool.


( b ) Effect of VCP silencing on cell growth . HeLa , HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter ( data represent mean values of two experiments ) .

(b) Effect of VCP silencing on cell growth. HeLa, HCT116 and U2OS cells were treated for 72 h with different VCP siRNA oligonucleotides and cell number was determined using a Coulter Counter (data represent mean values of two experiments).


( c ) Combination of VCP siRNA with standard - of - care therapeutics . HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose - response analysis with a variety of chemotherapeutic agents .

(c) Combination of VCP siRNA with standard-of-care therapeutics. HCT116 cells were treated for 24 h with siRNA to VCP and then were subjected to dose-response analysis with a variety of chemotherapeutic agents.


( b ) LC / MS analysis of VCP after 1 h incubation at 20 ° C with 1 % DMSO ( top ) or 100 μM NMS - 859 ( bottom ) , showing a 313 - Da increase corresponding to the addition of NMS - 859 lacking the Cl atom .

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(b) LC/MS analysis of VCP after 1 h incubation at 20 °C with 1% DMSO (top) or 100 μM NMS-859 (bottom), showing a 313-Da increase corresponding to the addition of NMS-859 lacking the Cl atom.


( a ) Activity of VCP inhibitors NMS - 249 and NMS - 873 on wild - type VCP and mutants . Data represent mean values of at least three independent experiments .

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(a) Activity of VCP inhibitors NMS-249 and NMS-873 on wild-type VCP and mutants. Data represent mean values of at least three independent experiments.


( b ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP in the presence of different allosteric inhibitors or ADP .

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(b) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP in the presence of different allosteric inhibitors or ADP.


( c ) Binding of the EDA - ADP - ATTO 590 probe to wild - type ( WT ) VCP and the K615V and N616F mutants in the presence of NMS - 249 . Minor differences in the mP values are due to the use of different enzyme batches .

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(c) Binding of the EDA-ADP-ATTO 590 probe to wild-type (WT) VCP and the K615V and N616F mutants in the presence of NMS-249. Minor differences in the mP values are due to the use of different enzyme batches.


( d ) Binding of the EDA - ADP - ATTO 590 probe to wild - type VCP and Walker A mutants in the presence of NMS - 249 . Data shown are representative results of one of three independent experiments : each determination was done in triplicate , data points denote the average value , and error bars represent s . d .

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(d) Binding of the EDA-ADP-ATTO 590 probe to wild-type VCP and Walker A mutants in the presence of NMS-249. Data shown are representative results of one of three independent experiments: each determination was done in triplicate, data points denote the average value, and error bars represent s.d.


( a ) Western blot analysis of biomarker modulation in the HCT116 cell line . HCT116 cells were treated with the indicated doses of inhibitors for 8 h ( top ) or 24 h ( bottom ) , and protein cell extracts were resolved on SDS - PAGE gel . Filters were probed with the indicated antibodies . C , control ; B , bortezomib ( 0 . 01 μM ) ; T , thapsigargin ( 0 . 1 μM ) ; Mg , MG132 ( 0 . 1 μM ) .

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(a) Western blot analysis of biomarker modulation in the HCT116 cell line. HCT116 cells were treated with the indicated doses of inhibitors for 8 h (top) or 24 h (bottom), and protein cell extracts were resolved on SDS-PAGE gel. Filters were probed with the indicated antibodies. C, control; B, bortezomib (0.01 μM); T, thapsigargin (0.1 μM); Mg, MG132 (0.1 μM).


( b ) NMS - 873 compound - mediated stabilization of Mcl - 1 in live cells . Top , confocal time - lapse images of live stable , inducible T - REx - 293 cells expressing a bicistronic construct of mCherry - BimS ( 2A ) ΔC ( red ) with eGFP - Mcl - 1 ( green ) after doxycycline treatment . Cells were treated with cycloheximide ( CHX , 50 μg ml − 1 ) ± 2 . 5 μM of NMS - 873 for 6 h . Bottom , total intensity of eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC was measured for time - lapse series shown at top . A decrease in eGFP - Mcl - 1 and mCherry - BimS ( 2A ) ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control . WT , wild type . Scale bar , 10 μm .

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(b) NMS-873 compound-mediated stabilization of Mcl-1 in live cells. Top, confocal time-lapse images of live stable, inducible T-REx-293 cells expressing a bicistronic construct of mCherry-BimS(2A)ΔC (red) with eGFP-Mcl-1 (green) after doxycycline treatment. Cells were treated with cycloheximide (CHX, 50 μg ml−1) ± 2.5 μM of NMS-873 for 6 h. Bottom, total intensity of eGFP-Mcl-1 and mCherry-BimS(2A)ΔC was measured for time-lapse series shown at top. A decrease in eGFP-Mcl-1 and mCherry-BimS(2A)ΔC total intensity was calculated for two different concentrations of indicated compounds and normalized to DMSO control. WT, wild type. Scale bar, 10 μm.


( c ) High - content analysis of poly - Ub and CHOP induction upon treatment with VCP inhibitors . HeLa cells were treated with increasing doses of the indicated compounds . Cells were then fixed and immunostained with the indicated antibodies . Cell numbers and mean fluorescence intensity ( MFI ) were determined using the ArrayScan platform . Data were collected in triplicate ( n = 3 ) , and error bars represent s . d . AU , arbitrary units .

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(c) High-content analysis of poly-Ub and CHOP induction upon treatment with VCP inhibitors. HeLa cells were treated with increasing doses of the indicated compounds. Cells were then fixed and immunostained with the indicated antibodies. Cell numbers and mean fluorescence intensity (MFI) were determined using the ArrayScan platform. Data were collected in triplicate (n = 3), and error bars represent s.d. AU, arbitrary units.


B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in ( A ) . 1cs , 1 - cell stage ; 2cs , 2 - cell stage ; st6 , 32 - cell stage ; st8 . 5 , blastula stage . Blots are representative of 3 independent experiments . C Densitometry analysis of Hwa bands from the western blot in ( B ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , * p < 0 . 05 , * * p < 0 . 01 by unpaired t test .

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B Western blot analysis for Hwa performed with lysates from embryos treated as indicated in (A). 1cs, 1-cell stage; 2cs, 2-cell stage; st6, 32-cell stage; st8.5, blastula stage. Blots are representative of 3 independent experiments. C Densitometry analysis of Hwa bands from the western blot in (B) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, *p < 0.05, **p < 0.01 by unpaired t test.


