Figure 2 ( A , Nodal - induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT - PCR . Animal caps ere treated with - ) , no injection of Co MO , Wip1 MO and / or Wip1 mRNA . The dose of reagent injected : Xnr1 ( 5 pg ) , Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) and Wip1 ( 1 ng ) . B ) Activin / Nodal - induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT - PCR . Animal caps in ( B ) were treated with Activin protein ( 5 ng / ml ) . ( - ) , no injection of Co MO , Wip1 MO and / or Wip1 mRNA . The dose of reagent injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng ) and Wip1 ( 1 ng ) . ( C ) Knockdown of Wip1 up - regulates the activity of Activin / Nodal - responsive promoter . HEK293T cells transfected with Co siRNA ( 50 nM ) or Wip1 siRNA ( 50 nM ) were stimulated with Activin protein ( 10 ng / ml ) for 10 h and then subjected to luciferase assays . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * * * P < 0 . 001 by unpaired Student ' s t - test . Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate . ( - ) , no injection of Noggin mRNA . The amount of MO and mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G and J ; 30 ng for F and I ) and Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm . Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate . Embryos in ( E - J ) are shown in dorsal views with anterior to the top . The amount of MO and mRNA injected : Co MO ( 60 ng ) , Wip1 MO ( 60 ng for D , G and J ; 30 ng for F and I ) and Noggin ( 5 , 10 , 20 pg ) . Scale bar , 150 μm . ( K , Activin induction of p21 is up - regulated upon Wip1 knockdown . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection , were stimulated with Activin ( 10 ng / ml ) for 3 to 12 h as indicated t = 0 in ( K ) is 48 h after siRNA transfection . P - Smad2 serves as an indicator of active Activin / Nodal signaling . Arrows ndicate non - specific bands . L ) TGF - β induction of p21 is up - regulated upon Wip1 knockdown . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection , were stimulated with increasing concentration of TGF - β1 ( 10 , 20 ng / ml ) for 3 h .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A, Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps ere treated with -), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Xnr1 (5 pg), Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).B) Activin/Nodal-induced gene responses are enhanced further in the absence of Wip1 as analyzed by RT-PCR. Animal caps in (B) were treated with Activin protein (5 ng/ml). (-), no injection of Co MO, Wip1 MO and/or Wip1 mRNA. The dose of reagent injected: Co MO (60 ng), Wip1 MO (60 ng) and Wip1 (1 ng).(C) Knockdown of Wip1 up-regulates the activity of Activin/Nodal-responsive promoter. HEK293T cells transfected with Co siRNA (50 nM) or Wip1 siRNA (50 nM) were stimulated with Activin protein (10 ng/ml) for 10 h and then subjected to luciferase assays. Data are expressed as the mean ± SEM (n=3 biological replicates). ***P<0.001 by unpaired Student's t-test.Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. (-), no injection of Noggin mRNA. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.Depletion of Wip1 promotes epidermal differentiation at the expense of neural fate. Embryos in (E-J) are shown in dorsal views with anterior to the top. The amount of MO and mRNA injected: Co MO (60 ng), Wip1 MO (60 ng for D, G and J; 30 ng for F and I) and Noggin (5, 10, 20 pg). Scale bar, 150 μm.(K, Activin induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with Activin (10 ng/ml) for 3 to 12 h as indicated t=0 in (K) is 48 h after siRNA transfection. P-Smad2 serves as an indicator of active Activin/Nodal signaling. Arrows ndicate non-specific bands.L) TGF-β induction of p21 is up-regulated upon Wip1 knockdown. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and 48 h after siRNA transfection, were stimulated with increasing concentration of TGF-β1 (10, 20 ng/ml) for 3 h.


Figure 3 ( A , Effects of the gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T cells were transfected or not with wt Wip1 , Wip1 ( D277A ) , as indicated and subjected to CCK - 8 assays before ( 0 h ) and after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - test . n . s . , not significant . B ) Effects of the gain - or loss - of - function of Wip1 on TGF - β - induced growth arrest . HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK - 8 assays before ( 0 h ) and after stimulation with TGF - β1 ( 40 ng / ml ) ( 24 h ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 by unpaired Student ' s t - test . n . s . , not significant . Depletion of Wip1 augments TGF - β - induced migration and invasion of MDA - MB231 breast cancer cells . MDA - MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20 - μL pipette tip and then incubated in the absence or presence of TGF - β1 for the indicated times ( C , D ) Depletion of Wip1 augments TGF - β - induced migration and invasion of MDA - MB231 breast cancer cells . MDA - MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF - β1 for 20 h ( E , F ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A, Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with wt Wip1, Wip1(D277A), as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.B) Effects of the gain- or loss-of-function of Wip1 on TGF-β-induced growth arrest. HEK293T cells were transfected or not with Co siRNA or Wip1 siRNA as indicated and subjected to CCK-8 assays before (0 h) and after stimulation with TGF-β1 (40 ng/ml) (24 h). Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05, **P<0.01, ***P<0.001 by unpaired Student's t-test. n.s., not significant.Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were scratched with a 20-μL pipette tip and then incubated in the absence or presence of TGF-β1 for the indicated times (C, D)Depletion of Wip1 augments TGF-β-induced migration and invasion of MDA-MB231 breast cancer cells. MDA-MB231 cells transfected with Co siRNA or Wip1 siRNA were subjected to transwell assay with or without TGF-β1 for 20 h (E, F).


Figure 4 ( A - D ) Knockdown of Wip1 expands the expression of dorsal mesodermal markers . Embryos injected dorsally with Co MO ( 40 ng ) or Wip1 MO ( 40 ng ) were subjected to in situ hybridization at stage 10 . 25 . Embryos are shown in dorso - vegetal views with dorsal to the top . Scale bar , 150 μm . ( E - I ) Morphological phenotypes of Wip1 morphants . Embryos were injected radially in the marginal zone with Co MO ( 80 ng ) , Wip1 MO ( 80 ng ) , wt Wip1 ( 1 ng ) and Wip1 ( D277A ) mRNA as indicated and cultured to tadpole stages . Embryos are shown in lateral views with anterior to the left . Un Co , uninjected control . Scale bar , 1 mm . ( J ) Quantification of the phenotypes shown in ( E - I ) . Severe defects include microcephaly , shortened and kinked body axis , and no eye . Mild defects indicate normal body axis with malformed eyes .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A-D) Knockdown of Wip1 expands the expression of dorsal mesodermal markers. Embryos injected dorsally with Co MO (40 ng) or Wip1 MO (40 ng) were subjected to in situ hybridization at stage 10.25. Embryos are shown in dorso-vegetal views with dorsal to the top. Scale bar, 150 μm.(E-I) Morphological phenotypes of Wip1 morphants. Embryos were injected radially in the marginal zone with Co MO (80 ng), Wip1 MO (80 ng), wt Wip1 (1 ng) and Wip1(D277A) mRNA as indicated and cultured to tadpole stages. Embryos are shown in lateral views with anterior to the left. Un Co, uninjected control. Scale bar, 1 mm. (J) Quantification of the phenotypes shown in (E-I). Severe defects include microcephaly, shortened and kinked body axis, and no eye. Mild defects indicate normal body axis with malformed eyes.


