Immunofluorescence analysis of p62 in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; TH = Thalamus ; CBX = Cerebellum ; Medulla = MY ) . Scale bar indicates 100 µm .

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Immunofluorescence analysis of p62 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; TH = Thalamus; CBX = Cerebellum; Medulla = MY). Scale bar indicates 100 µm.


Localization of p62 mainly in NeuN positive cells in the cortex ( CTX ) of 4 . 5 - month - old mice . Minor overlap of aggregates with microglial marker IBA1 . Scale bar indicates 15 µm .

Localization of p62 mainly in NeuN positive cells in the cortex (CTX) of 4.5-month-old mice. Minor overlap of aggregates with microglial marker IBA1. Scale bar indicates 15 µm.


Complete overlap of co - staining of p62 with NeuN in the cortex ( CTX ) and not with the reactive astrocycte marker GFAP . Scale bar indicates 15 µm .

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Complete overlap of co-staining of p62 with NeuN in the cortex (CTX) and not with the reactive astrocycte marker GFAP. Scale bar indicates 15 µm.


Immunofluorescence analysis of TDP - 43 in sagittal brain sections . Representative images of indicated brain regions ( CTX = Cortex ; MB = Midbrain ) . White arrow heads indicate TDP - 43 aggregation . Scale bar indicates 100 µm . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of the midbrain , scale bar zoom in indicates 50 μm . ( Animals 4 . 5 months of age ) .

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Immunofluorescence analysis of TDP-43 in sagittal brain sections. Representative images of indicated brain regions (CTX = Cortex; MB = Midbrain). White arrow heads indicate TDP-43 aggregation. Scale bar indicates 100 µm. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain, scale bar zoom in indicates 50 μm. (Animals 4.5 months of age).


Immunofluorescence analysis of total TDP - 43 and phosphorylated TDP - 43 ( p - TDP - 43 ) in sagittal brain sections by confocal microscopy . Representative images of one confocal layer in the midbrain . Zoom in on TDP - 43 aggregation in double knockout mouse brain sections of the midbrain . Scale bar indicates 25 µm . ( Animals 4 . 5 months of age ) .

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Immunofluorescence analysis of total TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sagittal brain sections by confocal microscopy. Representative images of one confocal layer in the midbrain. Zoom in on TDP-43 aggregation in double knockout mouse brain sections of the midbrain. Scale bar indicates 25 µm. (Animals 4.5 months of age).


Western blot analysis of TDP - 43 and phosphorylated TDP - 43 ( p - TDP - 43 ) in sequential high salt ( HS ) / RIPA / Urea fractionations of total brain lysates from 4 . 5 - month - old mice with the indicated genotype . Analysis of C - terminal fragments of TDP - 43 ( CTF ) in RIPA fraction . p - TDP - 43 analyzed in Urea fraction . Quantification of ( C ) Protein expression was normalized to levels in wild type animals . Data represent the mean ± SD .

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Western blot analysis of TDP-43 and phosphorylated TDP-43 (p-TDP-43) in sequential high salt (HS)/RIPA/Urea fractionations of total brain lysates from 4.5-month-old mice with the indicated genotype. Analysis of C-terminal fragments of TDP-43 (CTF) in RIPA fraction. p-TDP-43 analyzed in Urea fraction. Quantification of (C) Protein expression was normalized to levels in wild type animals. Data represent the mean ± SD.


A Turbidity and bright - field microscope images of indicated groups ( left ) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm ( right ) . 10 μM cGAS , 5 μM G3BP1 , 5 μM BSA and 100 nM dsDNA ( 60 bp ) were used in these assays . n = 3 biological replicates . Data information : Representative images are shown Error bars , mean with s . d . , * P < 0 . 05 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . Scale bars , 10

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A Turbidity and bright-field microscope images of indicated groups (left) and the quantitative analysis of turbidity of samples were measured by absorbance at 600 nm (right). 10 μM cGAS, 5 μM G3BP1, 5 μM BSA and 100 nM dsDNA (60 bp) were used in these assays. n = 3 biological replicates. Data information: Representative images are shown Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. Scale bars, 10 μm


F FRAP analysis of DNA - induced droplets of cGAS ( 1 μM dsDNA , 10 μM cGAS - mCherry ) , the yellow dotted circle indicated the region of photobleaching . G FRAP curve of ( F ) . n = 3 biological replicates . H FRAP analysis of cGAS - G3BP1 droplets . 10 μM cGAS - mCherry and 5 μM G3BP1 were used . The yellow dotted circle indicated the region of photobleaching . I FRAP curve of ( H ) . n = 3 biological replicates . Data information : Representative images are shown ( A , Error bars , mean with s . d . , * P < 0 . 05 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . FRAP , Fluorescence recovery after photobleaching . Scale bars , 2 μm ( F and H ) .

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F FRAP analysis of DNA-induced droplets of cGAS (1 μM dsDNA, 10 μM cGAS-mCherry), the yellow dotted circle indicated the region of photobleaching. G FRAP curve of (F). n = 3 biological replicates. H FRAP analysis of cGAS-G3BP1 droplets. 10 μM cGAS-mCherry and 5 μM G3BP1 were used. The yellow dotted circle indicated the region of photobleaching. I FRAP curve of (H). n = 3 biological replicates. Data information: Representative images are shown (A, Error bars, mean with s.d. , *P < 0.05, ****P < 0.0001, two-tailed t-test. NS, non-significant. FRAP, Fluorescence recovery after photobleaching. Scale bars, 2 μm (F and H).


B Immunofluorescent staining of cGAS in both WT and G3BP1 - / - U937 cells . 3D images were reconstituted with Leica LAS X software . C Quantitative analysis of total cGAS puncta number ( left ) and volume ( right ) per cell of ( B ) . n = 51 cells . D Data information : Representative images are shown Scale bars , 5 μm . Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . WT , wild - type .

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B Immunofluorescent staining of cGAS in both WT and G3BP1-/- U937 cells. 3D images were reconstituted with Leica LAS X software. C Quantitative analysis of total cGAS puncta number (left) and volume (right) per cell of (B). n = 51 cells. D Data information: Representative images are shown Scale bars, 5 μm. Hoechst (blue), nuclear staining. Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type.


F ELISA analysis of the secreted IFN - β in HT - DNA - treated WT or G3BP1 - / - U937 cells . n = 3 biological replicates . Data information : Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . WT , wild - type . HT - DNA , Herring testes DNA .

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F ELISA analysis of the secreted IFN-β in HT-DNA-treated WT or G3BP1-/- U937 cells. n = 3 biological replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. HT-DNA, Herring testes DNA.


G cGAMP production in ISD - treated WT or G3BP1 - / - U937 cells were analyzed by LC - MS / MRM . ND , not detected . n = 3 technical replicates . Data information : Error bars , mean with s . d , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant . WT , wild - type . ISD , interferon stimulatory DNA .

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G cGAMP production in ISD-treated WT or G3BP1-/- U937 cells were analyzed by LC-MS/MRM. ND, not detected. n = 3 technical replicates. Data information: Error bars, mean with s.d , *P < 0.05, **P < 0.01, ****P < 0.0001, two-tailed t-test. NS, non-significant. WT, wild-type. ISD, interferon stimulatory DNA.


B Fluorescence intensity distribution on cGAS - G3BP1 droplets . A quantitative analysis of a representative cGAS - G3BP1 droplet is shown . Along the white line on the merged image , the fluorescence intensity of both G3BP1 and cGAS channels were recorded . Data information : Representative images are shown Scale bars , 10 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP were used in the assays .

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B Fluorescence intensity distribution on cGAS-G3BP1 droplets. A quantitative analysis of a representative cGAS-G3BP1 droplet is shown. Along the white line on the merged image, the fluorescence intensity of both G3BP1 and cGAS channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP were used in the assays.


D cGAS , G3BP1 and Cy5 - dsDNA were incubated . A quantitative analysis of a representative cGAS - DNA droplet is shown . Along the white line on the merged image , the fluorescence intensity of G3BP1 , cGAS and dsDNA channels were recorded . Data information : Representative images are shown Scale bars , 10 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP , 2 μM dsDNA were used in the assays .

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D cGAS, G3BP1 and Cy5-dsDNA were incubated. A quantitative analysis of a representative cGAS-DNA droplet is shown. Along the white line on the merged image, the fluorescence intensity of G3BP1, cGAS and dsDNA channels were recorded. Data information: Representative images are shown Scale bars, 10 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsDNA were used in the assays.