D Western blot analysis of exogenous Hwa in HEK293T cells . Treatment of cells with lysosome inhibitors bafilomycin A1 ( Baf - A1 ) and NH4Cl for 24 hours , DMSO was used as control . Dosage of Baf - A1 : 10 nM , 50 nM , 100 nM ; Dosage of NH4Cl : 2 . 5 mM , 5 mM , 10 mM . Hwa with modification is indicated by white arrow ; Degraded Hwa is indicated by black arrow . Blots are representative of 3 independent experiments . E Densitometry analysis of Hwa bands from the western blot in ( D ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , * * p < 0 . 01 by unpaired t test .

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D Western blot analysis of exogenous Hwa in HEK293T cells. Treatment of cells with lysosome inhibitors bafilomycin A1 (Baf-A1) and NH4Cl for 24 hours, DMSO was used as control. Dosage of Baf-A1: 10 nM, 50 nM, 100 nM; Dosage of NH4Cl: 2.5 mM, 5 mM, 10 mM. Hwa with modification is indicated by white arrow; Degraded Hwa is indicated by black arrow. Blots are representative of 3 independent experiments. E Densitometry analysis of Hwa bands from the western blot in (D) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, **p < 0.01 by unpaired t test.


C Co - IP with HEK293T cells co - transfected with plasmids encoding Hwa - Flag and ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . Blots are representative of 3 independent experiments . D Co - IP with HEK293T cells co - transfected with plasmids encoding Hwa - Flag and ZNRF3 - HA or ZNRF3ΔRING . Lysates immunoprecipitated with anti - HA were immunoblotted with Flag and HA antibodies . Blots are representative of 3 independent experiments .

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C Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Blots are representative of 3 independent experiments. D Co-IP with HEK293T cells co-transfected with plasmids encoding Hwa-Flag and ZNRF3-HA or ZNRF3ΔRING. Lysates immunoprecipitated with anti-HA were immunoblotted with Flag and HA antibodies. Blots are representative of 3 independent experiments.


E Colocalization of Hwa - Flag ( red ) and ZNRF3 - HA ( green ) or ZNRF3ΔRING - HA ( green ) in Hela cells is indicated by yellow dots . Baf - A1 , 50 nM , 12 hrs . Magnification : 100 × ; scale bar : 10 μm .

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E Colocalization of Hwa-Flag (red) and ZNRF3-HA (green) or ZNRF3ΔRING-HA (green) in Hela cells is indicated by yellow dots. Baf-A1, 50 nM, 12 hrs. Magnification: 100 ×; scale bar: 10 μm.


B Whole mount in situ hybridization of Spemann organizer markers chrd , gsc , and xnr3 in gastrula embryos ( stage 10 . 5 ) . Oocytes were injected with AS or mRNAs as indicated , Dosage : znrf3 AS ( 3 ng AS4 + 3 ng AS9 ) , znrf3 mRNA 100 pg , znrf3ΔRING mRNA 50 pg . embryos were obtained through Host transfer technology . Scale bars : 1 mm .

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B Whole mount in situ hybridization of Spemann organizer markers chrd, gsc, and xnr3 in gastrula embryos (stage 10.5). Oocytes were injected with AS or mRNAs as indicated, Dosage: znrf3 AS (3 ng AS4 + 3 ng AS9), znrf3 mRNA 100 pg, znrf3ΔRING mRNA 50 pg. embryos were obtained through Host transfer technology. Scale bars: 1 mm.


E Relative levels of β - catenin ( left ) and Axin ( right ) in ( D ) normalized to GAPDH . n = 3 independent experiments , mean ± SEM , * p < 0 . 05 , * * * p < 0 . 005 , * * * * p < 0 . 0001 by unpaired t test .

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E Relative levels of β-catenin (left) and Axin (right) in (D) normalized to GAPDH. n = 3 independent experiments, mean ± SEM, *p < 0.05, ***p < 0.005, ****p < 0.0001 by unpaired t test.


H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos ( stage 10 . 5 ) . Embryos were injected and obtained as descripted in ( G ) . Scale bars : 1 mm .

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H Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos (stage 10.5). Embryos were injected and obtained as descripted in (G). Scale bars: 1 mm.


I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl ( gfp mRNA ) , znrf3FL , and znrf3 truncations as indicated at 4 - cell stage . Duplicated axes ( red arrowhead ) . znrf3FL , full - length znrf3 ; znrf3ΔRING , znrf3 lacking the RING domain ; znrf31 - 320 , amino acids 1 - 320 of znrf3 ; znrf3320 - 606 , amino acids 320 - 606 of znrf3 ; znrf3606 - 854 , amino acids 606 - 854 of znrf3 . Scale bars : 1 mm .

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I Representative phenotypes of tailbud stage Xenopus leavis embryos injected with 500 pg mRNAs of ctrl (gfp mRNA), znrf3FL, and znrf3 truncations as indicated at 4-cell stage. Duplicated axes (red arrowhead). znrf3FL, full-length znrf3; znrf3ΔRING, znrf3 lacking the RING domain; znrf31-320, amino acids 1-320 of znrf3; znrf3320-606, amino acids 320-606 of znrf3; znrf3606-854, amino acids 606-854 of znrf3. Scale bars: 1 mm.


B Hwa ubiquitination was analyzed under denaturing conditions . HEK293T cells co - transfected with wildtype His - HA - Ubiquitin ( His - HA - Ub WT ) or lysine - less mutant ( K0 ) , Hwa - Flag , and ZNRF3 - Myc . The lysates were immunoblotted with Myc , Flag , and α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . K0 , all lysine sites on ubiquitin were mutated . Blots are representative of 3 independent experiments .

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B Hwa ubiquitination was analyzed under denaturing conditions. HEK293T cells co-transfected with wildtype His-HA-Ubiquitin (His-HA-Ub WT) or lysine-less mutant (K0), Hwa-Flag, and ZNRF3-Myc. The lysates were immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. K0, all lysine sites on ubiquitin were mutated. Blots are representative of 3 independent experiments.


D Screen of ZNRF3 - mediated Hwa ubiquitination sites under denaturing conditions . HEK293T cells were co - transfected with His - HA - Ub , Hwa - Flag or Hwa10KR - Flag , and ZNRF3 or ZNRF3ΔRING . The lysate was immunoblotted with Myc , Flag , and α - tub ( α - tubulin ) . Lysates immunoprecipitated with anti - Flag were immunoblotted with HA and Flag antibodies . Hwa10KR - Flag mutant , all lysine sites in Hwa sequence were replaced with arginine . Denaturing conditions : before immunoprecipitation , lysates were denatured by adding SDS to a final concentration at 1 % and boiling at 95 ℃ for 15 min . Blots are representative of 3 independent experiments .