Figure 5 ( A Wip1 and Smad4 form an endogenous protein complex . Cell lysates of HEK293T cells , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) ( - ) , no treatment with protein ligand . IgG , control IgG . An arrow in ( A ) indicates non - specific bands . B ) Wip1 and Smad4 form an endogenous protein complex . Cell lysates of HEK293T cells , which were stimulated or not with Activin ( 10 ng / ml ) , TGF - β1 ( 40 ng / ml ) or BMP4 ( 20 ng / ml ) for 1 h , were immunoprecipitated with anti - Smad4 antibody followed by western blotting with anti - Wip1 antibody ( A ) and vice versa ( B ) . ( - ) , no treatment with protein ligand . IgG , control IgG . ( C ) Upon ligand stimulation , Wip1 forms a complex with Smad4 in the nucleus . HeLa cells were treated or not with TGF - β1 ( 40 ng / ml ) for 2 h , fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4 . Blue : DAPI ; Red : PLA signal . Scale bar , 20 μm . ( D ) Quantitative analysis of Wip1 / Smad4 interactions shown in ( C ) . Data are represented as the mean ± SEM ( n = 3 biological replicates ) . Twenty cells were analyzed per sample . * * * P < 0 . 001 by Student ' s t - test . ( E ) Wip1 interferes with the ability of Smad4 to bind to Smad2 . HEK293T cells co - transfected with the indicated constructs were treated or not with TGF - β1 for 1 h and then harvested . An arrow denotes Myc - tagged Wip1 protein . ( F ) Wip1 has a negative effect on nuclear accumulation of Smad4 . HEK293T cells were transfected with Co siRNA or Wip1 siRNA , treated with TGF - β1 ( 40 ng / ml , 1 h ) 48 h after siRNA transfection and subjected to nuclear / cytoplasmic fractionation and western blotting analysis . β - tubulin and lamin A / C serve as cytoplasmic and nuclear markers , respectively . C , cytoplasm ; N , nucleus . The experiment was repeated two times with similar results . ( G ) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in ( F ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) (-), no treatment with protein ligand. IgG, control IgG. An arrow in (A) indicates non-specific bands.B) Wip1 and Smad4 form an endogenous protein complex. Cell lysates of HEK293T cells, which were stimulated or not with Activin (10 ng/ml), TGF-β1 (40 ng/ml) or BMP4 (20 ng/ml) for 1 h, were immunoprecipitated with anti-Smad4 antibody followed by western blotting with anti-Wip1 antibody (A) and vice versa (B). (-), no treatment with protein ligand. IgG, control IgG.(C) Upon ligand stimulation, Wip1 forms a complex with Smad4 in the nucleus. HeLa cells were treated or not with TGF-β1 (40 ng/ml) for 2 h, fixed and then subjected to proximity ligation assays with primary antibodies against Wip1 and Smad4. Blue: DAPI; Red: PLA signal. Scale bar, 20 μm. (D) Quantitative analysis of Wip1/Smad4 interactions shown in (C). Data are represented as the mean ± SEM (n=3 biological replicates). Twenty cells were analyzed per sample. ***P<0.001 by Student's t-test. (E) Wip1 interferes with the ability of Smad4 to bind to Smad2. HEK293T cells co-transfected with the indicated constructs were treated or not with TGF-β1 for 1 h and then harvested. An arrow denotes Myc-tagged Wip1 protein.(F) Wip1 has a negative effect on nuclear accumulation of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA, treated with TGF-β1 (40 ng/ml, 1 h) 48 h after siRNA transfection and subjected to nuclear/cytoplasmic fractionation and western blotting analysis. β-tubulin and lamin A/C serve as cytoplasmic and nuclear markers, respectively. C, cytoplasm; N, nucleus. The experiment was repeated two times with similar results. (G) Quantification of the levels of nuclear Smad4 normalized to those of cytoplasmic Smad4 shown in (F).


Figure 6 ( A ) Overexpression of Wip1 down - regulates Smad4 phosphorylation at Thr277 . MOCK - or Flag - Wip1 - transfected HEK293T cells were treated with 1 h pulse of FGF2 ( 10 ng / ml ) , followed by addition of U0126 ( 40 μM ) and harvesting for western blotting at the indicated times . - FGF2 , no treatment with FGF2 . ( B ) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1 - depleted HEK293T cells , showing that knockdown of Wip1 up - regulates Smad4 phosphorylation at the MAPK site . A bracket denotes phospho - Smad4 . ( C ) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay . Myc - tagged Smad4 was expressed in HEK293T cells , which were then treated or not with FGF2 ( 40 ng / ml , 2 h ) , and the Myc - Smad4 was immunoprecipitated using anti - Myc antibody and subsequently incubated with or without recombinant human Wip1 protein ( 0 . 5 μg ) in the absence or presence of Mg2 + as indicated . ( D ) FGF - induced destabilization of Smad4 is enhanced further in Wip1 - depleted animal cap cells as analyzed by western blotting . The amount of reagent injected : Myc - Smad4 ( 400 pg ) , FGF8 ( 100 pg ) , Wnt8 ( 400 pg ) , Co MO ( 60 ng ) and Wip1 MO ( 60 ng ) . ( E ) Polyubiquitination of wt Smad4 is promoted in Wip1 - silenced cells . HEK293T cells were transfected with the indicated combinations of Flag - ubiquitin , Co siRNA , Wip1 siRNA , Myc - Smad4 and Myc - Smad4 ( T277A ) and then treated with FGF2 ( 10 ng / ml , 4 h ) in the presence of the proteasome inhibitor MG132 , and Myc - Smad4 and its phosphorylation - resistant mutant were immunoprecipitated with anti - Myc antibody . Ubiquitin - conjugated Smad4 ( Smad4 - Ubn ) was detected by western blotting with anti - Flag antibody . ( F ) Wip1 prolongs the half - life of Smad4 . HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide ( CHX , 100 ng / ml ) for the indicated times and harvested for western blotting . An arrow indicates non - specific bands .

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Figure 6(A) Overexpression of Wip1 down-regulates Smad4 phosphorylation at Thr277. MOCK- or Flag-Wip1-transfected HEK293T cells were treated with 1 h pulse of FGF2 (10 ng/ml), followed by addition of U0126 (40 μM) and harvesting for western blotting at the indicated times. - FGF2, no treatment with FGF2.(B) Time course of Smad4 phosphorylation at Thr277 induced by FGF2 in control and Wip1-depleted HEK293T cells, showing that knockdown of Wip1 up-regulates Smad4 phosphorylation at the MAPK site. A bracket denotes phospho-Smad4.(C) Wip1 directly dephosphorylates Smad4 at Thr277 as assayed by in vitro phosphatase assay. Myc-tagged Smad4 was expressed in HEK293T cells, which were then treated or not with FGF2 (40 ng/ml, 2 h), and the Myc-Smad4 was immunoprecipitated using anti-Myc antibody and subsequently incubated with or without recombinant human Wip1 protein (0.5 μg) in the absence or presence of Mg2+ as indicated.(D) FGF-induced destabilization of Smad4 is enhanced further in Wip1-depleted animal cap cells as analyzed by western blotting. The amount of reagent injected: Myc-Smad4 (400 pg), FGF8 (100 pg), Wnt8 (400 pg), Co MO (60 ng) and Wip1 MO (60 ng).(E) Polyubiquitination of wt Smad4 is promoted in Wip1-silenced cells. HEK293T cells were transfected with the indicated combinations of Flag-ubiquitin, Co siRNA, Wip1 siRNA, Myc-Smad4 and Myc-Smad4(T277A) and then treated with FGF2 (10 ng/ml, 4 h) in the presence of the proteasome inhibitor MG132, and Myc-Smad4 and its phosphorylation-resistant mutant were immunoprecipitated with anti-Myc antibody. Ubiquitin-conjugated Smad4 (Smad4-Ubn) was detected by western blotting with anti-Flag antibody.(F) Wip1 prolongs the half-life of Smad4. HEK293T cells were transfected with Co siRNA or Wip1 siRNA and then treated with FGF2 along with cycloheximide (CHX, 100 ng/ml) for the indicated times and harvested for western blotting. An arrow indicates non-specific bands.


Figure 7 ( A - J ) Embryos injected dorsally with wt Wip1 ( 1 ng ) , wt Smad4 ( 1 ng ) , Smad4 ( T277A ) ( 1 ng ) and Smad4 ( T277E ) ( 1 ng ) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10 . 25 . Control , control embryo injected with LacZ mRNA only . Scale bar , 150 μm . ( K ) Quantification of the results from in situ hybridization analysis in ( A - J ) . ( L ) Wip1 - mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho - mimetic amino acid at the MAPK site . ( - ) , no co - injection of wt or mutated Smad4 . ( M ) MDA - MB231 and MDA - MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4 ( T277A ) and then subjected to CCK - 8 assays to measure cell growth . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test . ( N ) Anchorage - independent colony - formation assays were performed in MDA - MB231 and MDA - MB468 cells transfected with Wip1 or negative control constructs . Scale bar , 100 μm . ( O ) Quantification of the assays shown in ( N ) . The number of colonies larger than 50 μm in diameter was scored . Data are expressed as the mean ± SEM ( n = 3 biological replicates ) . * P < 0 . 05 by unpaired Student ' s t - test .

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Figure 7(A-J) Embryos injected dorsally with wt Wip1 (1 ng), wt Smad4 (1 ng), Smad4(T277A) (1 ng) and Smad4 (T277E) (1 ng) as denoted were subjected to in situ hybridization against Chordin or Xbra at stage 10.25. Control, control embryo injected with LacZ mRNA only. Scale bar, 150 μm. (K) Quantification of the results from in situ hybridization analysis in (A-J). (L) Wip1-mediated neural induction is inhibited by Smad4 with a phosphorylatable or phospho-mimetic amino acid at the MAPK site. (-), no co-injection of wt or mutated Smad4.(M) MDA-MB231 and MDA-MB468 cells were transfected with wt Wip1 alone or with wt Smad4 or Smad4(T277A) and then subjected to CCK-8 assays to measure cell growth. Data are presented as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.(N) Anchorage-independent colony-formation assays were performed in MDA-MB231 and MDA-MB468 cells transfected with Wip1 or negative control constructs. Scale bar, 100 μm. (O) Quantification of the assays shown in (N). The number of colonies larger than 50 μm in diameter was scored. Data are expressed as the mean ± SEM (n=3 biological replicates). *P<0.05 by unpaired Student's t-test.