F cGAS , G3BP1 and Cy5 - dsRNA were incubated . Similar quantification analysis as in D was performed . Data information : Representative images are shown Scale bars , 5 μm 45 μM cGAS - mCherry , 10 μM G3BP1 - mEGFP , 2 μM dsRNA were used in the assays .

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F cGAS, G3BP1 and Cy5-dsRNA were incubated. Similar quantification analysis as in D was performed. Data information: Representative images are shown Scale bars, 5 μm 45 μM cGAS-mCherry, 10 μM G3BP1-mEGFP, 2 μM dsRNA were used in the assays.


A , B Recombinant cGAS ( 10 μM ) and the indicated amounts of Cy5 - labeled dsDNA were incubated , with or without G3BP1 ( 2 μM ) . The DNA - induced cGAS - DNA liquid droplets were analyzed with time - lapse imaging . Data information : Representative images are shown Scale bars , 10

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A, B Recombinant cGAS (10 μM) and the indicated amounts of Cy5-labeled dsDNA were incubated, with or without G3BP1 (2 μM). The DNA-induced cGAS-DNA liquid droplets were analyzed with time-lapse imaging. Data information: Representative images are shown Scale bars, 10 μm


D cGAMP production assays . Recombinant cGAS ( 10 μM ) was incubated with the indicated amounts of dsDNA , with or without G3BP1 ( 10 μM ) . The produced cGAMP was quantitatively analyzed by LC - MS / MRM . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * * P < 0 . 01 , two - tailed t - test .

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D cGAMP production assays. Recombinant cGAS (10 μM) was incubated with the indicated amounts of dsDNA, with or without G3BP1 (10 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.


G qPCR analysis of IFNB mRNA levels in WT and G3BP1 - / - U937 cells transfected with dsDNA as indicated . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * * P < 0 . 01 , two - tailed t - test .

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G qPCR analysis of IFNB mRNA levels in WT and G3BP1-/- U937 cells transfected with dsDNA as indicated. n = 3 technical replicates. Data information: Error bars, mean with s.d. , **P < 0.01, two-tailed t-test.


C cGAS - G3BP1 interaction analysis . Full - length and truncated G3BP1 was pulled - down using anti - Flag M2 beads . The cGAS - G3BP1 interaction was further analyzed with immunoblotting ( IB ) . 2 μg recombinant full - length cGAS and 2 μg each of truncated G3BP1 were used in the assay .

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C cGAS-G3BP1 interaction analysis. Full-length and truncated G3BP1 was pulled-down using anti-Flag M2 beads. The cGAS-G3BP1 interaction was further analyzed with immunoblotting (IB). 2 μg recombinant full-length cGAS and 2 μg each of truncated G3BP1 were used in the assay.


D , Time - lapse imaging of DNA - induced LLPS of cGAS , in the presence of full length or truncated G3BP1 ( 2 μM ) n = 3 biological replicates . 10 μM cGAS , 100 nM dsDNA were used in the assay Data information : Representative images are shown Scale bars , 10

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D, Time-lapse imaging of DNA-induced LLPS of cGAS, in the presence of full length or truncated G3BP1 (2 μM) n = 3 biological replicates.10 μM cGAS, 100 nM dsDNA were used in the assay Data information: Representative images are shown Scale bars, 10 μm


A Bright - field microscope images of indicated groups ( left ) and a quantitative analysis of total area of droplets ( right ) . n = 3 biological replicates . 10 μM cGAS , 5 μM G3BP1 and 50 μM KCl , 50 μM MgCl2 , 50 μM ZnCl2 were used . Data information : Representative images are shown Scale bars , 10 μm . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

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A Bright-field microscope images of indicated groups (left) and a quantitative analysis of total area of droplets (right). n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 50 μM KCl, 50 μM MgCl2, 50 μM ZnCl2 were used. Data information: Representative images are shown Scale bars, 10 μm. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


C Fluorescent images and analysis of DNA - induced cGAS LLPS in the presence of the indicated reagent . n = 3 biological replicates . 10 μM cGAS , 50 μM of each ion and 100 nM dsDNA were used in these assays . Data information : Representative images are shown Scale bars , 10 μm . The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

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C Fluorescent images and analysis of DNA-induced cGAS LLPS in the presence of the indicated reagent. n = 3 biological replicates. 10 μM cGAS, 50 μM of each ion and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


D Fluorescent images and analysis of DNA - induced LLPS of cGAS in the presence of different amounts of ZnCl2 . n = 3 biological replicates . 10 μM cGAS and 100 nM dsDNA were used in the assay . Data information : Representative images are shown Scale bars , 10 μm . The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

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D Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of different amounts of ZnCl2. n = 3 biological replicates. 10 μM cGAS and 100 nM dsDNA were used in the assay. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


E Fluorescent images and analysis of DNA - induced LLPS of cGAS in the presence of G3BP1 ( 2 μM ) and / or ZnCl2 ( 20 μM ) as indicated . n = 3 biological replicates . Data information : Representative images are shown Scale bars , 10 μm . The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst ( blue ) , nuclear staining . Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed t - test . NS , non - significant . NT , non - treated .

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E Fluorescent images and analysis of DNA-induced LLPS of cGAS in the presence of G3BP1 (2 μM) and/or ZnCl2 (20 μM) as indicated. n = 3 biological replicates. Data information: Representative images are shown Scale bars, 10 μm. The Partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background Hoechst (blue), nuclear staining. Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test. NS, non-significant. NT, non-treated.


A Fluorescent images of cGAS - G3BP1 droplets in the presence or absence of 10 μM EGCG ( left ) . The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background ( right ) , n = 3 biological replicates . 45 μM cGAS - mCherry and 10 μM G3BP1 - mEGFP were used in these assays . Data information : Representative images are shown Scale bars , 10 μm Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant .

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A Fluorescent images of cGAS-G3BP1 droplets in the presence or absence of 10 μM EGCG (left). The partition coefficient was calculated as the total fluorescence intensity of droplets / bulk fluorescence intensity of background (right), n = 3 biological replicates. 45 μM cGAS-mCherry and 10 μM G3BP1-mEGFP were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


D Fluorescent images of DNA - induced LLPS of cGAS , with or without G3BP1 , in the absence or presence of 10 μM EGCG ( left ) . The partition coefficient was calculated as in ( A ) , n = 3 biological replicates . 10 μM cGAS , 5 μM G3BP1 and 100 nM dsDNA were used in these assays . Data information : Representative images are shown Scale bars , 10 μm Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant .

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D Fluorescent images of DNA-induced LLPS of cGAS, with or without G3BP1, in the absence or presence of 10 μM EGCG (left). The partition coefficient was calculated as in (A), n = 3 biological replicates. 10 μM cGAS, 5 μM G3BP1 and 100 nM dsDNA were used in these assays. Data information: Representative images are shown Scale bars, 10 μm Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


E cGAMP production assays . Recombinant cGAS ( 10 μM ) and G3BP1 ( 10 μM ) were incubated , in the presence or absence of dsDNA ( 200 nM ) , with or without EGCG ( 20 μM ) . The produced cGAMP was quantitatively analyzed by LC - MS / MRM . n = 3 technical replicates . Data information : Error bars , mean with s . d . , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed t - test . NS , non - significant .

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E cGAMP production assays. Recombinant cGAS (10 μM) and G3BP1 (10 μM) were incubated, in the presence or absence of dsDNA (200 nM), with or without EGCG (20 μM). The produced cGAMP was quantitatively analyzed by LC-MS/MRM. n = 3 technical replicates. Data information: Error bars, mean with s.d. , *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed t-test. NS, non-significant.


F cGAS - G3BP1 interaction analysis . cGAS was precipitated using anti - cGAS antibodies in U937 cells treated with EGCG as indicated . IgG served as control . The cGAS - G3BP1 interaction was further analyzed by immunoblotting . Data information : Representative images are shown WCL , whole cell lysate . IP , immunoprecipitation .

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

F cGAS-G3BP1 interaction analysis. cGAS was precipitated using anti-cGAS antibodies in U937 cells treated with EGCG as indicated. IgG served as control. The cGAS-G3BP1 interaction was further analyzed by immunoblotting. Data information: Representative images are shown WCL, whole cell lysate. IP, immunoprecipitation.


( A ) Pictures of plants treated with BLE . Plants were grown vertically on 1 / 2 MS medium with or without 4 μM BLE for 9 days . Scale bar = 1 cm . ( B ) The relative root length of the indicated plants . The data are represented as means ± SD ( n = 10 plants ) relative to the values of Col - 0 under the control condition . The statistical significance was determined using two - way ANOVA analysis . * * * * , P < 0 . 0001 . The experiments were repeated three times with similar results .

