RNA - seq was conducted on day - 3 of differentiation induction of Tnfaip2 - / - EBs that were either treated with PA / PC or with a vehicle control ( n = 3 independent pools of 7 - 62 EBs per group ) . The heat maps depict the intersection of DEGs with the following gene sets ( G ) " ectoderm development " . Five other stem cell - related genes ( Tfcp2l1 , Dppa3 , Fbxo15 , Zfp42 and Klf2 ) were additionally incorporated based on literature searches . The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2 - / - EBs versus WT EBs The color scale represents the gene - wise z - score calculated from normalized gene expression levels .

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RNA-seq was conducted on day-3 of differentiation induction of Tnfaip2-/- EBs that were either treated with PA/PC or with a vehicle control (n=3 independent pools of 7-62 EBs per group). The heat maps depict the intersection of DEGs with the following gene sets (G) "ectoderm development". Five other stem cell-related genes (Tfcp2l1, Dppa3, Fbxo15, Zfp42 and Klf2) were additionally incorporated based on literature searches. The asterisks indicate the reverted changes in gene expression that were seen in Tnfaip2-/- EBs versus WT EBs The color scale represents the gene-wise z-score calculated from normalized gene expression levels.


( H ) Representative image of immunofluorescence staining against ectodermal marker ( SOX1 ) and the mesodermal marker ( T alias Brachyury ) on day - 3 of differentiation induction of Tnfaip2 - / - EBs + / - co - treatment with PA / PC . Three repeat experiments were conducted with a total number of 9 EBs per

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(H) Representative image of immunofluorescence staining against ectodermal marker (SOX1) and the mesodermal marker (T alias Brachyury) on day-3 of differentiation induction of Tnfaip2-/- EBs +/- co-treatment with PA/PC. Three repeat experiments were conducted with a total number of 9 EBs per group


Oct4 - eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co - infected with 4 reprogramming factors ( OSKM ) ( A ) Representative images of AP staining of iPSC

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Oct4-eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co-infected with 4 reprogramming factors (OSKM) (A) Representative images of AP staining of iPSC colonies


Oct4 - eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co - infected with 4 reprogramming factors ( OSKM ) and double - infected cells were FACS sorted and grown for 14 days , when reprogramming efficiencies were determined by FACS . ( B ) representative FACS profiles of Oct4 - positive iPSCs on day 14 , ( C ) quantification of the percentage of Oct4 - GFP + iPSCs in MEF cultures that were infected with 2 different shRNAs targeting Vim or a scrambled shRNA control [ n = 3 biological replicates per group ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test with Holm - Sidak correction multiple comparisons ] .

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Oct4-eGFP reporter MEFs or WT MEFs were infected with shRNAs Cells were co-infected with 4 reprogramming factors (OSKM) and double-infected cells were FACS sorted and grown for 14 days, when reprogramming efficiencies were determined by FACS. (B) representative FACS profiles of Oct4-positive iPSCs on day 14, (C) quantification of the percentage of Oct4-GFP+ iPSCs in MEF cultures that were infected with 2 different shRNAs targeting Vim or a scrambled shRNA control [n=3 biological replicates per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak correction multiple comparisons].


Oct4 - eGFP reporter MEFs or WT MEFs were infected with combinations of shRNAs . ( D ) Representative FACS profiles of mouse - specific pluripotency cell surface marker SSEA1 + iPSCs on day 14 of reprogramming and ( E ) quantification of the percentage of SSEA1 + iPSCs in MEF cultures that were infected with the indicated shRNAs targeting Vim and / or Tnfaip2 [ n = 5 biological replicates ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) but showed unequal variance ; data were analyzed by Brown - Forsythe and Welch ANOVA and Dunnett ' s multiple comparisons test ] .