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

D Screen of ZNRF3-mediated Hwa ubiquitination sites under denaturing conditions. HEK293T cells were co-transfected with His-HA-Ub, Hwa-Flag or Hwa10KR-Flag, and ZNRF3 or ZNRF3ΔRING. The lysate was immunoblotted with Myc, Flag, and α-tub (α-tubulin). Lysates immunoprecipitated with anti-Flag were immunoblotted with HA and Flag antibodies. Hwa10KR-Flag mutant, all lysine sites in Hwa sequence were replaced with arginine. Denaturing conditions: before immunoprecipitation, lysates were denatured by adding SDS to a final concentration at 1% and boiling at 95℃ for 15 min. Blots are representative of 3 independent experiments.


A Western blot analysis of Hwa in HEK293T cells co - transfected with Hwa - Flag , and ZNRF3 - HA ( 5 μg ) or ZNRF3ΔRING ( 2 . 5 μg ) . Lysate was immunoblotted with Flag , HA , and GAPDH antibodies . Blots are representative of 3 independent experiments . B Densitometry analysis of Hwa bands from the western blot in ( A ) for corresponding lanes normalized to GAPDH . n = 3 independent experiments , mean ± SEM , ns p > 0 . 05 , * * p < 0 . 01 by unpaired t test .

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A Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag, and ZNRF3-HA (5 μg) or ZNRF3ΔRING (2.5 μg). Lysate was immunoblotted with Flag, HA, and GAPDH antibodies. Blots are representative of 3 independent experiments. B Densitometry analysis of Hwa bands from the western blot in (A) for corresponding lanes normalized to GAPDH. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01 by unpaired t test.


C Western blot analysis of Hwa in HEK293T cells co - transfected with Hwa - Flag and ZNRF3 - HA . Cells were treated with or without Baf - A1 ( 50 nM ) or MG132 ( 20 μM ) for about 12 hours . Lysates were immunoblotted with Flag , HA , and α - tub ( α - tubulin ) antibodies . Blots are representative of 3 independent experiments . D Densitometry analysis of Hwa bands from the western blot in ( C ) for corresponding lanes normalized to α - tubulin . n = 3 independent experiments , mean ± SEM , ns p > 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 by unpaired t test .

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C Western blot analysis of Hwa in HEK293T cells co-transfected with Hwa-Flag and ZNRF3-HA. Cells were treated with or without Baf-A1 (50 nM) or MG132 (20 μM) for about 12 hours. Lysates were immunoblotted with Flag, HA, and α-tub (α-tubulin) antibodies. Blots are representative of 3 independent experiments. D Densitometry analysis of Hwa bands from the western blot in (C) for corresponding lanes normalized to α-tubulin. n = 3 independent experiments, mean ± SEM, ns p > 0.05, **p < 0.01, ****p < 0.0001 by unpaired t test.


F Colocalization by immunofluorescence of Hwa ( green signals ) with lysosome ( LAMP1 , red signals ) in Hela cells . Cells were co - transfected with Hwa - Flag , and ZNRF3 - HA or ZNRF3ΔRING - HA . Magnification , 100 × ; scale bars : 10 μm .

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F Colocalization by immunofluorescence of Hwa (green signals) with lysosome (LAMP1, red signals) in Hela cells. Cells were co-transfected with Hwa-Flag, and ZNRF3-HA or ZNRF3ΔRING-HA. Magnification, 100 ×; scale bars: 10 μm.


I Surface biotinylation and streptavidin pulldown of Hwa - Myc - Flag . HEK293T cells were co - transfected with Hwa - MF ( Hwa - Myc - Flag ) , and ZNRF3 - Myc or ZNRF3ΔRING - Myc . Lysate was immunoblotted with Myc , Flag , and GAPDH antibodies . Lysates immunoprecipitated with anti - Flag ( 2nd elution ) were immunoblotted with Flag and HA antibodies .

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I Surface biotinylation and streptavidin pulldown of Hwa-Myc-Flag. HEK293T cells were co-transfected with Hwa-MF (Hwa-Myc-Flag), and ZNRF3-Myc or ZNRF3ΔRING-Myc. Lysate was immunoblotted with Myc, Flag, and GAPDH antibodies. Lysates immunoprecipitated with anti-Flag (2nd elution) were immunoblotted with Flag and HA antibodies.


C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos . Embryos were obtained as descripted in ( B ) . Scale bars : 1mm .

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C Whole mount in situ hybridization of Spemann organizer markers chrd and gsc in gastrula embryos. Embryos were obtained as descripted in (B). Scale bars: 1mm.


E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage . 1 - cell stage embryos were co - injected with hwa ( 100 pg ) or hwa10KR ( 100 pg ) and znrf3 ( 1 ng ) or znrf3ΔRING ( 100 pg ) as indicated . Lysates were immunoblotted with HA , Flag , and α - tub ( α - tubulin ) .

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E Western blot analysis of Hwa or Hwa10KR in embryos at blastula stage. 1-cell stage embryos were co-injected with hwa (100 pg) or hwa10KR (100 pg) and znrf3 (1 ng) or znrf3ΔRING (100 pg) as indicated. Lysates were immunoblotted with HA, Flag, and α-tub (α-tubulin).


( A ) GFP - LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP ( + dbcAMP , panels d , e , and f ) for 30 min and then were treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) in the continuous presence of dbcAMP . Cells without any treatment were used as control ( − dbcAMP , panels a , b , and c ) . Cells were immediately analyzed by confocal microscopy . A quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions is shown in the right panel ( n = 100 cells / condition , * p < 0 . 05 paired Student ' s t - test ) . These data are representative of three independent experiments .

O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments.


( B ) GFP - LC3 CHO cells were incubated with complete medium in the absence ( − dbcAMP ) or presence of dbcAMP ( + dbcAMP ) and treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) with ( lower panel ) or without ( upper panel ) bafilomycin A1 to block lysosomal degradation . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of three independent experiments . ( C ) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program , and normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - test ) .

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

(B) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


( A ) GFP - LC3 CHO cells were preincubated for 30 min with 10 µM H89 , a PKA inhibitor , in the presence ( panels a , b , and c ) or absence ( panels d , e , and f ) of 1 mM dbcAMP . Subsequently , they were incubated for 2 h in starvation medium ( Stv , panels b and e ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla , panels c and f ) in full nutrient media . Cells without any treatment were used as control ( Ctr , panels a and d ) . Cells were finally analyzed by confocal microscopy . Images are representative of three independent experiments .