O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Pictures of plants treated with BLE. Plants were grown vertically on 1/2 MS medium with or without 4 μM BLE for 9 days . Scale bar = 1 cm. (B) The relative root length of the indicated plants. The data are represented as means ± SD (n = 10 plants) relative to the values of Col-0 under the control condition. The statistical significance was determined using two-way ANOVA analysis. ****, P < 0.0001. The experiments were repeated three times with similar results.


( H and I ) Representative GUS staining images of cotyledons ( H ) and quantification of the relative HR efficiency ( I ) . The reporter line and trigger line in either ddrm4 - 1 or Col - 0 background were crossed and the F1 seedlings were used for GUS staining analysis . Scale bar = 1 mm . The number of blue sectors was scored . The data are presented as means ± SD ( n > 132 cotyledons ) relative to the values of Col - 0 . The statistical significance was determined using two - tailed Students ' t - test . * * * * , P < 0 . 0001 .

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(H and I) Representative GUS staining images of cotyledons (H) and quantification of the relative HR efficiency (I). The reporter line and trigger line in either ddrm4-1 or Col-0 background were crossed and the F1 seedlings were used for GUS staining analysis. Scale bar = 1 mm. The number of blue sectors was scored. The data are presented as means ± SD (n > 132 cotyledons) relative to the values of Col-0. The statistical significance was determined using two-tailed Students' t-test. ****, P < 0.0001.


( A - F ) ( A , C , and E ) Representative images of CFP - PAF1 expressed in N . benthamiana leaves ( A ) , Arabidopsis protoplasts ( C ) , and transgenic Arabidopsis ( E ) . The N . benthamiana leaves and the transgenic seedlings were treated with 40 μM BLE for 4 h before imaging . The Arabidopsis protoplasts were treated with 20 μM BLE for 2 h before imaging . Scale bars in ( A and C ) = 2 . 5 μm . Bars in ( E ) = 10 μm . ( B , D , and F ) The percentage of cells with CFP - PAF1 foci in N . benthamiana leaves ( B ) , Arabidopsis protoplasts ( D ) , and transgenic Arabidopsis ( F ) . The data are represented as means ± SEM ( n = 3 biological replicates ) . For each sample , more than 35 cells were scored . The statistical significance was determined using two - tailed Students ' 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 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 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

(A-F) (A, C, and E) Representative images of CFP-PAF1 expressed in N. benthamiana leaves (A), Arabidopsis protoplasts (C), and transgenic Arabidopsis (E). The N. benthamiana leaves and the transgenic seedlings were treated with 40 μM BLE for 4 h before imaging. The Arabidopsis protoplasts were treated with 20 μM BLE for 2 h before imaging. Scale bars in (A and C) = 2.5 μm. Bars in (E) = 10 μm. (B, D, and F) The percentage of cells with CFP-PAF1 foci in N. benthamiana leaves (B), Arabidopsis protoplasts (D), and transgenic Arabidopsis (F). The data are represented as means ± SEM (n = 3 biological replicates). For each sample, more than 35 cells were scored. The statistical significance was determined using two-tailed Students' t-test.


( K ) eChIP - qPCR assays . The immunoprecipitated DNA and the input DNA were subjected to qPCR analysis . The ratios of eChIP and input are shown . UBQ5 serves as a negative control . The positions of the PCR fragment of 0 . 5 kb - 4 . 5 kb were shown in ( I ) . The data are represented as means ± SD ( n = 3 technical replicates ) relative to the values of Mock . The statistical significance was determined using two - tailed Students ' t - test . ns , not significant ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . The experiments were repeated three times with similar results .

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

(K) eChIP-qPCR assays. The immunoprecipitated DNA and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The positions of the PCR fragment of 0.5 kb-4.5 kb were shown in (I). The data are represented as means ± SD (n = 3 technical replicates) relative to the values of Mock. The statistical significance was determined using two-tailed Students' t-test. ns, not significant; **, P < 0.01; ***, P < 0.001. The experiments were repeated three times with similar results.


( A and B ) Yeast two - hybrid assays . PAF1 was fused with the activation domain ( AD ) . SMC5 and SMC6B were fused with the DNA binding domain ( BD ) . DDO , double dropout ( SD / - Trp / - Leu ) medium . QDO , quadruple dropout ( SD / - Trp / - Leu / - His / - Ade ) medium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) Yeast two-hybrid assays. PAF1 was fused with the activation domain (AD). SMC5 and SMC6B were fused with the DNA binding domain (BD). DDO, double dropout (SD/-Trp/-Leu) medium. QDO, quadruple dropout (SD/-Trp/-Leu/-His/-Ade) medium.


( G and H ) In vitro pull - down assays . The Dextrin beads coupled with MBP , MBP - SMC5 , or MBP - SMC6B were incubated with His - PAF1 , respectively . After washing , the beads were subjected to western blotting analysis using anti - His or anti - MBP antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) In vitro pull-down assays. The Dextrin beads coupled with MBP, MBP-SMC5, or MBP-SMC6B were incubated with His-PAF1, respectively. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies.


The PAF1 foci are dependent on SMC5 and SMC6B . ( A ) CFP - PAF1 was transfected alone or co - transfected with the RNAi construct of SMC5 ( SMC5 - Ri ) into Col - 0 protoplasts . ( C ) CFP - PAF1 was transfected into the protoplasts of Col - 0 or smc6b , respectively . The protoplasts were treated with 20 μM BLE for 2 h before imaging . The percentage of cells with foci . The data were represented as means ± SEM ( n = 3 biological replicates ) . For each sample , at least 32 cells were scored . The statistical significance was determined using two - way ANOVA analysis . * * , P < 0 . 01 ; * * * , P < 0 . 001 .

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

The PAF1 foci are dependent on SMC5 and SMC6B. (A) CFP-PAF1 was transfected alone or co-transfected with the RNAi construct of SMC5 (SMC5-Ri) into Col-0 protoplasts. (C) CFP-PAF1 was transfected into the protoplasts of Col-0 or smc6b, respectively. The protoplasts were treated with 20 μM BLE for 2 h before imaging. The percentage of cells with foci. The data were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 32 cells were scored. The statistical significance was determined using two-way ANOVA analysis. **, P < 0.01; ***, P < 0.001.


( G and H ) eChIP - qPCR assays . ( G ) The SMC5 - Ri construct was transfected into the protoplasts of SSDIS / CFP - PAF1 transgenic plants . ( H ) The 35S : CFP - PAF1 construct was transfected into the protoplasts of SSDIS / smc6b or SSDIS / Col - 0 transgenic plants . The protoplasts were treated with 100 μM DEX for 4 h to induce DSBs . The eChIP assays were carried out using an anti - GFP antibody . The immunoprecipitated DNA ( eChIP ) and the input DNA were subjected to qPCR analysis . The ratios of eChIP and input are shown . UBQ5 serves as a negative control . The data are represented as means ± SD ( n = 3 technical replicates ) . The statistical significance was determined using two - tailed Students ' t - test . ns , not significant ; * * * * , P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and H) eChIP-qPCR assays. (G) The SMC5-Ri construct was transfected into the protoplasts of SSDIS/CFP-PAF1 transgenic plants. (H) The 35S:CFP-PAF1 construct was transfected into the protoplasts of SSDIS/smc6b or SSDIS/Col-0 transgenic plants. The protoplasts were treated with 100 μM DEX for 4 h to induce DSBs. The eChIP assays were carried out using an anti-GFP antibody. The immunoprecipitated DNA (eChIP) and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD (n = 3 technical replicates). The statistical significance was determined using two-tailed Students' t-test. ns, not significant; ****, P < 0.0001.


( C and D ) BiFC assays . The proteins were fused to either the C - or N - terminal half of YFP ( cYFP or nYFP ) and were transiently expressed in N . benthamiana . The YFP fluoresce detected by confocal microscopy indicates interaction . Scale bars = 10 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) BiFC assays. The proteins were fused to either the C- or N-terminal half of YFP (cYFP or nYFP) and were transiently expressed in N. benthamiana. The YFP fluoresce detected by confocal microscopy indicates interaction. Scale bars = 10 μm.


( E and F ) CoIP assays . The fusion proteins were co - expressed in N . benthamiana leaves . The proteins precipitated by anti - GFP beads were subjected to western blotting analysis using anti - FLAG or anti - GFP antibodies .

O O O O O O O O O O O O O O O O O O O O O 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 and F) CoIP assays. The fusion proteins were co-expressed in N. benthamiana leaves. The proteins precipitated by anti-GFP beads were subjected to western blotting analysis using anti-FLAG or anti-GFP antibodies.