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Oct4-eGFP reporter MEFs or WT MEFs were infected with combinations of shRNAs. (D) Representative FACS profiles of mouse-specific pluripotency cell surface marker SSEA1+ iPSCs on day 14 of reprogramming and (E) quantification of the percentage of SSEA1+ iPSCs in MEF cultures that were infected with the indicated shRNAs targeting Vim and/or Tnfaip2 [n=5 biological replicates; data were normally distributed (P>0.05 as per Shapiro-Wilk test) but showed unequal variance; data were analyzed by Brown-Forsythe and Welch ANOVA and Dunnett's multiple comparisons test].


combinations of shRNAs . RT - qPCR was performed to determine ( F ) Vim mRNA expression in scrambled - shRNA infected MEFs versus shRNA - Tnfaip2 - infected MEFs or Infected cells were purified by BFP - sorting on day - 4 after infection [ n = 3 biological replicates ; log transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test with Holm - Sidak for multiple comparisons ] .

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combinations of shRNAs. RT-qPCR was performed to determine (F) Vim mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Tnfaip2-infected MEFs or Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].


RT - qPCR was performed to determine ( G ) Tnfaip2 mRNA expression in scrambled - shRNA infected MEFs versus shRNA - Vim - infected MEFs . G ) Infected cells were purified by BFP - sorting on day - 4 after infection [ n = 3 biological replicates ; log transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test with Holm - Sidak for multiple comparisons ] .

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RT-qPCR was performed to determine (G) Tnfaip2 mRNA expression in scrambled-shRNA infected MEFs versus shRNA-Vim-infected MEFs. G) Infected cells were purified by BFP-sorting on day-4 after infection [n=3 biological replicates; log transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test with Holm-Sidak for multiple comparisons].


Oct4 - eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA ( OE indicates overexpression ) , Cells were co - infected with 4 reprogramming factors ( 4F = Oct4 , Sox2 , Klf4 , c - myc = OSKM ) and double - infected cells were FACS sorted for analysis . ( A ) RT - qPCR analysis on day - 4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2 - depleted cells with ectopic expression of Vim ( left ) or in Vim - depleted cells with ectopic expression of Tnfaip2 ( right ) . Scramble shRNA ( shScr ) - infected MEFs served as a control [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test ] .

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Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), Cells were co-infected with 4 reprogramming factors (4F= Oct4, Sox2, Klf4, c-myc = OSKM) and double-infected cells were FACS sorted for analysis. (A) RT-qPCR analysis on day-4 after infection to determine RNA expression of Tnfaip2 or Vim in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test].


Oct4 - eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA ( OE indicates overexpression ) , ( B ) RT - qPCR analysis on day - 4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2 - depleted cells with ectopic expression of Vim ( left ) or in Vim - depleted cells with ectopic expression of Tnfaip2 ( right ) . Scramble shRNA ( shScr ) - infected MEFs served as a control [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by t - test ] .

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Oct4-eGFP reporter MEFs or WT MEFs were infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), (B) RT-qPCR analysis on day-4 after infection to determine RNA expression of Vim or Tnfaip2 in Tnfaip2-depleted cells with ectopic expression of Vim (left) or in Vim-depleted cells with ectopic expression of Tnfaip2 (right). Scramble shRNA (shScr)-infected MEFs served as a control [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by t-test].


Oct4 - eGFP reporter MEFs or WT MEFs were ( A - D ) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA ( OE indicates overexpression ) , ] . ( C ) Representative images of FACS profiles and ( D ) quantification of SSEA1 + iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs ( OE ) or scrambled shRNA control [ n = 3 independent cultures per group ; data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - way ANOVA and Dunnett ' s multiple comparison test ]

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Oct4-eGFP reporter MEFs or WT MEFs were (A-D) infected with a vector expressing the indicated shRNA in combination with the indicated cDNA (OE indicates overexpression), ]. (C) Representative images of FACS profiles and (D) quantification of SSEA1+ iPSCs on day 14 after infection of MEF cultures with the indicated combination of shRNAs plus cDNAs (OE) or scrambled shRNA control [n=3 independent cultures per group; data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test ]


Oct4 - eGFP reporter MEFs or WT MEFs were ( E , F ) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A . ( E ) Representative FACS profiles and ( F ) quantification of the percentage of SSEA1 + iPSCs in MEF cultures on day - 18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [ n = 5 biological replicates ; data were not normally distributed ( P < 0 . 05 as per Shapiro - Wilk test ) and analyzed by Mann - Whitney U test with Holm Sidak correction for multiple comparisons ] .