Figure 2E - H Experimental glucose uptake flux ( E ) , acetate flux ( F ) , and growth rate ( G ) of E . coli grown on glucose ( 15 mM ) with different concentrations of αMG ( 0 - 100 mM ) , at low ( 0 mM ) and high ( 30 mM ) acetate concentrations . These data were used to quantify the total carbon uptake flux as a function of the αMG concentration ( H ) . Each data point represents an independent biological replicate , and lines represent the best polynomial fits . I , J Total carbon uptake ( I ) and growth rate ( J ) as a function of the glucose uptake flux , at low ( 0 mM ) and high ( 30 mM ) acetate concentrations . Each data point represents an independent biological replicate , and lines represent the best linear fits . K , L Relationships between the acetate flux and glucose uptake ( K ) , and between the contribution of acetate to carbon uptake and glucose uptake ( L ) . Each data point represents an independent biological replicate , and lines represent the best polynomial fits .

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Figure 2E-H Experimental glucose uptake flux (E), acetate flux (F), and growth rate (G) of E. coli grown on glucose (15 mM) with different concentrations of αMG (0-100 mM), at low (0 mM) and high (30 mM) acetate concentrations. These data were used to quantify the total carbon uptake flux as a function of the αMG concentration (H). Each data point represents an independent biological replicate, and lines represent the best polynomial fits.I, J Total carbon uptake (I) and growth rate (J) as a function of the glucose uptake flux, at low (0 mM) and high (30 mM) acetate concentrations. Each data point represents an independent biological replicate, and lines represent the best linear fits. K, L Relationships between the acetate flux and glucose uptake (K), and between the contribution of acetate to carbon uptake and glucose uptake (L). Each data point represents an independent biological replicate, and lines represent the best polynomial fits.


Figure 3C Experimental acetate flux of E . coli K - 12 MG1655 grown on glucose ( 15 mM ) at different acetate concentrations in the presence of αMG ( 0 , 20 , 40 or 100 mM ) . Each data point represents an independent biological replicate , and the lines represent best fits using a logistic function . D Experimental acetate concentration threshold at which the acetate flux reverses as a function of the αMG concentration , determined from the best fits shown in panel ( C ) .

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Figure 3C Experimental acetate flux of E. coli K-12 MG1655 grown on glucose (15 mM) at different acetate concentrations in the presence of αMG (0, 20, 40 or 100 mM). Each data point represents an independent biological replicate, and the lines represent best fits using a logistic function.D Experimental acetate concentration threshold at which the acetate flux reverses as a function of the αMG concentration, determined from the best fits shown in panel (C).


Figure 4B Experimental growth rate of E . coli K - 12 MG1655 grown on glucose ( 15 mM ) as a function of the acetate concentration at high ( 0 mM αMG , light blue , low ( 100 mM αMG , dark blue ) glycolytic flux . Each data point represents an independent biological replicate and the lines represent best polynomial fits . C Relative change in the growth rate of E . coli K - 12 BW25113 wild - type in the presence of 10 mM acetate at high ( 0 mM αMG ) and low ( 100 mM αMG ) glycolytic flux . Mean values ± standard deviations ( error bars ) were estimated from n independent biological replicates , as indicated on the figure . D Relative change in the growth rates of E . coli K - 12 BW25113 Δpgi and ΔpfkA strains in the presence of 10 mM acetate . Mean values ± standard deviations ( error bars ) were estimated from three independent biological replicates .

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Figure 4B Experimental growth rate of E. coli K-12 MG1655 grown on glucose (15 mM) as a function of the acetate concentration at high (0 mM αMG, light blue, low (100 mM αMG, dark blue) glycolytic flux. Each data point represents an independent biological replicate and the lines represent best polynomial fits.C Relative change in the growth rate of E. coli K-12 BW25113 wild-type in the presence of 10 mM acetate at high (0 mM αMG) and low (100 mM αMG) glycolytic flux. Mean values ± standard deviations (error bars) were estimated from n independent biological replicates, as indicated on the figure.D Relative change in the growth rates of E. coli K-12 BW25113 Δpgi and ΔpfkA strains in the presence of 10 mM acetate. Mean values ± standard deviations (error bars) were estimated from three independent biological replicates.


Figure 6Relative change in the growth rates of wild - type , Δpta , and Δacs E . coli K - 12 BW25113 strains in the presence of 10 mM acetate at low glycolytic flux ( 100 mM αMG ) . Mean values and standard deviations ( error bars ) were estimated from n independent biological replicates , as indicated on the figure .

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Figure 6Relative change in the growth rates of wild-type, Δpta, and Δacs E. coli K-12 BW25113 strains in the presence of 10 mM acetate at low glycolytic flux (100 mM αMG). Mean values and standard deviations (error bars) were estimated from n independent biological replicates, as indicated on the figure.


Figure 7A , B Impact of the presence of 10 mM acetate on the growth rate of wild - type , Δpta , and Δacs E . coli K - 12 BW25113 strains grown on ( A ) 30 mM glycerol and ( B ) 15 mM galactose . Mean values and standard deviations ( error bars ) were estimated from three independent biological replicates .

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Figure 7A,B Impact of the presence of 10 mM acetate on the growth rate of wild-type, Δpta, and Δacs E. coli K-12 BW25113 strains grown on (A) 30 mM glycerol and (B) 15 mM galactose. Mean values and standard deviations (error bars) were estimated from three independent biological replicates.


Figure 1WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Lethality was followed - up . LPS - treated mice ( n = 5 ) are depicted as black circles and DEX / LPS - treated mice ( n = 5 ) are depicted as white circles . P - values for survival curves were analyzed with a Fisher ' s exact test . WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Blood samples were taken 2 hours after LPS and TNF levels were measured in serum ( n = 4 - 5 ) . WT C57BL / 6J mice were injected with 12 . 5 mg / kg LPS , with or without pre - treatment with 10 mg / kg DEX 30 minutes before LPS . Three hours after LPS challenge , dextran labeled with FITC was administered by oral gavage . Five hours after gavaging , blood plasma was collected and fluorescence was measured as a degree of intestinal permeability ( λexc / λem = 488 / 520 nm ) ( PBS : n = 3 , LPS and DEX / LPS : n = 5 ) . RNA sequencing ( RNAseq ) results of IECs of WT mice ( n = 3 ) treated with PBS or 12 . 5 mg / kg LPS for 6 hours . Heatmap with selection of LPS - induced genes . Color key shows the log2 of normalized counts . WT mice ( n = 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated and gene expression was measured via qPCR .

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Figure 1WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Lethality was followed-up. LPS-treated mice (n=5) are depicted as black circles and DEX/LPS-treated mice (n=5) are depicted as white circles. P-values for survival curves were analyzed with a Fisher's exact test.WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Blood samples were taken 2 hours after LPS and TNF levels were measured in serum (n=4-5).WT C57BL/6J mice were injected with 12.5 mg/kg LPS, with or without pre-treatment with 10 mg/kg DEX 30 minutes before LPS. Three hours after LPS challenge, dextran labeled with FITC was administered by oral gavage. Five hours after gavaging, blood plasma was collected and fluorescence was measured as a degree of intestinal permeability (λexc/λem = 488/520 nm) (PBS: n=3, LPS and DEX/LPS: n=5).RNA sequencing (RNAseq) results of IECs of WT mice (n=3) treated with PBS or 12.5 mg/kg LPS for 6 hours. Heatmap with selection of LPS-induced genes. Color key shows the log2 of normalized counts.WT mice (n=5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated and gene expression was measured via qPCR.


Figure 2WT mice ( n = 3 - 5 ) were treated with PBS or 12 . 5 mg / kg LPS with or without 10 mg / kg DEX pre - treatment . Ileum was isolated 6 hours after LPS challenge . ( A ) p - MLKL and MLKL protein levels and ( B ) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control . Expression levels were quantified using FIJI and normalized to ACTIN levels . P - values were calculated using 1 - way ANOVA . Gene expression ( Defa6 , lysozyme ) was measured via qPCR . P - values were calculated using 1 - way ANOVA . All bars represent mean ± SEM . * * * * P < 0 . 0001 ; * * * P < 0 . 001 ; * * P ≤ 0 . 01 ; * P ≤ 0 . 05 . H & E staining was performed on ileum samples . Scale bar : 50µm . Lysozyme expression ( in red ) was detected via IHC , with Hoechst counterstaining . Scale bars : 50µm . Quantification of lysozyme expression . For every picture ( n = 3 - 4 per treatment ) , a selection of 10 crypts was made . Lysozyme positive signal was quantified in this region of interest ( ROI ) using an automated script in QuPath . Values are the positive area relative to total area of ROI .

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Figure 2WT mice (n=3-5) were treated with PBS or 12.5 mg/kg LPS with or without 10 mg/kg DEX pre-treatment. Ileum was isolated 6 hours after LPS challenge. (A) p-MLKL and MLKL protein levels and (B) RIPK3 protein levels were analyzed via western blot using ACTIN as a loading control. Expression levels were quantified using FIJI and normalized to ACTIN levels. P-values were calculated using 1-way ANOVA.Gene expression (Defa6, lysozyme) was measured via qPCR. P-values were calculated using 1-way ANOVA. All bars represent mean ± SEM. ****P< 0.0001; *** P<0.001; ** P≤ 0.01; * P≤ 0.05.H&E staining was performed on ileum samples. Scale bar: 50µm.Lysozyme expression (in red) was detected via IHC, with Hoechst counterstaining. Scale bars: 50µm. Quantification of lysozyme expression. For every picture (n=3-4 per treatment), a selection of 10 crypts was made. Lysozyme positive signal was quantified in this region of interest (ROI) using an automated script in QuPath. Values are the positive area relative to total area of ROI.