( G and H ) In vitro pull - down assays . The Dextrin beads coupled with MBP or MBP - UBC2 were incubated with His - CDC73 or His - VIP5 . After washing , the beads were subjected to western blotting analysis using anti - His or anti - MBP antibodies .

(G and H) In vitro pull-down assays. The Dextrin beads coupled with MBP or MBP-UBC2 were incubated with His-CDC73 or His-VIP5. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies.


( I and J ) The UBC2 foci are dependent on VIP5 . UBC2 - mCherry and / or VIP5 - YFP were transfected into the vip5 protoplasts . The protoplasts were treated with 20 μM BLE for 2 h before imaging . Scale bars = 2 . 5 μm . The data in ( J ) were represented as means ± SEM ( n = 3 biological replicates ) . For each sample , at least 30 cells were scored . The statistical significance was determined using two - way ANOVA analysis . * * * * , P < 0 . 0001 . All experiments were repeated at least three times with similar results .

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

(I and J) The UBC2 foci are dependent on VIP5. UBC2-mCherry and/or VIP5-YFP were transfected into the vip5 protoplasts. The protoplasts were treated with 20 μM BLE for 2 h before imaging. Scale bars = 2.5 μm. The data in (J) were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 30 cells were scored. The statistical significance was determined using two-way ANOVA analysis. ****, P < 0.0001. All experiments were repeated at least three times with similar results.


( C and D ) In vitro pull - down assay . The Dextrin beads coupled with MBP , MBP - HUB1 , or MBP - HUB2 were incubated with His - UBC2 . After washing , the beads were subjected to western blotting analysis using anti - His or anti - MBP antibodies . The experiments were repeated three times with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and D) In vitro pull-down assay. The Dextrin beads coupled with MBP, MBP-HUB1, or MBP-HUB2 were incubated with His-UBC2. After washing, the beads were subjected to western blotting analysis using anti-His or anti-MBP antibodies. The experiments were repeated three times with similar results.


( E - J ) The HUB1 / 2 foci are dependent on UBC2 . VIP5 - CFP , HUB1 / 2 - YFP , and / or UBC2 - mCherry were transfected into the ubc1 ubc2 - c1 protoplasts . The protoplasts were treated with 20 μM BLE for 2 h before imaging . Bars = 2 . 5 μm . The data in ( F , G , I , and J ) were represented as means ± SEM ( n = 3 biological replicates ) . For each sample , at least 60 cells were scored . The statistical significance was determined using two - way ANOVA analysis . ns , not significant ; * * , P < 0 . 01 .

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

(E-J) The HUB1/2 foci are dependent on UBC2. VIP5-CFP, HUB1/2-YFP, and/or UBC2-mCherry were transfected into the ubc1 ubc2-c1 protoplasts. The protoplasts were treated with 20 μM BLE for 2 h before imaging. Bars = 2.5 μm. The data in (F, G, I, and J) were represented as means ± SEM (n = 3 biological replicates). For each sample, at least 60 cells were scored. The statistical significance was determined using two-way ANOVA analysis. ns, not significant; **, P < 0.01.


( A , C , E , and G ) Pictures of plants treated with BLE . Plants were grown vertically on 1 / 2 MS medium with or without 6 μM BLE ( A ) or 4 μM BLE ( C , E , and G ) for 8 days . Scale bar = 1 cm . ( B , D , F , and H ) The relative root length of the indicated plants . The data are represented as means ± SD ( n = 10 plants ) relative to the values of Col - 0 under the control condition . The statistical significance was determined using two - way ANOVA analysis . ns , not significant ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 . The experiments were repeated three times with similar results .

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 B-CONTROLLED_VAR 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

(A, C, E, and G) Pictures of plants treated with BLE. Plants were grown vertically on 1/2 MS medium with or without 6 μM BLE (A) or 4 μM BLE (C, E, and G) for 8 days. Scale bar = 1 cm. (B, D, F, and H) The relative root length of the indicated plants. The data are represented as means ± SD (n = 10 plants) relative to the values of Col-0 under the control condition. The statistical significance was determined using two-way ANOVA analysis. ns, not significant; ***, P < 0.001; ****, P < 0.0001. The experiments were repeated three times with similar results.


( I and J ) eChIP - qPCR assays . ( I ) The SSDIS / Col - 0 transgenic Arabidopsis was mock - treated or treated with 100 μM DEX for 4 h . ( J ) The SSDIS / Col - 0 and SSDIS / ddrm4 - 1 transgenic Arabidopsis were treated with 100 μM DEX for 4 h . The eChIP assays were carried out using an anti - H2Bub antibody . The immunoprecipitated DNA ( eChIP ) and the input DNA were subjected to qPCR analysis . The ratios of eChIP and input are shown . UBQ5 serves as a negative control . The data are represented as means ± SD ( n = 3 technical replicates ) . ( I ) The data are relative to the values of Mock . ( J ) The data are relative to the values of UBQ5 in Col - 0 . The statistical significance was determined using two - tailed Students ' t - test . ns , not significant ; * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 ; * * * * , P < 0 . 0001 . The experiments were repeated at least twice with similar results .

O O O O O O 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 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 O O O O O

(I and J) eChIP-qPCR assays. (I) The SSDIS/Col-0 transgenic Arabidopsis was mock-treated or treated with 100 μM DEX for 4 h. (J) The SSDIS/Col-0 and SSDIS/ddrm4-1 transgenic Arabidopsis were treated with 100 μM DEX for 4 h. The eChIP assays were carried out using an anti-H2Bub antibody. The immunoprecipitated DNA (eChIP) and the input DNA were subjected to qPCR analysis. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD (n = 3 technical replicates). (I) The data are relative to the values of Mock. (J) The data are relative to the values of UBQ5 in Col-0. The statistical significance was determined using two-tailed Students' t-test. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The experiments were repeated at least twice with similar results.


( A ) HEK293A cell lines stably expressing GFP , GFP - WIPI1a , and GFP - WIPI2b were incubated in full medium ( F ) or starvation medium ( EBSS ) ( S ) for 2 hr before being used for GFP - TRAP pull - down .

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(A) HEK293A cell lines stably expressing GFP, GFP-WIPI1a, and GFP-WIPI2b were incubated in full medium (F) or starvation medium (EBSS) (S) for 2 hr before being used for GFP-TRAP pull-down.


( B ) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP , at the indicated concentrations , before immunoblotting .

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(B) WIPI2 was immunoprecipitated from HEK293A cells treated with DSP, at the indicated concentrations, before immunoblotting.


( C ) WT or Atg16L1Δ / Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti - WIPI2 antibody . Scale bars , 10 μm . ( D ) WIPI2 puncta in ( C ) were counted , and a statistical analysis of WIPI2 puncta was performed using an unpaired Student ' s t test . ∗ p < 0 . 05 . SEM for n = 3 .

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(C) WT or Atg16L1Δ/Δ MEFs in fed medium or starved for 2 hr in EBSS were fixed and labeled with an anti-WIPI2 antibody. Scale bars, 10 μm.(D) WIPI2 puncta in (C) were counted, and a statistical analysis of WIPI2 puncta was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( E - H ) CLEM ( correlative light and electron microscopy ) of endogenous WIPI2 in Atg16L1Δ / Δ MEFs . ( E ) Merged phase and confocal section of Atg16L1Δ / Δ cells labeled with anti - WIPI2 antibody . Scale bar , 10 μm . ( F ) Low - magnification TEM of cell in ( E ) . Scale bar 10μm . ( G and H ) High magnification of boxed regions in ( E ) and ( F ) . Top box and bottom box indicate panels ( G ) and ( H ) , respectively . Arrows indicate open phagophores . M , mitochondria ; ER , endoplasmic reticulum . Scale bars , 1 μm ( G ) and 0 . 5 μm ( H ) .

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(E-H) CLEM (correlative light and electron microscopy) of endogenous WIPI2 in Atg16L1Δ/Δ MEFs.(E) Merged phase and confocal section of Atg16L1Δ/Δ cells labeled with anti-WIPI2 antibody. Scale bar, 10 μm.(F) Low-magnification TEM of cell in (E). Scale bar 10μm.(G and H) High magnification of boxed regions in (E) and (F). Top box and bottom box indicate panels (G) and (H), respectively. Arrows indicate open phagophores. M, mitochondria; ER, endoplasmic reticulum. Scale bars, 1 μm (G) and 0.5 μm (H).