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Oct4-eGFP reporter MEFs or WT MEFs were (E,F) infected with the indicated shRNA with or without combined treatment with Etomoxir - a chemical inhibitor of CPT1A. (E) Representative FACS profiles and (F) quantification of the percentage of SSEA1+ iPSCs in MEF cultures on day-18 after infection with shRNAs targeting Tnfaip2 or a scrambled shRNA control with or without continuous treatment with Etomoxir [n=5 biological replicates; data were not normally distributed (P<0.05 as per Shapiro-Wilk test) and analyzed by Mann-Whitney U test with Holm Sidak correction for multiple comparisons].


( A ) TAG levels in exoc3 ( RNAi ) - treated planarians vs . gfp ( RNAi ) - treated controls as determined by UPLC - MS / MS , [ n = 4 experimental replicates on pools of 25 planarians per group per experiment ; data were normally

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(A) TAG levels in exoc3(RNAi)-treated planarians vs. gfp(RNAi)-treated controls as determined by UPLC-MS/MS, [n=4 experimental replicates on pools of 25 planarians per group per experiment; data were normally distributed


( C ) RT - qPCR analysis of exoc3 mRNA expression in planarians of the indicated genotypes and treatment

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(C) RT-qPCR analysis of exoc3 mRNA expression in planarians of the indicated genotypes and treatment schedule


( D ) Representative images of regenerating planarians that were injected with gfp - RNAi , Smed - β - catenin - 1 - RNAi or Smed - β - catenin - 1 - RNAi in combination with PA / PC injection Heads and tails were cut on day - 10 , images were taken on day - 17 after amputation . Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype .

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(D) Representative images of regenerating planarians that were injected with gfp-RNAi, Smed-β-catenin-1-RNAi or Smed-β-catenin-1-RNAi in combination with PA/PC injection Heads and tails were cut on day-10, images were taken on day-17 after amputation. Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.


( E ) Images of body plan maintenance in non - injured planarians that were injected with gfp - RNAi , exoc3 - RNAi or exoc3 - RNAi in combination with PA / PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype .

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(E) Images of body plan maintenance in non-injured planarians that were injected with gfp-RNAi, exoc3-RNAi or exoc3-RNAi in combination with PA/PC injection Number ratios in the photographs indicate the total number of animals exhibiting the represented phenotype.


( F ) RT - qPCR analysis of marker genes of early stem cell differentiation ( prog - 1 and prog - 2 ) and late stem cell differentiation ( Smed - odc1 ) in planarians exposed to the indicated treatment regiments on day - 38 of the experiment as indicated in panel - B [ n = 3 biological replicates ; log - transformed data were normally distributed ( P > 0 . 05 as per Shapiro - Wilk test ) and analyzed by one - way ANOVA and Dunnett ' s multiple comparison test ] .

O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RT-qPCR analysis of marker genes of early stem cell differentiation (prog-1 and prog-2) and late stem cell differentiation (Smed-odc1) in planarians exposed to the indicated treatment regiments on day-38 of the experiment as indicated in panel-B [n=3 biological replicates; log-transformed data were normally distributed (P>0.05 as per Shapiro-Wilk test) and analyzed by one-way ANOVA and Dunnett's multiple comparison test].


( a ) Silver - stained gel image of the affinity - purified complexes of PSPC1 , SFPQ and NONO . The respective proteins are marked along with AcTEV protease used for the elution of purified proteins .

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(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.