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(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM H89, a PKA inhibitor, in the presence (panels a, b, and c) or absence (panels d, e, and f) of 1 mM dbcAMP. Subsequently, they were incubated for 2 h in starvation medium (Stv, panels b and e) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels c and f) in full nutrient media. Cells without any treatment were used as control (Ctr, panels a and d). Cells were finally analyzed by confocal microscopy. Images are representative of three independent experiments.


( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions . * * p < 0 . 01 ( paired Student ' s t - test , n = 100 cells / condition ) . These data are representative of two independent experiments .

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(B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. ** p<0.01 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.


( C ) GFP - LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89 , and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89 . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - phosphoCREB and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . The band intensities were quantified with the Adobe Photoshop program ( lower panel ) . These data are representative of two independent experiments .

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 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

(C) GFP-LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89, and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-phosphoCREB and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantified with the Adobe Photoshop program (lower panel). These data are representative of two independent experiments.


( A ) GFP - LC3 CHO cells were preincubated with 10 µM 8 - pCPT - 2 ′ - O - Me - cAMP ( 8 - pCPT ) for 30 min and then they were treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) or subjected to starvation conditions ( Stv ) . Cells without any treatment were used as control . Cells were immediately analyzed by confocal microscopy . Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel ( n = 50 cells / condition ) . These data are representative of two independent experiments .

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(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments.


( B ) CHO cells were cotransfected with GFP - LC3 and myc - EPAC wt ( left panel ) or myc - ΔEPAC ( right panel ) . Twenty - four hours after transfection they were incubated for 2 h in starvation medium ( Stv ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( C ) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions ( n = 20 cells / condition ) . These data are representative of two independent

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(B) CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (left panel) or myc-ΔEPAC (right panel). Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (C) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions (n = 20 cells/condition). These data are representative of two independent experiments


( A ) CHO cells were cotransfected with RFP - LC3 and GFP - Rap2b wt ( left panel ) or GFP - Rap2b ΔAAX ( right panel ) . Twenty - four hours after transfection , they were incubated for 2 h in starvation medium ( Stv ) , with 50 ng / µl of rapamycin ( Rapa ) in full nutrient media , or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of three independent experiments .

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(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments.


( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions is shown . * p < 0 . 05 , * * p < 0 . 01 ( paired Student ' s t - test , n = 50 cells / condition ) . These data are representative of three independent experiments .

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(B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.


( C ) CHO cells were transfected with GFP - Rap2b wt or GFP - Rap2b ΔAAX and incubated with complete medium without ( Ctr ) or with 10 µg / ml of α - hemolysin ( Hla ) for 4 h , with 50 ng / µl of rapamycin ( Rapa ) or subjected to starvation conditions ( Stv ) for 2 h in the absence or presence of bafilomycin A1 . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of two independent experiments .

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

(C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.


( D ) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program .

(D) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program.


( A ) HeLa cells were cotransfected with Rap2b siRNA and GFP - LC3 ( right panel ) or irrelevant siRNA and GFP - LC3 ( left panel ) . Forty - eight hours after transfection , they were incubated for 2 h in starvation medium ( Stv ) , with 50 ng / µl of rapamycin ( Rapa ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments .

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

(A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments.


( B ) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions . Data correspond to two independent experiments ( n = 50 cells / condition ) .

O O O O O O O O O O O O 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) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions. Data correspond to two independent experiments (n = 50 cells/condition).


( C ) Upper panel : The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods . Lower panel : The band intensities of two independent experiments were quantified with the Adobe Photoshop program , and normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - test ) .

(C) Upper panel: The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods. Lower panel: The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


( D ) HeLa cells were cotransfected with GFP - LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence ( Ctr ) or presence of 10 µg / ml of α - hemolysin ( Hla ) , with 50 ng / µl of rapamycin ( Rapa ) or subjected to starvation conditions ( Stv ) for 2 h . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody , and subsequently developed with an enhanced chemiluminescence detection kit . These data are representative of two independent experiments .

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

(D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.


( E ) The band intensities of two independent experiments were quantified with the Adobe Photoshop program , and normalized against tubulin . * p < 0 . 05 ( paired Student ' s t - test ) .

O O O O O O O O O O O O O O O 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) The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).


cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant . ( A ) CHO cells were cotransfected with RFP - LC3 and GFP - Rap2b ΔAAX . Twenty - four hours after transfection they were incubated for 2 h in starvation medium ( Stv ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla ) in the presence ( right panels ) or absence of 1 mM dbcAMP ( left panels ) . Cells without any treatment were used as control ( Ctr ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions . These data are representative of two independent experiments .

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

cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.


( A ) GFP - LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin ( a calpain inhibitor , panels b , f , and j ) , with 1 mM dbcAMP ( panels d , h and l ) , or with Calpeptin + dbcAMP ( panels c , g , and k ) . Afterwards , they were incubated for 2 h in starvation medium ( Stv , panels e , f , g , and h ) or treated for 4 h with 10 µg / ml of α - hemolysin ( Hla , panels i , j , k , and l ) . Cells without any treatment were used as control ( Ctr , panels a , b , c and d ) . Cells were analyzed by confocal microscopy . Images are representative of two independent experiments . ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation with the different conditions . * p < 0 . 05 ( paired Student ' s t - test , n = 100 cells / condition ) . These data are representative of two independent experiments .

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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin (a calpain inhibitor, panels b, f, and j), with 1 mM dbcAMP (panels d, h and l), or with Calpeptin+dbcAMP (panels c, g, and k). Afterwards, they were incubated for 2 h in starvation medium (Stv, panels e, f, g, and h) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels i, j, k, and l). Cells without any treatment were used as control (Ctr, panels a, b, c and d). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. * p<0.05 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.


( C ) Upper panel : CHO cells were preincubated for 30 min with 10 µM of calpeptin , with 1 mM dbcAMP , or with calpeptin + dbcAMP , and then they were incubated for 4 h in complete medium in the presence ( Hla ) or absence ( Ctr ) of α - hemolysin . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - LC3 and the corresponding HRP - labeled secondary antibody . The bands were subsequently developed with an enhanced chemiluminescence detection kit . Lower panel : Quantification of the band intensities with the Adobe Photoshop program . * p < 0 . 05 ( paired Student ' s t - test ) . These data are representative of three independent experiments .

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

(C) Upper panel: CHO cells were preincubated for 30 min with 10 µM of calpeptin, with 1 mM dbcAMP, or with calpeptin+dbcAMP, and then they were incubated for 4 h in complete medium in the presence (Hla) or absence (Ctr) of α-hemolysin. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. Lower panel: Quantification of the band intensities with the Adobe Photoshop program. * p<0.05 (paired Student's t-test). These data are representative of three independent experiments.