( I and J ) 2GL9 cells ( GFP - LC3 HEK293 cells ; Chan et al . , 2007 ) were starved for 2 hr before visualization using indicated antibodies . Scale bars , 10 μm .

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(I and J) 2GL9 cells (GFP-LC3 HEK293 cells; Chan et al., 2007) were starved for 2 hr before visualization using indicated antibodies. Scale bars, 10 μm.


( K ) Live - cell imaging demonstrates that Atg16L1 translocates to WIPI2b - positive puncta in starvation . HEK293 cells expressing GFP - WIPI2b and mCherry - Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope . See also Figure S1 and Movie S1 .

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(K) Live-cell imaging demonstrates that Atg16L1 translocates to WIPI2b-positive puncta in starvation. HEK293 cells expressing GFP-WIPI2b and mCherry-Atg16L1 were starved in EBSS and imaged every 2 s using a spinning disk microscope. See also Figure S1 and Movie S1.


( B ) Untransfected ( UN ) or FLAG - Atg16L1 full - length ( FL ) , 79 - 623 , or 1 - 265 constructs were expressed in HEK293A cells stably expressing GFP - WIPI2b and immunoprecipitated using GFP - TRAP . Tags were visualized by immunoblotting . I , input ; UB , unbound .

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(B) Untransfected (UN) or FLAG-Atg16L1 full-length (FL), 79-623, or 1-265 constructs were expressed in HEK293A cells stably expressing GFP-WIPI2b and immunoprecipitated using GFP-TRAP. Tags were visualized by immunoblotting. I, input; UB, unbound.


( C ) GFP , CFP - Atg5 , GFP - WIPI1a , and GFP - WIPI2b were transiently expressed in HEK293A cells . GFP - tagged proteins were isolated using GFP - TRAP and incubated with in vitro translated 35S - labeled FLAG - Atg16L1 constructs 1 - 265 , 1 - 242 , 1 - 230 , and 1 - 207 before washing and analysis by autoradiography . Protein expression was validated by immunoblot ( bottom panel ) .

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(C) GFP, CFP-Atg5, GFP-WIPI1a, and GFP-WIPI2b were transiently expressed in HEK293A cells. GFP-tagged proteins were isolated using GFP-TRAP and incubated with in vitro translated 35S-labeled FLAG-Atg16L1 constructs 1-265, 1-242, 1-230, and 1-207 before washing and analysis by autoradiography. Protein expression was validated by immunoblot (bottom panel).


( D ) Lysates from HEK293A cells transiently expressing GFP , GFP - WIPI1a , or GFP - WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG - Atg16L1 or Atg16L2 . Protein complexes were immunoprecipitated using GFP - TRAP , followed by immunoblot . See also Figure S2 and Table S1 .

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(D) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, or GFP-WIPI2b were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 or Atg16L2. Protein complexes were immunoprecipitated using GFP-TRAP, followed by immunoblot. See also Figure S2 and Table S1.


( B ) Lysates from HEK293A cells transfected with GFP - WIPI2b were mixed with lysates from HEK293A cells transfected with FLAG - Atg16L2 , FLAG - Atg16L1 , or Flag - Atg16L1 mutants , immunoprecipitated , and analyzed by immunoblotting . ( C ) Statistical analysis of FLAG - Atg16L1 binding in ( B ) was performed by Student ' s t test . SEM for n = 4 . ∗ p < 0 . 05 .

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(B) Lysates from HEK293A cells transfected with GFP-WIPI2b were mixed with lysates from HEK293A cells transfected with FLAG-Atg16L2, FLAG-Atg16L1, or Flag-Atg16L1 mutants, immunoprecipitated, and analyzed by immunoblotting.(C) Statistical analysis of FLAG-Atg16L1 binding in (B) was performed by Student's t test. SEM for n = 4. ∗p < 0.05.


( D ) Cell lysates from HEK293A cells transiently expressing FLAG - Atg16L2 , FLAG - Atg16L1 , or FLAG - Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads . Protein complexes were analyzed using immunoblotting . ( E ) Statistical analysis of FIP200 binding in ( D ) was performed by Student ' s t test . SEM for n = 3 . ∗ p < 0 . 05 .

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(D) Cell lysates from HEK293A cells transiently expressing FLAG-Atg16L2, FLAG-Atg16L1, or FLAG-Atg16L1 mutants were subjected to immunoprecipitation using FLAG M2 agarose beads. Protein complexes were analyzed using immunoblotting.(E) Statistical analysis of FIP200 binding in (D) was performed by Student's t test. SEM for n = 3. ∗p < 0.05.


( F ) WIPI2 was immunoprecipitated from lysates from WT or FIP200 − / − MEFS after treatment with 0 . 5 mM DSP . Bound Atg16 and Atg12 - 5 were detected by immunoblotting .

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(F) WIPI2 was immunoprecipitated from lysates from WT or FIP200−/− MEFS after treatment with 0.5 mM DSP. Bound Atg16 and Atg12-5 were detected by immunoblotting.


( G ) HEK293 cells stably expressing GFP - WIPI2b were and transiently transfected either FLAG - Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) . Bound and input ( bottom ) were analyzed by immunoblotting .

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(G) HEK293 cells stably expressing GFP-WIPI2b were and transiently transfected either FLAG-Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR). Bound and input (bottom) were analyzed by immunoblotting.


( A ) Lysates from HEK293A cells transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2a , GFP - WIPI2b , or GFP - WIPI2b FTTG mutant were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( B ) Statistical analysis of ( A ) was performed by one - way ANOVA with Tukey ' s posttest . SEM for n = 3 . ∗ p < 0 . 05 .

(A) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2a, GFP-WIPI2b, or GFP-WIPI2b FTTG mutant were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 3. ∗p < 0.05.


( D ) GFP - Trap from HEK293A cells transiently expressing GFP , GFP - WIPI1a , GFP - WIPI2b , GFP - WIPI2b R108E , GFP - WIPI2b R125E , or GFP - WIPI2b R108E R125E was mixed with in vitro translated 35S - labeled FLAG - Atg16L1 and analyzed by autoradiography .

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(D) GFP-Trap from HEK293A cells transiently expressing GFP, GFP-WIPI1a, GFP-WIPI2b, GFP-WIPI2b R108E, GFP-WIPI2b R125E, or GFP-WIPI2b R108E R125E was mixed with in vitro translated 35S-labeled FLAG-Atg16L1 and analyzed by autoradiography.


( E ) Lysates from HEK293A cells transiently expressing GFP , GFP - WIPI2b , GFP - WIPI2b ΔCT , GFP - WIPI2a , GFP - WIPI1a , or GFP - WIPI1a ΔCT were used for GFP - Trap . Endogenous Atg16L1 binding was analyzed by immunoblot . ( F ) Statistical analysis of ( E ) was performed by one - way ANOVA with Tukey ' s posttest . SEM for n = 2 . ∗ p < 0 . 05 .

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(E) Lysates from HEK293A cells transiently expressing GFP, GFP-WIPI2b, GFP-WIPI2b ΔCT, GFP-WIPI2a, GFP-WIPI1a, or GFP-WIPI1a ΔCT were used for GFP-Trap. Endogenous Atg16L1 binding was analyzed by immunoblot.(F) Statistical analysis of (E) was performed by one-way ANOVA with Tukey's posttest. SEM for n = 2. ∗p < 0.05.


( G ) Lysates from HEK293A cells transiently expressing GFP - WIPI2b WT , GFP - WIPI2b R108E , R125E , or R108E R125E were mixed with lysates from HEK293A cells transiently expressing FLAG - Atg16L1 WT , E226R , E230R , or E226R E230R in all possible permutations . Protein complexes from mixed lysates were immunoprecipitated using GFP - Trap , followed by immunoblot analysis . ( H ) Statistical analysis of ( G ) was performed by one - way ANOVA with Tukey ' s posttest . SEM from n = 3 . ∗ p < 0 . 05 .

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(G) Lysates from HEK293A cells transiently expressing GFP-WIPI2b WT, GFP-WIPI2b R108E, R125E, or R108E R125E were mixed with lysates from HEK293A cells transiently expressing FLAG-Atg16L1 WT, E226R, E230R, or E226R E230R in all possible permutations. Protein complexes from mixed lysates were immunoprecipitated using GFP-Trap, followed by immunoblot analysis.(H) Statistical analysis of (G) was performed by one-way ANOVA with Tukey's posttest. SEM from n = 3. ∗p < 0.05.