Figure 3TNFR1 + / + ( n = 9 ) and TNFR1 - / - mice ( n = 11 ) were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . TLR4fl / fl mice ( n = 7 ) and TLR4VillinKO ( n = 9 ) mice were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . TNFR1fl / fl mice ( n = 13 ) and TNFR1VillinKO ( n = 10 ) mice were treated with 12 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . TNFR1 + / + and TNFR1 - / - mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are shown on the graph . TNFR1fl / fl and TNFR1VillinKO mice ( n = 4 - 6 ) were treated with 12 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are shown on the graph .

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Figure 3TNFR1+/+ (n=9) and TNFR1-/- mice (n=11) were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TLR4fl/fl mice (n=7) and TLR4VillinKO (n=9) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNFR1fl/fl mice (n=13) and TNFR1VillinKO (n=10) mice were treated with 12.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNFR1+/+ and TNFR1-/- mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.TNFR1fl/fl and TNFR1VillinKO mice (n=4-6) were treated with 12.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are shown on the graph.


Figure 4GRfl / fl ( n = 8 , ) and GRLysMKO mice ( n = 4 ) were treated with 2 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . GRLysMKO mice were treated with PBS ( n = 12 ) or 10 mg / kg DEX ( n = 13 ) before a lethal dose of LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRLysMKO mice ( n = 3 ) . Mice were treated with 2 . 5 mg / kg LPS and blood was drawn 2 hours later . TNF concentration was measured with ELISA . TNF measurement in the serum of GRfl / fl and GRLysMKO ( n = 4 ) . Mice were treated with PBS or a lethal dose of LPS ( 12 . 5 and 2 . 5 mg / kg for GRfl / fl and GRLysMKO mice respectively ) , with or without pre - treatment with 10 mg / kg DEX and blood was drawn 2 hours later . TNF concentration was measured with ELISA . GRfl / fl ( n = 4 ) and GRLysMKO mice ( n = 3 - 4 ) were treated with 2 . 5 mg / kg LPS . Ileum was isolated after 6 hours and gene expression was measured via qPCR . Fold changes are depicted on the graphs . GRfl / fl and GRLysMKO mice ( n = 4 ) were treated with a PBS or LPS ( 12 . 5 and 2 . 5 mg / kg for GRfl / fl and GRLysMKO mice respectively ) with or without 10 mg / kg DEX pre - treatment of 30 minutes . Ileum was isolated after 6 hours and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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Figure 4GRfl/fl (n=8,) and GRLysMKO mice (n=4) were treated with 2.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.GRLysMKO mice were treated with PBS (n=12) or 10 mg/kg DEX (n=13) before a lethal dose of LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNF measurement in the serum of GRfl/fl (n=4) and GRLysMKO mice (n=3). Mice were treated with 2.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.TNF measurement in the serum of GRfl/fl and GRLysMKO (n=4). Mice were treated with PBS or a lethal dose of LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) , with or without pre-treatment with 10 mg/kg DEX and blood was drawn 2 hours later. TNF concentration was measured with ELISA.GRfl/fl (n=4) and GRLysMKO mice (n=3-4) were treated with 2.5 mg/kg LPS. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.GRfl/fl and GRLysMKO mice (n=4) were treated with a PBS or LPS (12.5 and 2.5 mg/kg for GRfl/fl and GRLysMKO mice respectively) with or without 10 mg/kg DEX pre-treatment of 30 minutes. Ileum was isolated after 6 hours and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


Figure 5GRfl / fl ( n = 8 ) and GRVillinKO mice ( n = 10 ) were treated with 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . GRVillinKO mice were treated with PBS ( n = 9 ) or 10 mg / kg DEX ( n = 10 ) before 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) . Mice were treated with 5 mg / kg LPS and blood was drawn 2 hours later . TNF measurement in the serum of GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) . Mice were treated with PBS or LPS ( 12 . 5 and 5 mg / kg for GRfl / fl and GRVillinKO mice respectively ) with or without pre - treatment with 10 mg / kg DEX for 30 minutes . Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA . GRVillinKO mice were treated with PBS ( n = 8 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 9 ) 2 hours before and 8 hours after 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . Ileum was isolated from non - stimulated GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) and gene expression was measured via qPCR . Expression levels are shown as fold induction with GRfl / fl expression values set at 1 . P - values were calculated using a student ' s t - test . GRfl / fl ( n = 4 ) and GRVillinKO mice ( n = 4 ) were treated with 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR .

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Figure 5GRfl/fl (n=8) and GRVillinKO mice (n=10) were treated with 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.GRVillinKO mice were treated with PBS (n=9) or 10 mg/kg DEX (n=10) before 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n= 4). Mice were treated with 5 mg/kg LPS and blood was drawn 2 hours later.TNF measurement in the serum of GRfl/fl (n=4) and GRVillinKO mice (n=4). Mice were treated with PBS or LPS (12.5 and 5 mg/kg for GRfl/fl and GRVillinKO mice respectively) with or without pre-treatment with 10 mg/kg DEX for 30 minutes. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.GRVillinKO mice were treated with PBS (n=8) or a neutralizing P55 (TNFR1) antibody (n=9) 2 hours before and 8 hours after 5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.Ileum was isolated from non-stimulated GRfl/fl (n=4) and GRVillinKO mice (n=4) and gene expression was measured via qPCR. Expression levels are shown as fold induction with GRfl/fl expression values set at 1. P-values were calculated using a student's t-test.GRfl/fl (n=4) and GRVillinKO mice (n=4) were treated with 5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR.


Figure 6Left : LPS dose - response curves of GRwt / wt ( n = 3 - 7 per LPS dose ) and GRdim / dim mice ( n = 3 - 7 per LPS dose ) treated with 1 . 5 mg / kg LPS . Right : LD50 values of LPS are depicted on top of each bar for each group . GRdim / dim mice were treated with PBS ( n = 5 ) or 10 mg / kg DEX ( n = 5 ) before 1 . 5 mg / kg LPS and survival was monitored . P - values were analyzed with a Fisher ' s exact test . TNF measurement in the serum of GRwt / wt ( n = 5 ) and GRdim / dim mice ( n = 5 ) . Mice were treated with 1 . 5 mg / kg LPS and blood was drawn 2 hours later . TNF concentration was measured with ELISA . TNF measurement in the serum of GRwt / wt and GRdim / dim mice ( n = 4 ) treated with PBS or LPS ( 12 . 5 and 1 . 5 mg / kg in GRwt / wt and GRdim / dim mice respectively ) with or without pretreatment with 10 mg / kg DEX . Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA . GRdim / dim mice were treated with PBS ( n = 6 ) or a neutralizing P55 ( TNFR1 ) antibody ( n = 8 ) 2 hours before and 8 hours after 1 . 5 mg / kg LPS and lethality was monitored . P - values were analyzed with a Fisher ' s exact test . Tofacitinib ( n = 6 ) or vehicle ( n = 7 ) was given orally to GRdim / dim mice . After 1 . 5 mg / kg LPS injection survival was monitored and p - values were analyzed with a Fisher ' s exact test . GRwt / wt ( n = 4 ) and GRdim / dim ( n = 4 ) mice were treated with 1 . 5 mg / kg LPS . Ileum was isolated and gene expression was measured via qPCR . Fold changes are depicted on the graphs . GRdim / dim mice ( n = 4 ) were treated with PBS or 1 . 5 mg / kg LPS with or without 10 mg / kg DEX pretreatment . Ileum was isolated and gene expression was measured via qPCR . Fold changes are depicted on the graphs .

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Figure 6Left: LPS dose-response curves of GRwt/wt (n=3-7 per LPS dose) and GRdim/dim mice (n=3-7 per LPS dose) treated with 1.5 mg/kg LPS. Right: LD50 values of LPS are depicted on top of each bar for each group.GRdim/dim mice were treated with PBS (n=5) or 10 mg/kg DEX (n=5) before 1.5 mg/kg LPS and survival was monitored. P-values were analyzed with a Fisher's exact test.TNF measurement in the serum of GRwt/wt (n=5) and GRdim/dim mice (n= 5). Mice were treated with 1.5 mg/kg LPS and blood was drawn 2 hours later. TNF concentration was measured with ELISA.TNF measurement in the serum of GRwt/wt and GRdim/dim mice (n=4) treated with PBS or LPS (12.5 and 1.5 mg/kg in GRwt/wt and GRdim/dim mice respectively) with or without pretreatment with 10 mg/kg DEX. Blood was taken 2 hours after LPS challenge and TNF concentrations were measured with ELISA.GRdim/dim mice were treated with PBS (n=6) or a neutralizing P55 (TNFR1) antibody (n=8) 2 hours before and 8 hours after 1.5 mg/kg LPS and lethality was monitored. P-values were analyzed with a Fisher's exact test.Tofacitinib (n=6) or vehicle (n=7) was given orally to GRdim/dim mice. After 1.5 mg/kg LPS injection survival was monitored and p-values were analyzed with a Fisher's exact test.GRwt/wt (n=4) and GRdim/dim (n=4) mice were treated with 1.5 mg/kg LPS. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.GRdim/dim mice (n=4) were treated with PBS or 1.5 mg/kg LPS with or without 10 mg/kg DEX pretreatment. Ileum was isolated and gene expression was measured via qPCR. Fold changes are depicted on the graphs.