( A ) GFP - LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP ( + dbcAMP ) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S . aureus ( wt ) , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . Cells without any treatment were used as control ( − dbcAMP ) . The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods , and immediately visualized by confocal microscopy . Images are representative of two independent experiments .

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

(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments.


. ( B ) Quantification of the percentage of cells presenting LC3 puncta ( i . e . , stimulated cells ) upon incubation in the different conditions . * p < 0 . 05 , * * p < 0 . 01 ( paired Student ' s t - test , n = 50 cells / condition ) . These data are representative of two independent experiments .

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

. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation in the different conditions. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of two independent experiments.


( C ) Quantification of the number of CFU / ml of CHO cells infected for 3 h with the wt strain of S . aureus , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid in the absence or presence of cAMP . * p < 0 . 05 ( paired Student ' s t - test ) . These data are representative of two independent experiments .

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

(C) Quantification of the number of CFU/ml of CHO cells infected for 3 h with the wt strain of S. aureus, the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid in the absence or presence of cAMP. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.


CHO cells were cotransfected with GFP - LC3 and myc - EPAC wt ( upper panel ) or myc - ΔEPAC ( lower panel ) . Twenty - four hours after transfection , they were infected for 4 h with the wt strain of S . aureus ( wt ) , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy . Images are representative of three independent experiments .

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

CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (upper panel) or myc-ΔEPAC (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. Images are representative of three independent experiments.


B ) Quantification of the percentage of cells presenting LC3 puncta upon the infection . These data are representative of three independent experiments .

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

B) Quantification of the percentage of cells presenting LC3 puncta upon the infection. These data are representative of three independent experiments.


( C ) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S . aureus . * * p < 0 . 01 ( paired Student ' s t - test , n = 20 cells / condition ) . These data are representative of three independent experiments .

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

(C) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S. aureus. ** p<0.01 (paired Student's t-test, n = 20 cells/condition). These data are representative of three independent experiments.


( A ) CHO cells were cotransfected with RFP - LC3 and GFP - Rap2b wt ( upper panel ) or GFP - Rap2b ΔAAX ( lower panel ) . Twenty - four hours after transfection , they were infected for 4 h with the wt strain of S . aureus ( wt ) , the mutant deficient for α - hemolysin ( Hla − ) , or the Hla ( − ) mutant expressing an α - hemolysin plasmid ( Hla ( − ) + pHla ) . The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy . These data are representative of three independent experiments .

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

(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. These data are representative of three independent experiments.


( B ) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection . * p < 0 . 05 , * * * p < 0 . 001 ( paired Student ' s t - test , n = 50 cells / condition ) . These data are representative of three independent experiments .

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

(B) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p<0.05, *** p<0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.


( C ) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection . * * p < 0 . 01 ( paired Student ' s t - test , n = 30 cells / condition ) . These data are representative of three independent

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

(C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p<0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experim


( A ) HeLa cells were incubated for 4 h with complete medium in the presence ( Hla ) or absence ( Ctr ) of 10 µg / ml α - hemolysin or they were infected for 4 h with the wt strain of S . aureus ( wt ) or the α - hemolysin deficient mutant ( Hla − ) . Afterwards , cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti - Rap2b and the corresponding HRP - labeled secondary antibody . The bands were subsequently developed with an enhanced chemiluminescence detection kit . A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel . These data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments.


B ) HeLa cells were incubated for 4 h with complete medium in the presence ( Hla ) or absence ( Ctr ) of 10 µg / ml α - hemolysin or they were infected for 4 h with the wt strain of S . aureus ( wt ) or the α - hemolysin deficient mutant ( Hla − ) . Cells were disrupted and whole cell lysates were subjected to pull - down assays using GST - Ral - GDS - RBD - sepharose . The levels of GTP - bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti - Rap2b and the corresponding HRP - labeled secondary antibody . The bands were subsequently developed with an enhanced chemiluminescence detection kit . The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel . * p < 0 . 05 ( paired Student ' s t - test ) . These data are representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.


( E ) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 ( LxxAxA ) . Binding of YFP - B56α to indicated proteins was determined and quantified by LiCor .

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

(E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor.


( G ) YFP - B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins . Representative of 4 independent experiments .

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

(G) YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.


( B ) IP of YFP - B56α from cells stably expressing the B56α WT , R222E , and 5A followed by immunoblotting of indicated proteins . Representative blots are shown ( top ) . hSgo1 signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).


( C ) Reciprocal IP of ( B ) . YFP - hSgo1 expression construct was transfected into cells stably expressing FLAG - B56α WT , R222E and 5A , followed by YFP IP and immunoblotting of indicated proteins . Representative blots are shown ( top ) . B56α signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) .

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

(C) Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).


( D ) Representative images of chromosome spreads from the indicated conditions . Scale bar , 5μm . ( E ) Quantification of ( D ) . The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 4 independent experiments and the mean and SD are indicated .

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

(D) Representative images of chromosome spreads from the indicated conditions. Scale bar, 5μm. (E) Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated.


( F ) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere - targeting domain ( CB ) . Representative images of chromosome spreads are shown . CB targets all the rescue constructs ( green ) to the centromere . Scale bar , 5μm . ( G ) Quantification of ( F ) . The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 3 independent experiments and the mean and SD are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5μm. (G) Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.


( D ) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP - hSgo1 variants . Representative immunofluorescent images are shown . Scale bar , 5μm . ( E ) B56α signal intensity was quantified . B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP - hSgo1 signal . Each circle represents an individual cell , and the average and SD are indicated .

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

(D) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP-hSgo1 variants. Representative immunofluorescent images are shown. Scale bar, 5μm. (E) B56α signal intensity was quantified. B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP-hSgo1 signal. Each circle represents an individual cell, and the average and SD are indicated.


( F ) Mitotic U2OS LacO Haspin CM cells expressing hSgo1 - LacI - GFP variants or LacI - GFP ( control ) were stained for PP2A - C . Scale bar , 5μm . ( G ) PP2A - C signal intensity was quantified , normalized to GFP , and then plotted . Each circle represents an individual cell , and the mean fluorescent intensity is indicated . Representative of at least 3 independent experiments .

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

(F) Mitotic U2OS LacO Haspin CM cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C. Scale bar, 5μm. (G) PP2A-C signal intensity was quantified, normalized to GFP, and then plotted. Each circle represents an individual cell, and the mean fluorescent intensity is indicated. Representative of at least 3 independent experiments.