( I ) CLEM analysis of HEK293 cells treated with siRNA to WIPI2 were transfected with GFP - WIPI2b RERE and starved in EBSS . Bright - field image ( top ) , GFP - WIPI2 RERE signal ( middle ) , TEM of selected cell ( bottom ) . Boxed area shown in ( J ) . Scale bars represent 10 μM for bright - field and confocal and 5 μM for TEM . ( J ) High - magnification TEM of boxed area in ( I ) . Arrows indicate open phagophores in the vicinity of ER and mitochondria ( M ) , which contain GFP - WIPI2b RERE . See also Figures S4 and S5 and Table S1 .

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(I) CLEM analysis of HEK293 cells treated with siRNA to WIPI2 were transfected with GFP-WIPI2b RERE and starved in EBSS. Bright-field image (top), GFP-WIPI2 RERE signal (middle), TEM of selected cell (bottom). Boxed area shown in (J). Scale bars represent 10 μM for bright-field and confocal and 5 μM for TEM.(J) High-magnification TEM of boxed area in (I). Arrows indicate open phagophores in the vicinity of ER and mitochondria (M), which contain GFP-WIPI2b RERE. See also Figures S4 and S5 and Table S1.


( A ) GFP , siRNA - resistant GFP - WIPI2b , or GFP - WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC - free ( RF ) or WIPI2 siRNA . Cells were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot analysis . ( B ) Statistical analysis of ( A ) . SEM for n = 3 . Statistical analysis was performed by one - way ANOVA with Tukey ' s posttest . ∗ p < 0 . 05 .

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(A) GFP, siRNA-resistant GFP-WIPI2b, or GFP-WIPI2b RERE was expressed in HEK293A cells treated for 72 hr with either RISC-free (RF) or WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(B) Statistical analysis of (A). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Tukey's posttest. ∗p < 0.05.


( C ) GFP , siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA . Cells were left in full medium ( F ) or starved for 2 hr with EBSS ( S ) or EBSS with BafA ( B ) before immunoblot analysis . ( D ) Statistical analysis of ( C ) was performed by one - way ANOVA with Tukey ' s posttest . The SEM for LC3 ( n = 2 ) and p62 ( n = 4 ) are shown . ∗ p < 0 . 05 .

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(C) GFP, siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were left in full medium (F) or starved for 2 hr with EBSS (S) or EBSS with BafA (B) before immunoblot analysis.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's posttest. The SEM for LC3 (n = 2) and p62 (n = 4) are shown. ∗p < 0.05.


( E ) siRNA - resistant GFP - WIPI2b , GFP - WIPI2b RERE , GFP - WIPI2b FTTG , or GFP - WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA . Cells were starved in EBSS for 2 hr without or with BafA , fixed , and labeled , and LC3 was visualized by confocal microscopy . Scale bars , 10 μm . ( F ) Quantification of WIPI2 puncta per cell from ( E ) . SEM from 10 cells per condition ; ∗ ∗ ∗ p < 0 . 001 using one - way ANOVA . ( G ) Statistical analysis of LC3 puncta from ( E ) with GFP control ( not shown in E ) . SEM for n = 3 . Statistical analysis was performed by one - way ANOVA with Dunn ' s posttest t test . ∗ p < 0 . 05 . See also Table S1 .

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

(E) siRNA-resistant GFP-WIPI2b, GFP-WIPI2b RERE, GFP-WIPI2b FTTG, or GFP-WIPI2b FTTG RERE was expressed in HEK293A cells treated for 72 hr with WIPI2 siRNA. Cells were starved in EBSS for 2 hr without or with BafA, fixed, and labeled, and LC3 was visualized by confocal microscopy. Scale bars, 10 μm.(F) Quantification of WIPI2 puncta per cell from (E). SEM from 10 cells per condition; ∗∗∗p < 0.001 using one-way ANOVA.(G) Statistical analysis of LC3 puncta from (E) with GFP control (not shown in E). SEM for n = 3. Statistical analysis was performed by one-way ANOVA with Dunn's posttest t test. ∗p < 0.05. See also Table S1.


( A ) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium ( F ) , starvation medium ( S ) , or starvation medium with wortmannin ( W ) for 2 hr before immunoblot analysis . ( B ) Statistical analysis of ( A ) was performed by one - way ANOVA with Tukey ' s post hoc test . SEM for n = 3 . ∗ p < 0 . 05 .

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(A) HEK293A cells transiently expressing the indicated mCherry constructs were treated in either full medium (F), starvation medium (S), or starvation medium with wortmannin (W) for 2 hr before immunoblot analysis.(B) Statistical analysis of (A) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( C ) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP - Trap before analysis by immunoblotting .

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(C) Complexes from HEK293A cells transiently expressing the indicated mCherry constructs were immunoprecipitated using RFP-Trap before analysis by immunoblotting.


( D ) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in ( A ) were fixed and visualized by confocal microscopy .

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(D) 2GL9 cells transiently expressing the indicated mCherry constructs treated as in (A) were fixed and visualized by confocal microscopy.


( E ) CLEM of MCF7 cells expressing GFP - LC3 and mCherry - WIPI2b CAAX . Bright - field and confocal image merged ( left ) , confocal of expressing cell ( middle ) , low - magnification TEM of cell of interest ( right ) . Boxed area indicates plasma membrane region showing colocalization of GFP - LC3 and mCherry - WIPI2b . Scale bar , 10 μm ( n = 3 ) . ( F ) High - magnification TEM showing plasma membrane region boxed in ( E ) . Magnified insets , in top right , show small vesicular clusters under the plasma membrane detected in mCherry - WIPI2b CAAX , GFP - LC3 expressing cells but not in untransfected cells from a control experiment .

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(E) CLEM of MCF7 cells expressing GFP-LC3 and mCherry-WIPI2b CAAX. Bright-field and confocal image merged (left), confocal of expressing cell (middle), low-magnification TEM of cell of interest (right). Boxed area indicates plasma membrane region showing colocalization of GFP-LC3 and mCherry-WIPI2b. Scale bar, 10 μm (n = 3).(F) High-magnification TEM showing plasma membrane region boxed in (E). Magnified insets, in top right, show small vesicular clusters under the plasma membrane detected in mCherry-WIPI2b CAAX, GFP-LC3 expressing cells but not in untransfected cells from a control experiment.


( G ) HEK293A cells treated with either RISC - free ( RF ) , Atg16L1 siRNA , or FIP200 siRNA for 72 hr before transfection with HA - WIPI2b - CAAX were incubated in full medium ( F ) , EBSS ( S ) , or EBSS with wortmannin ( W ) for 2 hr before immunoblot analysis . Please note that all WIPI2 constructs are FTTG mutants . See also Figure S6 and Table S1 .

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(G) HEK293A cells treated with either RISC-free (RF), Atg16L1 siRNA, or FIP200 siRNA for 72 hr before transfection with HA-WIPI2b-CAAX were incubated in full medium (F), EBSS (S), or EBSS with wortmannin (W) for 2 hr before immunoblot analysis. Please note that all WIPI2 constructs are FTTG mutants. See also Figure S6 and Table S1.


( A ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , ERER , or DRDR . After 2 hr in EBSS , cells were fixed and labeled with anti - LC3 and anti - FLAG antibodies and visualized by confocal microscopy . Scale bars , 20 μm . ( B ) Statistical analysis of ( A ) was performed using one - way ANOVA with Dunn ' s posttest . ∗ p < 0 . 05 . SEM for n = 3 .

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(A) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, ERER, or DRDR. After 2 hr in EBSS, cells were fixed and labeled with anti-LC3 and anti-FLAG antibodies and visualized by confocal microscopy. Scale bars, 20 μm.(B) Statistical analysis of (A) was performed using one-way ANOVA with Dunn's posttest. ∗p < 0.05. SEM for n = 3.


( C ) Atg16L1Δ / Δ MEFs were transiently transfected with either mock , FLAG - tagged Atg16L1 WT , E226R E230R ( ERER ) , or D237R D239R ( DRDR ) before analysis by immunoblotting . ( D ) Statistical analysis of ( C ) was performed by one - way ANOVA with Tukey ' s post hoc test . SEM for n = 3 . ∗ p < 0 . 05 .

(C) Atg16L1Δ/Δ MEFs were transiently transfected with either mock, FLAG-tagged Atg16L1 WT, E226R E230R (ERER), or D237R D239R (DRDR) before analysis by immunoblotting.(D) Statistical analysis of (C) was performed by one-way ANOVA with Tukey's post hoc test. SEM for n = 3. ∗p < 0.05.