Figure 1d / e : Density gradient centrifugation analysis of virus - containing supernatant . Forty milliliters of culture supernatant was harvested at 72 hours post ZIKV infection ( MOI = 0 . 01 ) and concentrated to 1 ml , followed by gradient centrifugation as described in the methods . Fractions were analyzed by Western blot with the indicated antibodies ( d ) . The ZIKV titer of each fraction was measured by plaque assay , and the viral genome was determined via RT - PCR ( e ) . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Dashed line represents infectivity detection limit . f / g : Density gradient centrifugation analysis of UC - EVs . UC - EVs ( 0 . 1 ml ) were loaded onto the top of a density gradient in a centrifugation tube as described in the methods . The fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies ( f ) , the ZIKV titer of each fraction was measured by plaque assay , and the viral genome was quantified via RT - PCR ( g ) . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Dashed line represents infectivity detection limit .

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Figure 1d/e: Density gradient centrifugation analysis of virus-containing supernatant. Forty milliliters of culture supernatant was harvested at 72 hours post ZIKV infection (MOI=0.01) and concentrated to 1 ml, followed by gradient centrifugation as described in the methods. Fractions were analyzed by Western blot with the indicated antibodies (d). The ZIKV titer of each fraction was measured by plaque assay, and the viral genome was determined via RT-PCR (e). Data represent the means from three biological replicates; error bars represent standard deviations from the means. Dashed line represents infectivity detection limit.f/g: Density gradient centrifugation analysis of UC-EVs. UC-EVs (0.1 ml) were loaded onto the top of a density gradient in a centrifugation tube as described in the methods. The fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies (f), the ZIKV titer of each fraction was measured by plaque assay, and the viral genome was quantified via RT-PCR (g). Data represent the means from three biological replicates; error bars represent standard deviations from the means. Dashed line represents infectivity detection limit.


Figure 2b : Western blots of ZIKV - infected cell - derived UC - EVs or IC - EVs . Samples during purification were subjected to western blot analysis with the indicated antibodies against cellular proteins . Total lysate of ZIKV - infected cells was served as positive control . d : Density gradient centrifugation analysis of IC - EVs . ZIKV - infected cell - derived UC - EVs ( 0 . 1 ml ) were subjected to density gradient analysis similarly . Fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies .

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Figure 2b: Western blots of ZIKV-infected cell-derived UC-EVs or IC-EVs. Samples during purification were subjected to western blot analysis with the indicated antibodies against cellular proteins. Total lysate of ZIKV-infected cells was served as positive control.d: Density gradient centrifugation analysis of IC-EVs. ZIKV-infected cell-derived UC-EVs (0.1 ml) were subjected to density gradient analysis similarly. Fractions were obtained from the top of gradient and analyzed by Western blot with the indicated antibodies.


Figure 3Detection of infectivity ( d e ) of UC - EVs pre - and post CD9 - capture or E - capture . UC - EV was subjected to CD9 - capture and E - capture respectively . After 1hour incubation , ZIKV infectivity of pre - incubation and post - incubation samples was measured . f : The ZIKV infectious titer of each fraction was measured by plaque assay , and the number of viral genomes was determined via RT - PCR . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Dashed line represents infectivity detection limit .

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Figure 3Detection of infectivity(d e) of UC-EVs pre- and post CD9-capture or E-capture. UC-EV was subjected to CD9-capture and E-capture respectively. After 1hour incubation, ZIKV infectivity of pre-incubation and post-incubation samples was measured.f: The ZIKV infectious titer of each fraction was measured by plaque assay, and the number of viral genomes was determined via RT-PCR. Data represent the means from three biological replicates; error bars represent standard deviations from the means. Dashed line represents infectivity detection limit.


Figure 4d : Western blots of ZIKV - infected cell - derived UC - EVs or IC - EVs using indicated antibodies against viral proteins and EV proteins . Total lysate of ZIKV - infected cells was the positive control .

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Figure 4d: Western blots of ZIKV-infected cell-derived UC-EVs or IC-EVs using indicated antibodies against viral proteins and EV proteins. Total lysate of ZIKV-infected cells was the positive control.


Figure 5a : ZIKV E and CD9 distribution in a density gradient . After density gradient centrifugation , all fractions were analyzed by Western blotting with anti - ZIKV E and anti - CD9 antibodies . CD9 blots was reused from Figure 2d . d : Quantification of the number of ZIKV E proteins on the EV surface . The percentage of 10nm gold sphere - labeled IC - EVs was counted based on the immuno - EM images of IC - EVs . N indicates the total number of counted IC - EVs .

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Figure 5a: ZIKV E and CD9 distribution in a density gradient. After density gradient centrifugation, all fractions were analyzed by Western blotting with anti-ZIKV E and anti-CD9 antibodies. CD9 blots was reused from Figure 2d.d: Quantification of the number of ZIKV E proteins on the EV surface. The percentage of 10nm gold sphere-labeled IC-EVs was counted based on the immuno-EM images of IC-EVs. N indicates the total number of counted IC-EVs.


Figure 6c : Detection of ADE - induced ZIKV infection in the presence of the ZIKV - specific neutralizing antibody 2B10 and IC - EVs derived from ZIKV - infected cells by ADE assay . K562 cells were incubated with a mixture of EV - free ZIKV , neutralizing antibody , and IC - EVs . Forty - eight hours post incubation ; cells were stained for ZIKV E and analyzed by FACS . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Differences between treatments were analyzed using the two - tailed , unpaired Student t test available in the GraphPad Prism 5 software package . P values of less than 0 . 05 ( indicated by asterisks ) were considered statistically significant . The following categories were used : * * * , P 0 . 001 ; * * , P 0 . 01 ; * , P 0 . 05 .

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Figure 6c: Detection of ADE-induced ZIKV infection in the presence of the ZIKV-specific neutralizing antibody 2B10 and IC-EVs derived from ZIKV-infected cells by ADE assay. K562 cells were incubated with a mixture of EV-free ZIKV, neutralizing antibody, and IC-EVs. Forty-eight hours post incubation; cells were stained for ZIKV E and analyzed by FACS. Data represent the means from three biological replicates; error bars represent standard deviations from the means. Differences between treatments were analyzed using the two-tailed, unpaired Student t test available in the GraphPad Prism 5 software package. P values of less than 0.05 (indicated by asterisks) were considered statistically significant. The following categories were used: ***, P 0.001; **, P 0.01; *, P 0.05.


Figure 7c / d : IC - EVs affected the ADE of DENV2 ( c ) or ZIKV ( d ) mediated by DENV3 patient serum . Diluted sera were mixed with EV - free viruses ( DENV2 or ZIKV ) and IC - EVs derived from DENV2 - or ZIKV - infected cells and then incubated with K562 cells as described in the methods . At 48 hours post incubation , cells were stained with an antibody against flavivirus E and analyzed by FACS . Data represent the means from three biological replicates ; error bars represent standard deviations from the means . Differences between treatments were analyzed using the two - tailed , unpaired Student t test available in the GraphPad Prism 5 software package . P values of less than 0 . 05 ( indicated by asterisks ) were considered statistically significant . The following categories were used : * * * , P 0 . 001 ; * * , P 0 . 01 ; * , P 0 . 05 .

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Figure 7c/d: IC-EVs affected the ADE of DENV2 (c) or ZIKV (d) mediated by DENV3 patient serum. Diluted sera were mixed with EV-free viruses (DENV2 or ZIKV) and IC-EVs derived from DENV2- or ZIKV-infected cells and then incubated with K562 cells as described in the methods. At 48 hours post incubation, cells were stained with an antibody against flavivirus E and analyzed by FACS. Data represent the means from three biological replicates; error bars represent standard deviations from the means. Differences between treatments were analyzed using the two-tailed, unpaired Student t test available in the GraphPad Prism 5 software package. P values of less than 0.05 (indicated by asterisks) were considered statistically significant. The following categories were used: ***, P 0.001; **, P 0.01; *, P 0.05.


Figure 1 . The structure of SARS - CoV - 2 monomer .

Figure 1.The structure of SARS-CoV-2 monomer.


Figure 1SDS - PAGE of SecYEG - ND sample after size - exclusion chromatography . Asterisks indicate translocon - enriched fractions used for forming the RNC FtsQ : SecYEG - ND complex . Lipid - loaded " empty " nanodiscs elute at larger volumes and so can be separated .

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Figure 1SDS-PAGE of SecYEG-ND sample after size-exclusion chromatography. Asterisks indicate translocon-enriched fractions used for forming the RNC FtsQ:SecYEG-ND complex. Lipid-loaded "empty" nanodiscs elute at larger volumes and so can be separated.