( A ) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP - hSgo1 variants . All the YFP - hSgo1 rescue constructs ( green ) localize to the centromeres ( CREST , red ) . Scale bar , 5μm . ( B ) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in ( A ) and averaged for a single cell . The data are from 3 independent experiments and the mean and SD are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP-hSgo1 variants. All the YFP-hSgo1 rescue constructs (green) localize to the centromeres (CREST, red). Scale bar, 5μm. (B) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in (A) and averaged for a single cell. The data are from 3 independent experiments and the mean and SD are indicated.


( C ) IP of YFP - hSgo1 from cells stably expressing hSgo1 WT , 3A , and 4A followed by immunoblotting of cohesin components , Smc1 and Smc3 . Representative blots are shown ( top ) . Signals were quantification by LiCor , normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 for Smc1 and n = 3 for Smc3 ) .

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

(C) IP of YFP-hSgo1 from cells stably expressing hSgo1 WT, 3A, and 4A followed by immunoblotting of cohesin components, Smc1 and Smc3. Representative blots are shown (top). Signals were quantification by LiCor, normalized to YFP and plotted (bottom). Error bars represent SD (n=4 for Smc1 and n=3 for Smc3).


( F ) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi - resistant constructs was performed . Representative still images captured during the live cell imaging showing DIC and YFP - hSgo1 WT , 3A , and 4A localization during mitosis . Time ( min ) from nuclear envelop breakdown ( NEBD ) is indicated . Scale bar , 15μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi-resistant constructs was performed. Representative still images captured during the live cell imaging showing DIC and YFP-hSgo1 WT, 3A, and 4A localization during mitosis. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15μm.


Mutations in epg - 7 cause defective degradation of SQST - 1 aggregates . ( A and B ) In wild - type embryos , SQST - 1 : : GFP is weakly expressed and diffusely localized in the cytoplasm . ( A ) DIC image of the embryo shown in B . ( C ) Elevated expression level and accumulation of many SQST - 1 : : GFP aggregates in epg - 7 mutant embryos .

Mutations in epg-7 cause defective degradation of SQST-1 aggregates. (A and B) In wild-type embryos, SQST-1::GFP is weakly expressed and diffusely localized in the cytoplasm. (A) DIC image of the embryo shown in B. (C) Elevated expression level and accumulation of many SQST-1::GFP aggregates in epg-7 mutant embryos.


( D ) Compared with wild type and epg - 2 mutants , levels of SQST - 1 are dramatically elevated in atg - 3 and epg - 7 mutant embryos in an immunoblotting assay .

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(D) Compared with wild type and epg-2 mutants, levels of SQST-1 are dramatically elevated in atg-3 and epg-7 mutant embryos in an immunoblotting assay.


( E - G ) Compared with wild - type embryos ( E and F ) , endogenous SQST - 1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg - 7 mutant embryos ( G ) . ∼ 200 - cell stage embryos are shown in E - G . ( E ) DAPI image of the embryo shown in F .

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(E-G) Compared with wild-type embryos (E and F), endogenous SQST-1 shows dramatically elevated levels and accumulates into a large number of aggregates in epg-7 mutant embryos (G). ∼200-cell stage embryos are shown in E-G. (E) DAPI image of the embryo shown in F.


( H ) Number of SQST - 1 aggregates per focal plane in wild type , atg - 3 mutants , and epg - 7 mutants .

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(H) Number of SQST-1 aggregates per focal plane in wild type, atg-3 mutants, and epg-7 mutants.


( I - L ) At the ∼ 100 - cell stage , far fewer SQST - 1 aggregates are formed in epg - 7 mutants than in atg - 3 mutants . ( I and K ) DAPI images of the embryos shown in J and L , respectively .

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(I-L) At the ∼100-cell stage, far fewer SQST-1 aggregates are formed in epg-7 mutants than in atg-3 mutants. (I and K) DAPI images of the embryos shown in J and L, respectively.


( M ) SQST - 1 directly interacts with ZK1053 . 4 in a GST pull - down assay .

(M) SQST-1 directly interacts with ZK1053.4 in a GST pull-down assay.


( N ) Compared with wild - type animals , levels of ZK1053 . 4 : : GFP are dramatically elevated in atg - 3 and epg - 7 mutants .

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(N) Compared with wild-type animals, levels of ZK1053.4::GFP are dramatically elevated in atg-3 and epg-7 mutants.


( O - R ) ZK1053 . 4 : : GFP is weakly expressed in wild - type embryos ( O - P ) and sqst - 1 mutants ( R ) , but forms a large number of aggregates in epg - 7 mutants ( Q ) . ( O ) DIC image of the embryo shown in P .

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(O-R) ZK1053.4::GFP is weakly expressed in wild-type embryos (O-P) and sqst-1 mutants (R), but forms a large number of aggregates in epg-7 mutants (Q). (O) DIC image of the embryo shown in P.


( S ) Survival curve of L1 larvae under food depletion conditions in wild type , epg - 1 mutants , and epg - 7 mutants . This experiment was completed once ( wild type : n = 2 , 964 ; epg - 7 : n = 602 ; epg - 1 : n = 215 ) . Statistical analysis of L1 survival shows that epg - 7 mutants exhibit significant difference to wild type and also to epg - 1 mutants ( log - rank test , P = 0 . 000 ) .

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(S) Survival curve of L1 larvae under food depletion conditions in wild type, epg-1 mutants, and epg-7 mutants. This experiment was completed once (wild type: n = 2,964; epg-7: n = 602; epg-1: n = 215). Statistical analysis of L1 survival shows that epg-7 mutants exhibit significant difference to wild type and also to epg-1 mutants (log-rank test, P = 0.000).


( T ) Loss of function of epg - 7 does not ameliorate the degeneration of mec - 4 : : gfp - labeled touch neurons caused by mec - 4 ( u231 ) .

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(T) Loss of function of epg-7 does not ameliorate the degeneration of mec-4::gfp-labeled touch neurons caused by mec-4(u231).


( U ) Survival curves of wild type and various mutants . sqst - 1 ; epg - 7 mutants have a shortened mean and maximum life span . ( P = 0 . 000 for epg - 7 or wild type vs . sqst - 1 ; epg - 7 in all three repeats ; log - rank test ) .

(U) Survival curves of wild type and various mutants. sqst-1;epg-7 mutants have a shortened mean and maximum life span. (P = 0.000 for epg-7 or wild type vs. sqst-1;epg-7 in all three repeats; log-rank test).


( V ) Survival of hydrogen peroxide - treated animals . ( In all three repeats , P = 0 . 000 for epg - 7 vs . sqst - 1 ; epg - 7 ; P > 0 . 2 for wild type vs . sqst - 1 ; epg - 7 ; log - rank test ) .