( f ) Western blotting of affinity purified complexes of PSPC1 , SFPQ and NONO with SWI / SNF antibodies . cBAF , PBAF , ncBAF specific , as well as common subunits , are marked .

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(f) Western blotting of affinity purified complexes of PSPC1, SFPQ and NONO with SWI/SNF antibodies. cBAF, PBAF, ncBAF specific, as well as common subunits, are marked.


( a ) Silver - stained gel image of the affinity - purified complexes of ARID1A and ARID1B . The bait and a few other SWI / SNF subunits are marked as per their molecular weight .

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(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.


Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( c ) SWI / SNF

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Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (c) SWI/SNF subunits


Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( e ) PSPs .

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Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (e) PSPs.


( f ) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components . SMARCA4 was used as a positive control for purification .

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(f) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components. SMARCA4 was used as a positive control for purification.


( a ) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo - FLAG - tagged ARID1B . Input and eluted samples were resolved on a gel followed by western blotting with various antibodies . Western with SWI / SNF subunits - SMARCA4 and SMARCD1 were used as the control for purification , while beta - actin was used as a loading control .

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(a) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo-FLAG-tagged ARID1B. Input and eluted samples were resolved on a gel followed by western blotting with various antibodies. Western with SWI/SNF subunits- SMARCA4 and SMARCD1 were used as the control for purification, while beta-actin was used as a loading control.


( e , f ) ( e ) RIP qPCR and ( f ) PCR analysis performed using an antibody against endogenous ARID1B . for monitoring NEAT1 _ 2 levels . RIP with NONO and IgG was used as a positive and negative control respectively . GAPDH levels were also assessed in the assay . Data are expressed as a percent of Input .

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(e, f) (e) RIP qPCR and (f) PCR analysis performed using an antibody against endogenous ARID1B. for monitoring NEAT1_2 levels. RIP with NONO and IgG was used as a positive and negative control respectively. GAPDH levels were also assessed in the assay. Data are expressed as a percent of Input.


( c ) smFISH analysis was performed to detect NEAT1 signal in the nucleus . The scale bar corresponds to 10 µm . Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control . The number of nuclei ( n ) used for quantification is mentioned .

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(c) smFISH analysis was performed to detect NEAT1 signal in the nucleus. The scale bar corresponds to 10 µm. Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control. The number of nuclei (n) used for quantification is mentioned.


Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs . Passage 6 cells represent the proliferating state , and passages 21 and 34 the senescent state for HUVECs and IMR90 , respectively .

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Western blots showing changing protein levels on the path to senescence in IMR90 and HUVECs. Passage 6 cells represent the proliferating state, and passages 21 and 34 the senescent state for HUVECs and IMR90, respectively.


Super - resolution ( gSTED ) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI . Bar : 2μm .

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Super-resolution (gSTED) imaging of HMGB1 distribution in proliferating HUVEC nuclei counterstained with DAPI. Bar: 2μm.


Representative immunofluorescence images of IMR90 and HUVECs ( left ) showing reduced HMGB1 levels in senescent nuclei ; bean plots quantify this reduction ( right ; N is the number of cells analyzed per each condition / cell type ) . Bars : 5 μm . * : P < 0 . 01 ; Wilcoxon - Mann - Whitney test .

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Representative immunofluorescence images of IMR90 and HUVECs (left) showing reduced HMGB1 levels in senescent nuclei; bean plots quantify this reduction (right; N is the number of cells analyzed per each condition/cell type). Bars: 5 μm. *: P<0.01; Wilcoxon-Mann-Whitney test.


Genome browser view of HMGB1 ChIP - seq ( normalized to input ; mean from two replicates ) from proliferating HUVEC ( red ) or proliferating / senescent IMR90 ( grey / green ) in the TMEM92 locus .

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Genome browser view of HMGB1 ChIP-seq (normalized to input; mean from two replicates) from proliferating HUVEC (red) or proliferating/senescent IMR90 (grey/green) in the TMEM92 locus.