Figure 2Representative cryo - EM micrograph of RNC FtsQ : SecYEG - ND . Exemplary individual ribosomes are encircled . Examples of two - dimensional classes of imaged particles . RNC : nanodisc assemblies can be seen at different view angles .

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Figure 2Representative cryo-EM micrograph of RNC FtsQ:SecYEG-ND. Exemplary individual ribosomes are encircled. Examples of two-dimensional classes of imaged particles. RNC:nanodisc assemblies can be seen at different view angles.


Figure 5Western - blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross - linking product of ~ 80 kDa in presence of nanodisc - reconstituted SecYC282EG and SecYC283EG , presumably assigned to FtsQxSecY adduct . The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa - histidine tag by 3C protease . In - gel fluorescence imaging of SecYCF488A reveals a cross - linking product of ~ 80 kDa assigned to the FtsQC40xSecYC283 adduct ( " SecYxFtsQ " ) . Bands for covalently cross - linked SecY dimer ( " SecYxSecY " ) , and adducts of SecY and ribosomal proteins ( " SecYxRiboP " ) are indicated . The nascent chain - specific adduct " SecYxFtsQ " does not depend on the presence of PE lipids . FtsQ : SecY cross - linking is position specific . FtsQ TMH C - terminal residues 40 - 42 could be cross - linked to the lateral gate ( SecY residue 283 ) , while no adduct could be detected at the upstream position 35 .

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Figure 5Western-blot against the hemagglutinin tag within the nascent chain FtsQC40 reveals a cross-linking product of ~80 kDa in presence of nanodisc-reconstituted SecYC282EG and SecYC283EG, presumably assigned to FtsQxSecY adduct. The double band for FtsQC40 likely indicates the incomplete cleavage of the hexa-histidine tag by 3C protease.In-gel fluorescence imaging of SecYCF488A reveals a cross-linking product of ~80 kDa assigned to the FtsQC40xSecYC283 adduct ("SecYxFtsQ"). Bands for covalently cross-linked SecY dimer ("SecYxSecY"), and adducts of SecY and ribosomal proteins ("SecYxRiboP") are indicated. The nascent chain-specific adduct "SecYxFtsQ" does not depend on the presence of PE lipids.FtsQ:SecY cross-linking is position specific. FtsQ TMH C-terminal residues 40-42 could be cross-linked to the lateral gate (SecY residue 283), while no adduct could be detected at the upstream position 35.


Figure 1D . Relative FCR activities in the roots of wild - type plants and photoreceptor mutants grown under white light with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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Figure 1D. Relative FCR activities in the roots of wild-type plants and photoreceptor mutants grown under white light with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


Figure 2D - F . Fe contents of whole plants , FER expression in roots and relative FCR activities in the roots of plants grown under Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Plants were exposed to high ( H - , 2 . 0 ) or low ( L - , 0 . 5 ) red ( R ) to far red ( FR ) ratios .

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Figure 2D-F. Fe contents of whole plants, FER expression in roots and relative FCR activities in the roots of plants grown under Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d. Plants were exposed to high (H-, 2.0) or low (L-, 0.5) red (R) to far red (FR) ratios.


Figure 3C . Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light ( 200 µmol m - 2s - 1 ) for 7 d . Rubisco and β - Actin were used as loading controls for leaves and roots in the western blot analysis , respectively . The values shown above each lane indicate the relative abundance of the HY5 protein . D . Accumulation of HY5 protein in the roots of WT and phyB plants under high ( H , 2 . 0 ) or low ratio of ( L , 0 . 5 ) red ( R ) to far red ( FR ) light conditions . β - Actin was used as loading controls in the western blot analysis . The values shown above each lane indicate the relative abundance of the HY5 protein .

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Figure 3C. Accumulation of HY5 protein in leaves and roots of reciprocal grafts of phyB and WT plants grown under white light (200 µmol m-2s-1) for 7 d. Rubisco and β-Actin were used as loading controls for leaves and roots in the western blot analysis, respectively. The values shown above each lane indicate the relative abundance of the HY5 protein. D. Accumulation of HY5 protein in the roots of WT and phyB plants under high (H, 2.0) or low ratio of (L, 0.5) red (R) to far red (FR) light conditions. β-Actin was used as loading controls in the western blot analysis. The values shown above each lane indicate the relative abundance of the HY5 protein.


Figure 4D . Fe content of whole plants . WT and hy5 plants were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) light conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . E . FER expression in roots . WT and hy5 plants were grown under high ( H ) or low ratios of ( L ) red ( R ) to far red ( FR ) light conditions with either Fe - sufficient ( 20 µM , + Fe ) or Fe - deficient ( 2 µM , - Fe ) conditions for 7 d .

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Figure 4D. Fe content of whole plants. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.E. FER expression in roots. WT and hy5 plants were grown under high (H) or low ratios of (L) red (R) to far red (FR) light conditions with either Fe-sufficient (20 µM, +Fe) or Fe-deficient (2 µM, -Fe) conditions for 7 d.


Figure 5E . Direct binding of HY5 to the FER promoter analyzed using ChIP - qPCR in 35S - HY5 - 3HA - overexpressing ( HY5 - OE ) tomato plants . WT and HY5 - OE plants were exposed to white light ( 200 µmol m - 2s - 1 ) grown under Fe - deficient ( 2 µM , - Fe ) conditions for 7 d . Input chromatin was isolated from root samples at 7 d . The epitope - tagged HY5 - chromatin complex was immunoprecipitated with an anti - HA antibody . A control reaction was processed side - by - side using mouse IgG . Input - and ChIP - DNA samples were quantified by RT - qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in ( D ) . The ChIP results are presented as percentage of the input DNA .

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Figure 5E. Direct binding of HY5 to the FER promoter analyzed using ChIP-qPCR in 35S-HY5-3HA-overexpressing (HY5-OE) tomato plants. WT and HY5-OE plants were exposed to white light (200 µmol m-2s-1) grown under Fe-deficient (2 µM, -Fe) conditions for 7 d. Input chromatin was isolated from root samples at 7 d. The epitope-tagged HY5-chromatin complex was immunoprecipitated with an anti-HA antibody. A control reaction was processed side-by-side using mouse IgG. Input- and ChIP-DNA samples were quantified by RT-qPCR using primers specific for the promoter and exon fragment of the FER gene as indicated in (D). The ChIP results are presented as percentage of the input DNA.


Figure 6A - C . Fe content of whole plants ( A ) and the photochemical efficiencies of PSI and PSII [ Y ( I ) and Y ( II ) ] ( B and C ) in young leaves of grafted tomato plants with grafts between WT / WT and WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d . D - F . Transcript levels of FER , FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT / WT and WT / fer grown under H - R / FR ( 2 . 0 ) or L - R / FR ( 0 . 5 ) conditions for 7 d .

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Figure 6A-C. Fe content of whole plants (A) and the photochemical efficiencies of PSI and PSII [Y(I) and Y(II)] (B and C) in young leaves of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.D-F. Transcript levels of FER, FRO1 and IRT1 in the roots of grafted tomato plants with grafts between WT/WT and WT/fer grown under H-R/FR (2.0) or L-R/FR (0.5) conditions for 7 d.


Figure 7B . The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe - EDTA in nutrient solution in a growth room . D . Fe content in the fruits of WT and phyB plants . Plants were grown in soil in a greenhouse .

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Figure 7B. The abundance of FER transcripts in the roots of WT and phyB plants grown under 20 μM Fe-EDTA in nutrient solution in a growth room.D. Fe content in the fruits of WT and phyB plants. Plants were grown in soil in a greenhouse.


Figure 1 ( A ) Coomassie ‐ stained gels showing the results of liposome co ‐ sedimentation assays using Folch liposomes and the indicated proteins . Liposome binding allows the protein to be pelleted . The star indicates a degradation product of the Atg5 - Atg12 conjugate . ( B , D ) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16 , respectively , and subjected to fractionation experiments . The proteins were detected by anti ‐ Myc western blotting . The presence of the protein in the pellet fraction indicated membrane binding . ( C ) Quantification showing the relative amounts of Atg5 - 9 × Myc and Atg5 - 9 × Myc - Atg12 in the pellet fractions relative to Pex30 . The amount of Atg5 - 9 × Myc and Atg5 - 9 × Myc - Atg12 in atg5Δ cells was set to 1 . See Supplementary Figure 1 for graph based on non ‐ normed data . The quantification is based on three independent experiments and the averages and the standard deviations are shown . The numbers next to the blots indicate the molecular weight in kDa . P , pellet ; S , supernatant . Figure source data can be found with the Supplementary data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Coomassie‐stained gels showing the results of liposome co‐sedimentation assays using Folch liposomes and the indicated proteins. Liposome binding allows the protein to be pelleted. The star indicates a degradation product of the Atg5-Atg12 conjugate.(B, D) Yeast cells of the indicated genotype were transformed with plasmids coding for Atg5 and Atg16, respectively, and subjected to fractionation experiments. The proteins were detected by anti‐Myc western blotting. The presence of the protein in the pellet fraction indicated membrane binding.(C) Quantification showing the relative amounts of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in the pellet fractions relative to Pex30. The amount of Atg5-9 × Myc and Atg5-9 × Myc-Atg12 in atg5Δ cells was set to 1. See Supplementary Figure 1 for graph based on non‐normed data. The quantification is based on three independent experiments and the averages and the standard deviations are shown. The numbers next to the blots indicate the molecular weight in kDa. P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