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(V) Survival of hydrogen peroxide-treated animals. (In all three repeats, P = 0.000 for epg-7 vs. sqst-1;epg-7; P > 0.2 for wild type vs. sqst-1;epg-7; log-rank test).


( W ) Survival of animals at the indicated time after heat shock treatment . ( P < 0 . 05 for wild type vs . epg - 7 or epg - 7 vs . sqst - 1 ; epg - 7 at 5 , 6 , and 7 h ) .

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(W) Survival of animals at the indicated time after heat shock treatment. (P < 0.05 for wild type vs. epg-7 or epg-7 vs. sqst-1;epg-7 at 5, 6, and 7 h).


( X ) Dauer formation of daf - 2 ( e1370 ) mutants in different backgrounds .

(X) Dauer formation of daf-2(e1370) mutants in different backgrounds.


( D ) A large number of SQST - 1 : : GFP aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del Atg11 ) transgene .

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(D) A large number of SQST-1::GFP aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del Atg11) transgene.


( E and F ) No SQST - 1 aggregates are detected in epg - 7 mutants carrying an epg - 7 : : gfp reporter . ( E ) DAPI image of the embryo shown in F .

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(E and F) No SQST-1 aggregates are detected in epg-7 mutants carrying an epg-7::gfp reporter. (E) DAPI image of the embryo shown in F.


( G ) EPG - 7 : : GFP is weakly expressed and diffusely localized in the cytoplasm in wild - type embryos .

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(G) EPG-7::GFP is weakly expressed and diffusely localized in the cytoplasm in wild-type embryos.


( H - I ) EPG - 7 : : GFP is expressed in pharyngeal muscles ( H , arrow ) and cells in the tail region ( I ) at larval stages .

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(H-I) EPG-7::GFP is expressed in pharyngeal muscles (H, arrow) and cells in the tail region (I) at larval stages.


( J ) Compared with wild - type animals , levels of EPG - 7 : : GFP are dramatically elevated in epg - 4 mutants , but remain unchanged in sqst - 1 mutants .

(J) Compared with wild-type animals, levels of EPG-7::GFP are dramatically elevated in epg-4 mutants, but remain unchanged in sqst-1 mutants.


( K ) The expression level of EPG - 7 : : GFP is dramatically elevated and many EPG - 7 : : GFP aggregates are formed in lgg - 1 mutant embryos .

(K) The expression level of EPG-7::GFP is dramatically elevated and many EPG-7::GFP aggregates are formed in lgg-1 mutant embryos.


( L and M ) No EPG - 7 aggregates , detected by anti - EPG - 7 antibody , are formed in wild - type embryos . ( L ) DAPI image of the embryo shown in M . ( N ) Levels of EPG - 7 are dramatically elevated and a large number of EPG - 7 aggregates are formed in atg - 3 mutants . ( O ) No EPG - 7 aggregates are formed in sqst - 1 mutant embryos .

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(L and M) No EPG-7 aggregates, detected by anti-EPG-7 antibody, are formed in wild-type embryos. (L) DAPI image of the embryo shown in M. (N) Levels of EPG-7 are dramatically elevated and a large number of EPG-7 aggregates are formed in atg-3 mutants. (O) No EPG-7 aggregates are formed in sqst-1 mutant embryos.


( A ) EPG - 7 colocalizes with SQST - 1 aggregates in epg - 8 mutant embryos . Colocalization was defined as two aggregates showing > 70 % overlap in their fluorescence signals . Insets show a magnified view .

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(A) EPG-7 colocalizes with SQST-1 aggregates in epg-8 mutant embryos. Colocalization was defined as two aggregates showing >70% overlap in their fluorescence signals. Insets show a magnified view.


( B ) Percentage of EPG - 7 aggregates colocalized with SQST - 1 in indicated autophagy mutants .

(B) Percentage of EPG-7 aggregates colocalized with SQST-1 in indicated autophagy mutants.


( C ) A coimmunoprecipitation assay reveals that EPG - 7 associates with SQST - 1 .

(C) A coimmunoprecipitation assay reveals that EPG-7 associates with SQST-1.


( D ) EPG - 7 interacts with SQST - 1 in a pull - down assay .

(D) EPG-7 interacts with SQST-1 in a pull-down assay.


( E ) EPG - 7 self - associates in a pull - down assay .

(E) EPG-7 self-associates in a pull-down assay.


( F ) SDS - PAGE and blue - native PAGE of purified EPG - 7 ( 520 - 714 ) proteins . Lane M shows molecular weight marker . Lanes 1 , 2 , 3 , and 4 contain 10 , 5 , 2 . 5 , and 1 . 25 µg of proteins , respectively .

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(F) SDS-PAGE and blue-native PAGE of purified EPG-7(520-714) proteins. Lane M shows molecular weight marker. Lanes 1, 2, 3, and 4 contain 10, 5, 2.5, and 1.25 µg of proteins, respectively.


( G ) An EPG - 7 fragment containing amino acids 531 - 631 self - interacts .

(G) An EPG-7 fragment containing amino acids 531-631 self-interacts.


( H and I ) SQST - 1 aggregates accumulate in epg - 7 mutant embryos carrying an epg - 7 ( del 531 - 631 ) : : gfp transgene . ( H ) DAPI image of the embryo shown in I .

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(H and I) SQST-1 aggregates accumulate in epg-7 mutant embryos carrying an epg-7(del 531-631)::gfp transgene. (H) DAPI image of the embryo shown in I.


( J and K ) EPG - 7 ( del 531 - 631 ) : : GFP is weakly expressed and diffusely localized in epg - 7 ( tm2508 ) embryos . Its expression is not elevated in lgg - 1 ( RNAi ) animals . H - K show embryos carrying the same transgene . Images in J and K were taken using the same exposure time .

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(J and K) EPG-7(del 531-631)::GFP is weakly expressed and diffusely localized in epg-7(tm2508) embryos. Its expression is not elevated in lgg-1(RNAi) animals. H-K show embryos carrying the same transgene. Images in J and K were taken using the same exposure time.


( L and M ) SQST - 1 self - interacts ( L ) and the self - interaction is mediated by a fragment containing amino acids 26 - 181 ( M ) .

(L and M) SQST-1 self-interacts (L) and the self-interaction is mediated by a fragment containing amino acids 26-181 (M).


( N ) Levels of SQST - 1 ( del 26 - 181 ) : : GFP are dramatically elevated in epg - 7 and atg - 3 mutants .

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(N) Levels of SQST-1(del 26-181)::GFP are dramatically elevated in epg-7 and atg-3 mutants.