( E ) HEK293A cells were treated with either RISC - free control or WIPI2 siRNA before infection with Salmonella ( moi = 100 ) for 1 hr , labeled with anti - LC3 , WIPI2 , and p62 antibodies , and followed by confocal analysis . Scale bars , 5 μm . ( F ) Statistical analysis for ( E ) was performed using an unpaired Student ' s t test . ∗ p < 0 . 05 . SEM for n = 3 .

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(E) HEK293A cells were treated with either RISC-free control or WIPI2 siRNA before infection with Salmonella (moi = 100) for 1 hr, labeled with anti-LC3, WIPI2, and p62 antibodies, and followed by confocal analysis. Scale bars, 5 μm.(F) Statistical analysis for (E) was performed using an unpaired Student's t test. ∗p < 0.05. SEM for n = 3.


( G ) Atg16L1Δ / Δ MEFs were transiently transfected with FLAG - Atg16L1 WT , FLAG - Atg16L1 ERER , or FLAG - Atg16L2 DRDR 24 hr before being infected with Salmonella ( moi = 25 ) for 1 hr , labeled with anti - LC3 , anti - p62 , and anti - FLAG antibodies , and followed by analysis by confocal microscopy . Scale bars , 5 μm . ( H ) Statistical analysis of ( G ) was performed by one - way ANOVA with Dunn ' s post hoc test . SEM for n = 4 . ∗ p < 0 . 05 . See also Figure S7 .

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(G) Atg16L1Δ/Δ MEFs were transiently transfected with FLAG-Atg16L1 WT, FLAG-Atg16L1 ERER, or FLAG-Atg16L2 DRDR 24 hr before being infected with Salmonella (moi = 25) for 1 hr, labeled with anti-LC3, anti-p62, and anti-FLAG antibodies, and followed by analysis by confocal microscopy. Scale bars, 5 μm.(H) Statistical analysis of (G) was performed by one-way ANOVA with Dunn's post hoc test. SEM for n = 4. ∗p < 0.05. See also Figure S7.


( B ) Starvation - induced autophagosomes ( AP ) and autolysosomes ( AL ) in the fat body ( the functional analogue of the liver ) of a fruit fly larva . Note that APs contain intact cytoplasm , whereas the contents of ALs show various stages of degradation .

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(B) Starvation-induced autophagosomes (AP) and autolysosomes (AL) in the fat body (the functional analogue of the liver) of a fruit fly larva. Note that APs contain intact cytoplasm, whereas the contents of ALs show various stages of degradation.


( C ) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene , labeling cup - shaped and ring - shaped structures that correspond to IMs and autophagosomes , respectively . Images courtesy of Ryan Scott ( B ) and Dr . Noboru Mizushima ( C ) .

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(C) Liver cells of starved mice carrying a fluorescently tagged LC3 transgene, labeling cup-shaped and ring-shaped structures that correspond to IMs and autophagosomes, respectively. Images courtesy of Ryan Scott (B) and Dr. Noboru Mizushima (C).


A The mRNA value of AKT2 was analyzed in the GEO Profiles from the liver explant of Hepatitis B virus ( HBV ) - associated acute liver failure ( ALF ) patients ( GDS4387 / 225471 _ s _ at ) , and from bronchial epithelial cells with pandemic and seasonal H1N1 influenza virus infections in vitro ( GDS4855 / 203808 _ at ) . normal , n = 10 ; HBV , n = 17 ; control , n = 3 ; influenza A , n = 9 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using unpaired t - test Error bars mean ±

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A The mRNA value of AKT2 was analyzed in the GEO Profiles from the liver explant of Hepatitis B virus (HBV)-associated acute liver failure (ALF) patients (GDS4387/225471_s_at), and from bronchial epithelial cells with pandemic and seasonal H1N1 influenza virus infections in vitro (GDS4855/203808_at). normal, n=10; HBV, n=17; control, n=3; influenza A, n=9. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using unpaired t-test Error bars mean ± SD


C The qRT - PCR analysis of Akt2 mRNA in PEMs stimulated with lipo - poly ( I : C ) ( 1 μg / mL ) , lipo - poly ( A : T ) ( 1 μg / mL ) , lipo - ISD ( 3 μg / mL ) or HSV - 1 ( MOI , 1 ) for 6 hours . n = 3 , respectively . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using one - way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM ) .

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

C The qRT-PCR analysis of Akt2 mRNA in PEMs stimulated with lipo-poly(I:C) (1 μg/mL), lipo-poly(A:T) (1 μg/mL), lipo-ISD (3 μg/mL) or HSV-1 (MOI, 1) for 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using one-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).


D The mRNA of Akt2 ( red line ) and Ifnb1 ( black line ) were analyzed by qRT - PC in the mock and VSV ( MOI , 1 ) or LM ( MOI , 1 ) - stimulated PEMs or BMDMs at the indicated time . Mock and VSV , n = 4 ; mock and LM , n ≥ 3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The mRNA of Akt2 (red line) and Ifnb1 (black line) were analyzed by qRT-PC in the mock and VSV (MOI, 1) or LM (MOI, 1)-stimulated PEMs or BMDMs at the indicated time. Mock and VSV, n=4; mock and LM, n≥3.


G - H PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 ( 10 μM ) ( G ) for 2 hours , or knocked down of Akt2 with siAkt2 - 1 ( 20 nM ) for 48 hours ( H ) , then the mRNA level of Ifnb1 was measured by qRT - PCR after lipo - poly ( I : C ) , lipo - poly ( A : T ) , lipo - ISD , VSV , or HSV - 1 stimulation for another 6 hours . n = 3 , respectively . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM ) .

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

G-H PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 (10 μM) (G) for 2 hours, or knocked down of Akt2 with siAkt2-1 (20 nM) for 48 hours (H), then the mRNA level of Ifnb1 was measured by qRT-PCR after lipo-poly(I:C), lipo-poly(A:T), lipo-ISD, VSV, or HSV-1 stimulation for another 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).


A - B qRT - PCR detection for the mRNA expression of Ifnb1 ( A ) , Ifna4 , Cxcl10 and Ccl5 ( B ) in WT ( n = 3 ) and Akt2 KO ( n = 3 ) PEMs stimulated with lipo - poly ( I : C ) , VSV , lipo - poly ( A : T ) , lipo - ISD or HSV - 1 for 6 hours . C The expression of Ifnb1 mRNA level by qRT - PCR in WT ( n = 3 ) and Akt2 KO ( n = 3 ) PEMs treated with poly ( I : C ) ( 10 μg / mL ) for 6 hours ( left panel ) or LPS ( 1 μg / mL ) for 2 hours ( right panel ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA test Data are from three independent experiments Error bars mean ±

O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B qRT-PCR detection for the mRNA expression of Ifnb1 (A), Ifna4, Cxcl10 and Ccl5 (B) in WT (n=3) and Akt2 KO (n=3) PEMs stimulated with lipo-poly(I:C), VSV, lipo-poly(A:T), lipo-ISD or HSV-1 for 6 hours. C The expression of Ifnb1 mRNA level by qRT-PCR in WT (n=3) and Akt2 KO (n=3) PEMs treated with poly(I:C) (10 μg/mL) for 6 hours (left panel) or LPS (1 μg/mL) for 2 hours (right panel). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM


H Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours , then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT - PCR ( left panel ) . Every dot represents three zebrafish embryos . Horizontal square bracket shows the statistical analysis of the comparison with ' PBS mock ' , the rest shows the comparison with ' PBS VSV ' . PBS mock , n = 5 ; PBS VSV , n = 13 ; AKT2 VSV , n = 7 ; AKT2 - T309A / S474A VSV , n = 9 . H & E staining ( middle panel ) and survival rates ( Kaplan - Meier curve ) ( right panel ) were collected from zebrafish larvae after VSV micro - injection for 18 hours or longer . The arrows indicated the VSV - infected eye and skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA test or log - rank ( Mantel - Cox ) test Data are from three independent biological replicates Error bars mean ± SD ) .

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

H Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours, then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT-PCR (left panel). Every dot represents three zebrafish embryos. Horizontal square bracket shows the statistical analysis of the comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=5; PBS VSV, n=13; AKT2 VSV, n=7; AKT2-T309A/S474A VSV, n=9. H&E staining (middle panel) and survival rates (Kaplan-Meier curve) (right panel) were collected from zebrafish larvae after VSV micro-injection for 18 hours or longer. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test or log-rank (Mantel-Cox) test Data are from three independent biological replicates Error bars mean ± SD).