Figure 2 ( A ) Coomassie ‐ stained gels of liposome co ‐ sedimentation assays using either Atg5 - Atg12 / Atg16 ( upper panel ) or Atg5 / Atg16 ( lower panel ) and liposomes with the indicated lipid composition ( Folch : Folch lipids , PE / PI3P : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI3P , PE / PI : 40 % POPC , 35 % POPS , 20 % POPE , 5 % PI , DAG / PI3P : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI3P , DAG / PI : 40 % POPC , 35 % POPS , 20 % DAG , 5 % PI ) . Note that the Atg5 - Atg12 / Atg16 and the Atg5 / Atg16 complexes show almost identical binding behaviours . ( B ) Quantification showing the amounts of Atg5 - Atg12 / Atg16 and Atg5 / Atg16 in the pellet fractions . The amount of protein in the pellet fraction in the absence of liposomes ( background ) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes . Data were obtained from three independent experiments , one of which is shown in ( A ) . The Atg5 - Atg12 and Atg5 bands were used for quantification . ( C ) Coomassie ‐ stained gel of a liposome co ‐ sedimentation assay showing the lipid requirements of the Atg5 - Atg12 / Atg16 complex . Liposomes were composed of 40 % POPC , 35 % POPS , 20 % POPE and 5 % PI3P . In the liposomes containing no POPE or POPS , these lipids were replaced with POPC . ( D ) Quantification of Atg5 - Atg12 / Atg16 in the pellet fraction in liposome co ‐ sedimentation , one of which is shown in ( C ) . Data were obtained from six independent experiments . The Atg5 - Atg12 was used for quantification . The average value is shown and the error bars represent the standard deviation . P ‐ values were calculated using Student ' s t ‐ test . The numbers next to the gels indicate the molecular weight in kDa . DAG , diacylglycerol ; P , pellet ; PI , phosphatidylinositol ; PI3P , phosphatidylinositol ‐ 3 ‐ phosphate ; ( PO ) PC , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylcholine ; ( PO ) PE , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylethanolamine ; ( PO ) PS , ( 1 ‐ palmitoyl ‐ 2 ‐ oleoyl ‐ ) phosphatidylserine ; S , supernatant . Figure source data can be found with the Supplementary data .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Coomassie‐stained gels of liposome co‐sedimentation assays using either Atg5-Atg12/Atg16 (upper panel) or Atg5/Atg16 (lower panel) and liposomes with the indicated lipid composition (Folch: Folch lipids, PE/PI3P: 40% POPC, 35% POPS, 20% POPE, 5% PI3P, PE/PI: 40% POPC, 35% POPS, 20% POPE, 5% PI, DAG/PI3P: 40% POPC, 35% POPS, 20% DAG, 5% PI3P, DAG/PI: 40% POPC, 35% POPS, 20% DAG, 5% PI). Note that the Atg5-Atg12/Atg16 and the Atg5/Atg16 complexes show almost identical binding behaviours.(B) Quantification showing the amounts of Atg5-Atg12/Atg16 and Atg5/Atg16 in the pellet fractions. The amount of protein in the pellet fraction in the absence of liposomes (background) was subtracted from the amount of protein in the pellet fractions in the presence of liposomes. Data were obtained from three independent experiments, one of which is shown in (A). The Atg5-Atg12 and Atg5 bands were used for quantification.(C) Coomassie‐stained gel of a liposome co‐sedimentation assay showing the lipid requirements of the Atg5-Atg12/Atg16 complex. Liposomes were composed of 40% POPC, 35% POPS, 20% POPE and 5% PI3P. In the liposomes containing no POPE or POPS, these lipids were replaced with POPC.(D) Quantification of Atg5-Atg12/Atg16 in the pellet fraction in liposome co‐sedimentation, one of which is shown in (C). Data were obtained from six independent experiments. The Atg5-Atg12 was used for quantification. The average value is shown and the error bars represent the standard deviation. P‐values were calculated using Student's t‐test. The numbers next to the gels indicate the molecular weight in kDa. DAG, diacylglycerol; P, pellet; PI, phosphatidylinositol; PI3P, phosphatidylinositol‐3‐phosphate; (PO)PC, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylcholine; (PO)PE, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylethanolamine; (PO)PS, (1‐palmitoyl‐2‐oleoyl‐) phosphatidylserine; S, supernatant. Figure source data can be found with the Supplementary data.


Figure 3 ( A ) Coomassie ‐ stained gels showing co ‐ sedimentation assays using Folch liposomes and the indicated proteins . Atg5 - Atg12 / Atg16 was most efficient in recruiting Atg3 to the pellet fraction . ( B ) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins . The quantification is based on three independent experiments . ( C ) Coomassie ‐ stained gels showing the results of Atg8 - PE conjugation assays using the indicated proteins and E . coli lipids derived liposomes . Atg8 - PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea ‐ SDS gels . The numbers above the gels indicate the time in minutes . Note that at least under these conditions almost no Atg8 - PE conjugation was detected in the absence of the Atg5 - Atg12 conjugate or the Atg5 - Atg12 / Atg16 complex . The numbers next to the gels indicate the molecular weight in kDa . The averages and the error bars representing the standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ test . NS , non ‐ significant ; P , pellet ; S , supernatant . Figure source data can be found with the Supplementary data .

O O B-PANEL_START O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Coomassie‐stained gels showing co‐sedimentation assays using Folch liposomes and the indicated proteins. Atg5-Atg12/Atg16 was most efficient in recruiting Atg3 to the pellet fraction.(B) Quantification of Atg3 in the pellet fraction in the presence of the indicated proteins. The quantification is based on three independent experiments.(C) Coomassie‐stained gels showing the results of Atg8-PE conjugation assays using the indicated proteins and E. coli lipids derived liposomes. Atg8-PE conjugation was detected as characteristic band shift as the conjugated form runs faster on urea‐SDS gels. The numbers above the gels indicate the time in minutes. Note that at least under these conditions almost no Atg8-PE conjugation was detected in the absence of the Atg5-Atg12 conjugate or the Atg5-Atg12/Atg16 complex. The numbers next to the gels indicate the molecular weight in kDa. The averages and the error bars representing the standard deviations are shown. P‐values were calculated using Student's t‐test. NS, non‐significant; P, pellet; S, supernatant. Figure source data can be found with the Supplementary data.


Figure 4 ( A ) Confocal microscopy images showing eGFP - Atg8 recruitment to GUVs is dependent on the Atg5 - Atg12 / Atg16 complex and PE . The membrane was labelled by incorporation of rhodamine ‐ PE . ( B ) Single confocal section showing colocalization of Atg5 - Atg12 / Atg16 - eGFP and mCherry - Atg8 on GUVs . ( C ) Quantification of GUV ‐ associated eGFP - Atg8 in the presence of the Atg5 - Atg12 / Atg16 complex and the Atg5 - Atg12 conjugate . The first graph shows the percentage of GUVs associated with eGFP - Atg8 fluorescence . The second graph shows the intensity of eGFP - Atg8 on the GUVs . The quantification is based on four independent experiments . Inset : anti ‐ Atg12 western blot using the rabbit anti ‐ Atg12 antiserum showing the amounts of Atg5 - Atg12 / Atg16 complex and Atg5 - Atg12 conjugate in one of the assays used for the quantification shown in ( C ) . ( D ) Quantification of membrane recruitment of Atg3 - mCherry by the Atg5 - Atg12 / Atg16 complex , the Atg5 - Atg12 conjugate and a buffer control . The graph is based on two independent experiments . ( E ) Widefield images showing individual frames of a time series of GUVs incubated with Atg3 , Atg7 , Atg8 and the Atg5 - Atg12 / Atg16 complex ( see Materials and methods for details ) . The membrane was labelled by incorporation of rhodamine ‐ PE . The time is indicated in minutes . ( F ) Widefield images of GUVs incubated with Atg3 , Atg7 , eGFP - Atg8 and the Atg5 - Atg12 / Atg16 complex for 35 min ( left ) or 70 min ( right ) . The membrane was labelled by incorporation of rhodamine ‐ PE . Note the massive accumulation of eGFP - Atg8 at the interface between individual GUVs . The arrows point to some of these interfaces . For the quantifications , the averages and standard deviations are shown . P ‐ values were calculated using Student ' s t ‐ test . Scale bars : 10 μm . Figure source data can be found with the Supplementary data .