( O and P ) SQST - 1 ( del 26 - 181 ) : : GFP is weakly expressed and diffusely localized in sqst - 1 ( ok2892 ) mutant embryos . ( O ) DIC image of the embryo shown in P .

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(O and P) SQST-1(del 26-181)::GFP is weakly expressed and diffusely localized in sqst-1(ok2892) mutant embryos. (O) DIC image of the embryo shown in P.


( Q and R ) SQST - 1 ( del 26 - 181 ) : : GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg - 1 ( Q ) and epg - 7 ( R ) mutants .

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(Q and R) SQST-1(del 26-181)::GFP is expressed at dramatically elevated levels and accumulates into a large number of aggregates in lgg-1 (Q) and epg-7 (R) mutants.


( A - D ) Expression of LGG - 1 in wild - type embryos at the ∼ 100 - cell ( A and B ) , the ∼ 200 - cell ( C ) , and the fourfold ( D ) stages . ( A ) DAPI image of the embryo shown in B .

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(A-D) Expression of LGG-1 in wild-type embryos at the ∼100-cell (A and B), the ∼200-cell (C), and the fourfold (D) stages. (A) DAPI image of the embryo shown in B.


( E ) Loss of function of epg - 7 does not increase levels of LGG - 1 and also does not suppress accumulation of LGG - 1 in epg - 3 mutant embryos .

(E) Loss of function of epg-7 does not increase levels of LGG-1 and also does not suppress accumulation of LGG-1 in epg-3 mutant embryos.


( F - H ) Expression of LGG - 1 in epg - 7 mutant embryos before the ∼ 100 - cell stage ( F ) , at the ∼ 200 - cell stage ( G ) , and at the fourfold stage ( H ) . Images in B - D and F - H were taken using the same exposure time .

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(F-H) Expression of LGG-1 in epg-7 mutant embryos before the ∼100-cell stage (F), at the ∼200-cell stage (G), and at the fourfold stage (H). Images in B-D and F-H were taken using the same exposure time.


( I ) SQST - 1 aggregates are separable from LGG - 1 puncta in epg - 7 mutant embryos . Insets show a magnified view . ( J ) In epg - 6 mutant embryos , LGG - 1 puncta and SQST - 1 aggregates are enlarged and colocalized . ( K ) LGG - 1 and SQST - 1 aggregates are smaller in size and largely separable in epg - 6 ; epg - 7 double mutants . ( I - K ) DAPI images of the embryos shown in the same row .

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(I) SQST-1 aggregates are separable from LGG-1 puncta in epg-7 mutant embryos. Insets show a magnified view. (J) In epg-6 mutant embryos, LGG-1 puncta and SQST-1 aggregates are enlarged and colocalized. (K) LGG-1 and SQST-1 aggregates are smaller in size and largely separable in epg-6;epg-7 double mutants. (I-K) DAPI images of the embryos shown in the same row.


( L - O ) Enlarged LGG - 1 and SQST - 1 aggregates are colocalized in epg - 4 mutants ( L and M ) . Simultaneously depleting epg - 7 reduces the size of LGG - 1 puncta and SQST - 1 aggregates , which become largely separable ( N and O ) . ( L and N ) DAPI images of the embryos shown in M and O , respectively .

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(L-O) Enlarged LGG-1 and SQST-1 aggregates are colocalized in epg-4 mutants (L and M). Simultaneously depleting epg-7 reduces the size of LGG-1 puncta and SQST-1 aggregates, which become largely separable (N and O). (L and N) DAPI images of the embryos shown in M and O, respectively.


( P ) In atg - 9 mutant embryos , LGG - 1 puncta largely colocalize with SQST - 1 aggregates at the comma stage . Notably , there are more SQST - 1 aggregates than LGG - 1 puncta . ( Q ) SQST - 1 aggregates are separable from LGG - 1 puncta in atg - 9 ; epg - 7 mutants at the comma stage .

(P) In atg-9 mutant embryos, LGG-1 puncta largely colocalize with SQST-1 aggregates at the comma stage. Notably, there are more SQST-1 aggregates than LGG-1 puncta. (Q) SQST-1 aggregates are separable from LGG-1 puncta in atg-9;epg-7 mutants at the comma stage.


( R ) Percentage of LGG - 1 puncta colocalized with SQST - 1 aggregates in indicated autophagy mutants . Comma - stage embryos were examined for atg - 9 and atg - 9 ; epg - 7 mutants . ∼ 200 - cell stage embryos were examined for other autophagy mutants .

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(R) Percentage of LGG-1 puncta colocalized with SQST-1 aggregates in indicated autophagy mutants. Comma-stage embryos were examined for atg-9 and atg-9;epg-7 mutants. ∼200-cell stage embryos were examined for other autophagy mutants.


( A ) EPG - 7 coimmunoprecipitates with ATG - 9 : : HA .

(A) EPG-7 coimmunoprecipitates with ATG-9::HA.


( B ) EPG - 7 directly interacts with ATG - 9 in a GST pull - down assay .

(B) EPG-7 directly interacts with ATG-9 in a GST pull-down assay.


( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 3 ( C )

(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-3 (C)


( C and D ) Coimmunoprecipitation reveals that EPG - 7 associates with LGG - 1 ( D ) .

(C and D) Coimmunoprecipitation reveals that EPG-7 associates with LGG-1 (D).


( E ) EPG - 7 directly interacts with LGG - 3 .

(E) EPG-7 directly interacts with LGG-3.


( F ) EPG - 7 fragments containing amino acids 852 - 1003 or 1004 - 1142 , but not 762 - 851 , interact with LGG - 1 .

(F) EPG-7 fragments containing amino acids 852-1003 or 1004-1142, but not 762-851, interact with LGG-1.


( G ) Fragments of EPG - 7 containing amino acids 762 - 851 or 852 - 1003 directly interact with ATG - 18 .

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(G) Fragments of EPG-7 containing amino acids 762-851 or 852-1003 directly interact with ATG-18.


( H ) Fragments of EPG - 7 containing amino acids 1 - 704 or 1143 - 1330 directly interact with UNC - 51 .

(H) Fragments of EPG-7 containing amino acids 1-704 or 1143-1330 directly interact with UNC-51.


( I ) SQST - 1 associates with LGG - 1 in vivo . Embryonic extracts from epg - 7 mutants show that interaction of SQST - 1 with LGG - 1 is EPG - 7 independent .

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(I) SQST-1 associates with LGG-1 in vivo. Embryonic extracts from epg-7 mutants show that interaction of SQST-1 with LGG-1 is EPG-7 independent.