D WT and Akt2 KO PEMs were infected with or without VSV for 6 hours . Then , cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti - AKT2 antibody , followed by immunoblotting using anti - IRF3 antibody ( left panel ) . Alternatively , cell lysates were prepared for immunoprecipitation using anti - IRF3 antibody , followed by immunoblotting using anti - AKT2 antibody ( right panel ) to detect the endogenous interaction between AKT2 and IRF3 . Data information : * Data are representative of two or three independent biological

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WT and Akt2 KO PEMs were infected with or without VSV for 6 hours. Then, cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti-AKT2 antibody, followed by immunoblotting using anti-IRF3 antibody (left panel). Alternatively, cell lysates were prepared for immunoprecipitation using anti-IRF3 antibody, followed by immunoblotting using anti-AKT2 antibody (right panel) to detect the endogenous interaction between AKT2 and IRF3. Data information: * Data are representative of two or three independent biological replicates


G Immunofluorescent microscopic imaging for ( left panel ) and statistics analysis ( right panel ) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post - VSV infection . IRF3 ( red ) , Nuclei ( Hoechst , green ) . The white arrows indicate the nuclei with IRF3 translocation . Mock , n = 2 ; VSV , n = 3 . Bar , 20 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunofluorescent microscopic imaging for (left panel) and statistics analysis (right panel) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post-VSV infection. IRF3 (red), Nuclei (Hoechst, green). The white arrows indicate the nuclei with IRF3 translocation. Mock, n=2; VSV, n=3. Bar, 20 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM).


H Immunoassay of IRF3 , AKT2 , LaminB1 and GAPDH in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 hours . Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively . Data information : * Data are representative of two or three independent biological

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

H Immunoassay of IRF3, AKT2, LaminB1 and GAPDH in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 hours. Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates


I Immunoassay of IRF3 , SP1 and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 hours and stimulated with VSV for 6 hours . Tubulin and SP1 served as cytoplasmic and nucleic protein loading control respectively . Data information : * Data are representative of two or three independent biological

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

I Immunoassay of IRF3, SP1 and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 hours and stimulated with VSV for 6 hours. Tubulin and SP1 served as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates


D Immunofluorescence microscopy ( top panel ) of AKT2 - , Flag - IRF3 - or Flag - IRF3 - T207A - overexpressed MEF cells after poly ( A : T ) stimulation for 6 hours . Flag ( IRF3 , red ) and Hoechst ( nuclei , blue ) . Immunoassay of nuclear - cytoplasm extractions ( bottom panel ) from HEK293T - IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours . GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively . Bar , 20 μm . Data information : * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunofluorescence microscopy (top panel) of AKT2-, Flag-IRF3- or Flag-IRF3-T207A-overexpressed MEF cells after poly(A:T) stimulation for 6 hours. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay of nuclear-cytoplasm extractions (bottom panel) from HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours. GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively. Bar, 20 μm. Data information: * Data are representative of three independent experiments


F Survival rates ( left panel ) until 72 hours and H & E staining ( right panel ) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge . The arrows indicated the VSV - infected eye and skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) test 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Survival rates (left panel) until 72 hours and H&E staining (right panel) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


G qRT - PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later . Every dot represents three zebrafish embryos . Horizontal vertical square bracket shows the statistical analysis of comparison with ' PBS mock ' , the rest shows the comparison with ' PBS VSV ' . PBS mock , n = 15 ; PBS VSV , n = 9 ; IRF3 VSV , n = 16 ; IRF3 - T207A VSV , n = 11 ; AKT2 VSV , n = 14 ; AKT2 - T309A / S474A VSV , n = 9 ; IRF3 + AKT2 VSV , n = 9 ; IRF3 + AKT2 - T309A / S474A VSV , n = 13 ; IRF3 - T207A + AKT2 VSV , n = 10 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA test Data are representative of three independent experiments Error bars mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 qRT-PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=15; PBS VSV, n=9; IRF3 VSV, n=16; IRF3-T207A VSV, n=11; AKT2 VSV, n=14; AKT2-T309A/S474A VSV, n=9; IRF3 + AKT2 VSV, n=9; IRF3 + AKT2-T309A/S474A VSV, n=13; IRF3-T207A + AKT2 VSV, n=10. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are representative of three independent experiments Error bars mean ± SD).


H The survival rates of over - expressed zebrafish larvae as long as 72 hours after the VSV challenge . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) test 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H The survival rates of over-expressed zebrafish larvae as long as 72 hours after the VSV challenge. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) test 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

B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


E WT and Akt2 KO mice were injected with VSV for 24 hours ( 1 × 106 PFU / g , i . v . ) . Representative images of the VSV infected cells ( GFP ) were collected by fluorescence microscope ( left panel ) and the VSV copies ( WT , n = 6 ; Akt2 KO n = 7 ) were measured by qRT - PCR ( right panel ) in the liver of mice . Bar , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a unpaired t - test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 WT and Akt2 KO mice were injected with VSV for 24 hours (1 × 106 PFU/g, i.v.). Representative images of the VSV infected cells (GFP) were collected by fluorescence microscope (left panel) and the VSV copies (WT, n=6; Akt2 KO n=7) were measured by qRT-PCR (right panel) in the liver of mice. Bar, 100 μm. Data information: *P < 0.05, **P < 0.01; using a unpaired t-test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD).


Bone marrow cells from WT and Akt2 KO mice ( CD45 . 2 ) were adoptively transferred to lethally irradiated CD45 . 1 mice and infected with VSV . representative images were analyzed Data information : * 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

Bone marrow cells from WT and Akt2 KO mice (CD45.2) were adoptively transferred to lethally irradiated CD45.1 mice and infected with VSV. representative images were analyzed Data information: * Data are representative of three independent experiments respectively


I Survival rates of WT mice that were intraperitoneal injected with CCT128930 ( 20 mg / kg ) twice every other day before VSV infection ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent experiments

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

I Survival rates of WT mice that were intraperitoneal injected with CCT128930 (20 mg/kg) twice every other day before VSV infection (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


immunoblot analysis of IRF3 , LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions ( C ) from WT and Akt2 KO mice treated with TMPD for 2 weeks . GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively . Data information : * 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 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

immunoblot analysis of IRF3, LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions (C) from WT and Akt2 KO mice treated with TMPD for 2 weeks. GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of three independent experiments


H & E - staining ( E left panel ) and immunofluorescence of IgG deposits ( E right panel ) in the kidney sections were collected from WT and Akt2 KO bone marrow - reconstituted CD45 . 1 mice Bar , 50 μm . Data information : * 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 O O O O

H&E-staining (E left panel) and immunofluorescence of IgG deposits (E right panel) in the kidney sections were collected from WT and Akt2 KO bone marrow-reconstituted CD45.1 mice Bar, 50 μm. Data information: * Data are representative of three independent experiments


J . qRT - PCR analysis of the relative mRNA expression levels of AKT2 ( left panel ) and IFNA ( middle panel ) or IFNβ1 ( right panel ) in CD14 positive monocytes isolated from SLE patients ( n = 9 ) or healthy donors ( n = 9 ) . Data information : * P < 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 ; using unpaired t - test or correlation analyses Data are pooled from different individuals Error bars mean ± SD ) .

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

J. qRT-PCR analysis of the relative mRNA expression levels of AKT2 (left panel) and IFNA (middle panel) or IFNβ1 (right panel) in CD14 positive monocytes isolated from SLE patients (n=9) or healthy donors (n=9). Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using unpaired t-test or correlation analyses Data are pooled from different individuals Error bars mean ± SD).


K Immunofluorescence of IRF3 ( Red ) , AKT2 ( Green ) , DAPI ( Blue , nucleus ) in CD14 + cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software . Cells ( n = 39 ) were collected 3 patients . Bar , 2 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 ; using correlation analyses Data are pooled from different individuals or 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Immunofluorescence of IRF3 (Red), AKT2 (Green), DAPI (Blue, nucleus) in CD14+ cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software. Cells (n=39) were collected 3 patients. Bar, 2 μm. Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using correlation analyses Data are pooled from different individuals or representative of three independent experiments


Representative images of GINs cultures showing GABA and TUJ1 expression . Scale bar = 100 μm .

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

Representative images of GINs cultures showing GABA and TUJ1 expression. Scale bar = 100 μm.


The proportion of GABA + cells of TUJ1 + neurons from both CTRL and sMDD iPSCs derived GINs at day 35 . ( CTRL , n = 120 fields were counted from 5 cell lines ; sMDD , n = 134 fields were counted from 6 cell lines ) .

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

The proportion of GABA+ cells of TUJ1+ neurons from both CTRL and sMDD iPSCs derived GINs at day 35. (CTRL, n=120 fields were counted from 5 cell lines; sMDD, n=134 fields were counted from 6 cell lines).