O O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Confocal microscopy images showing eGFP-Atg8 recruitment to GUVs is dependent on the Atg5-Atg12/Atg16 complex and PE. The membrane was labelled by incorporation of rhodamine‐PE.(B) Single confocal section showing colocalization of Atg5-Atg12/Atg16-eGFP and mCherry-Atg8 on GUVs.(C) Quantification of GUV‐associated eGFP-Atg8 in the presence of the Atg5-Atg12/Atg16 complex and the Atg5-Atg12 conjugate. The first graph shows the percentage of GUVs associated with eGFP-Atg8 fluorescence. The second graph shows the intensity of eGFP-Atg8 on the GUVs. The quantification is based on four independent experiments. Inset: anti‐Atg12 western blot using the rabbit anti‐Atg12 antiserum showing the amounts of Atg5-Atg12/Atg16 complex and Atg5-Atg12 conjugate in one of the assays used for the quantification shown in (C).(D) Quantification of membrane recruitment of Atg3-mCherry by the Atg5-Atg12/Atg16 complex, the Atg5-Atg12 conjugate and a buffer control. The graph is based on two independent experiments.(E) Widefield images showing individual frames of a time series of GUVs incubated with Atg3, Atg7, Atg8 and the Atg5-Atg12/Atg16 complex (see Materials and methods for details). The membrane was labelled by incorporation of rhodamine‐PE. The time is indicated in minutes.(F) Widefield images of GUVs incubated with Atg3, Atg7, eGFP-Atg8 and the Atg5-Atg12/Atg16 complex for 35 min (left) or 70 min (right). The membrane was labelled by incorporation of rhodamine‐PE. Note the massive accumulation of eGFP-Atg8 at the interface between individual GUVs. The arrows point to some of these interfaces. For the quantifications, the averages and standard deviations are shown. P‐values were calculated using Student's t‐test. Scale bars: 10 μm. Figure source data can be found with the Supplementary data.


Figure 5 ( A ) Frames of widefield images taken from a time series of rhodamine ‐ PE labelled GUVs in the presence of the Atg5 - Atg12 / Atg16 complex . For the control condition buffer but no protein was added . Note the increase in clustered GUVs over time . The numbers indicate the time in minutes . ( B ) Confocal images of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP complex . Note the accumulation of the complex at the interface between GUVs ( arrows ) . ( C ) Individual confocal microscopy frames of a time series of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . The arrows point to the GUV - GUV interface where the protein initially accumulated . ( D ) Confocal microscopy images of rhodamine ‐ PE labelled GUVs incubated with Atg5 - Atg12 / Atg16 - eGFP . The arrows point to the concentrations of Atg5 - Atg12 / Atg16 on the GUV membrane . Scale bars : 10 μm ( A - C ) and 3 μm ( D ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Frames of widefield images taken from a time series of rhodamine‐PE labelled GUVs in the presence of the Atg5-Atg12/Atg16 complex. For the control condition buffer but no protein was added. Note the increase in clustered GUVs over time. The numbers indicate the time in minutes.(B) Confocal images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP complex. Note the accumulation of the complex at the interface between GUVs (arrows).(C) Individual confocal microscopy frames of a time series of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the GUV-GUV interface where the protein initially accumulated.(D) Confocal microscopy images of rhodamine‐PE labelled GUVs incubated with Atg5-Atg12/Atg16-eGFP. The arrows point to the concentrations of Atg5-Atg12/Atg16 on the GUV membrane. Scale bars: 10 μm (A-C) and 3 μm (D).


Figure 6 ( A ) Widefield images of rhodamine ‐ PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5 - Atg12 / Atg16 and Atg5 / Atg16 complexes . ( B ) SUV aggregation assay using Folch liposomes and the indicated proteins . The proteins were added after a short delay . The symbols above , below the images or next to the curves represent Atg5 ( blue oval ) , Atg12 ( red circle ) and Atg16 ( green Y ) . The overall shape of the Atg5 - Atg12 / Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al ( 2010 ) . Scale bars : 10 μm .

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Figure 6(A) Widefield images of rhodamine‐PE labelled GUVs 30 min GUVs cluster only in the presence of the Atg5-Atg12/Atg16 and Atg5/Atg16 complexes.(B) SUV aggregation assay using Folch liposomes and the indicated proteins. The proteins were added after a short delay. The symbols above, below the images or next to the curves represent Atg5 (blue oval), Atg12 (red circle) and Atg16 (green Y). The overall shape of the Atg5-Atg12/Atg16 complex is according to the structural model of the complex as proposed in Fujioka et al (2010). Scale bars: 10 μm.


Figure 7 ( A ) Liposome co ‐ sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N ‐ terminal fragment of Atg16 ( amino acids 1 - 46 ) . ( B ) Quantification based on three co ‐ sedimentation assays as shown in ( A ) . The amount of protein pelleted in the absence of liposomes was subtracted . The averages and the standard deviations are shown . ( C ) Yeast cells were transformed with the indicated expression constructs and Atg5 - 2 × Myc was immunoprecipitated with an anti ‐ Myc antibody . The precipitated protein was subjected to anti ‐ Atg5 and anti ‐ Atg12western blotting . The substitution of R171 and K160 to glutamate caused the mutant protein to run higher . The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3 ) . ( D ) Anti ‐ Atg12 western blot of cell fractions prepared from rapamycin ‐ treated yeast cells . The anti ‐ Pgk1 and anti ‐ Pex30 served as control for the cytosolic and membrane fractions , respectively . ( E ) Anti ‐ Myc and anti ‐ Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E , R171E . ( F ) Starvation assay with the indicated yeast strains and the indicated rescue constructs . GFP was detected by western blotting . Pgk1 served as loading control . ( G ) Same assay as shown in ( F ) but using an anti ‐ Ape1 antiserum for western blotting . ( H ) Western blots of yeast cell lysates using an anti ‐ Atg8 antiserum . ( I ) Ape1 protease protection assay with the indicated yeast strains . The percentages below the blots indicate the percent of protected ApeI in the + protease K , − Triton X ‐ 100 lanes and are the average of three independent experiments ± the standard deviation . The numbers next to the blots indicate the molecular weight in kDa . Figure source data can be found with the Supplementary data .

O O B-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Liposome co‐sedimentation assay using Folch liposomes and the indicated Atg5 proteins stabilized with an N‐terminal fragment of Atg16 (amino acids 1-46).(B) Quantification based on three co‐sedimentation assays as shown in (A). The amount of protein pelleted in the absence of liposomes was subtracted. The averages and the standard deviations are shown.(C) Yeast cells were transformed with the indicated expression constructs and Atg5-2 × Myc was immunoprecipitated with an anti‐Myc antibody. The precipitated protein was subjected to anti‐Atg5 and anti‐Atg12western blotting. The substitution of R171 and K160 to glutamate caused the mutant protein to run higher. The same phenomenon was observed for the recombinant protein ( Supplementary Figure 3).(D) Anti‐Atg12 western blot of cell fractions prepared from rapamycin‐treated yeast cells. The anti‐Pgk1 and anti‐Pex30 served as control for the cytosolic and membrane fractions, respectively.(E) Anti‐Myc and anti‐Pex30western blots of yeast cell fractions from cells of the indicated genotype and expressing Atg16 and Atg5 or Atg5 K160E, R171E.(F) Starvation assay with the indicated yeast strains and the indicated rescue constructs. GFP was detected by western blotting. Pgk1 served as loading control.(G) Same assay as shown in (F) but using an anti‐Ape1 antiserum for western blotting.(H) Western blots of yeast cell lysates using an anti‐Atg8 antiserum.(I) Ape1 protease protection assay with the indicated yeast strains. The percentages below the blots indicate the percent of protected ApeI in the +protease K, −Triton X‐100 lanes and are the average of three independent experiments±the standard deviation. The numbers next to the blots indicate the molecular weight in kDa. Figure source data can be found with the Supplementary data.


Figure 8 ( A ) Yeast cells of the indicated genotype expressing Atg5 - 3 × mCherry or Atg5 K160E , R171E - 3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope . Maximum projections of z ‐ stacks are shown . ( B ) Graph showing the percentage of mCherry ‐ positive cells that display Atg5 - 3 × mCherry dots . In total , 3 independent experiments were conducted and > 400 cells were counted . ( C ) Graph showing the percentage of cells that display Atg16 - 3 × mCherry dots . In total , 3 independent experiments were conducted and > 750 cells were counted . The graphs show the averages and the error bars representing the standard deviations . P ‐ values were calculated using Student ' s t ‐ test . Scale bars : 5 μm .

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Figure 8(A) Yeast cells of the indicated genotype expressing Atg5-3 × mCherry or Atg5 K160E, R171E-3 × mCherry were treated with rapamycin and imaged using a Deltavision microscope. Maximum projections of z‐stacks are shown.(B) Graph showing the percentage of mCherry‐positive cells that display Atg5-3 × mCherry dots. In total, 3 independent experiments were conducted and >400 cells were counted.(C) Graph showing the percentage of cells that display Atg16-3 × mCherry dots. In total, 3 independent experiments were conducted and >750 cells were counted. The graphs show the averages and the error bars representing the standard deviations. P‐values were calculated using Student's t‐test. Scale bars: 5 μm.