Heatmap showing protein ( SASP , from http : / / www . saspatlas . com / ; log fold ratio ) and gene expression levels ( log2FC ) of IMR90 SASP - related genes embedded in high - HMGB1 ( red ) TADs like those in panel C . * * : genes bound by HMGB1 in ChIP - seq data are more than expected by chance ; P > 0 . 001 , hypergeometric test .

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Heatmap showing protein (SASP, from http://www.saspatlas.com/; log fold ratio)and gene expression levels (log2FC) of IMR90 SASP-related genes embedded in high-HMGB1 (red) TADs like those in panel C. **: genes bound by HMGB1 in ChIP-seq data are more than expected by chance; P>0.001, hypergeometric test.


Immunofluorescence ( top ) , western blot ( middle ) and RT - qPCR analyses ( bottom ; mean fold - change ± S . D . from two biological replicates ) confirm HMGB1 knockdown in IMR90 .

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Immunofluorescence (top), western blot (middle) and RT-qPCR analyses (bottom; mean fold-change ±S.D. from two biological replicates) confirm HMGB1 knockdown in IMR90.


Representative images of IMR90 overexpressing HMGB1 - GFP , immunostained for p21 and CTCF , and counterstained with DAPI . IMR90 transfected with empty vectors provide a control . Bar : 5 μm .

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Representative images of IMR90 overexpressing HMGB1-GFP, immunostained for p21 and CTCF, and counterstained with DAPI. IMR90 transfected with empty vectors provide a control. Bar: 5 μm.


Genome browser views showing HMGB1 sCLIP data ( black ) along the ASH1L , CCNL2 and TET2 loci ; input tracks ( grey ) provide background levels . * : significantly - enriched peaks . The consensus motif for HMGB1 binding on RNA is also shown ( bottom right ) .

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Genome browser views showing HMGB1 sCLIP data (black) along the ASH1L, CCNL2 and TET2 loci; input tracks (grey) provide background levels. *: significantly-enriched peaks. The consensus motif for HMGB1 binding on RNA is also shown (bottom right).


Bar graphs showing mean fold - change of selected mRNAs ( over ± S . D . from two biological replicates ) from ILF3 - ( purple ) , RBMX - ( white ) or PNN - knockdown experiments ( blue ) in proliferating IMR90 . * : significantly different to siRNA controls ; P < 0 . 01 , unpaired two - tailed Student ' s t - test . Dashed line indicates no change in expression .

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Bar graphs showing mean fold-change of selected mRNAs (over ±S.D. from two biological replicates) from ILF3- (purple), RBMX- (white) or PNN-knockdown experiments (blue) in proliferating IMR90. *: significantly different to siRNA controls; P<0.01, unpaired two-tailed Student's t-test. Dashed line indicates no change in expression.


A . RNA - seq of whole blood from symptomatic ( n = 26 ; median 8 days PIO ) and asymptomatic ( n = 30 ; median 3 days post - hospital admission ) COVID - 19 patients at the acute phase of infection ( SARS - CoV - 2 PCR - positive ) was performed . Only samples with RNA integrity number > 6 were sent for sequencing and included in the analysis . Volcano plot indicating DEGs between asymptomatic and symptomatic COVID - 19 patients , with thresholds of FDR < 0 . 05 and | FC | > 2 . Numbers of over - expressed and under - expressed genes are indicated .

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A. RNA-seq of whole blood from symptomatic (n=26; median 8 days PIO) and asymptomatic (n=30; median 3 days post-hospital admission) COVID-19 patients at the acute phase of infection (SARS-CoV-2 PCR-positive) was performed. Only samples with RNA integrity number >6 were sent for sequencing and included in the analysis. Volcano plot indicating DEGs between asymptomatic and symptomatic COVID-19 patients, with thresholds of FDR < 0.05 and |FC| > 2. Numbers of over-expressed and under-expressed genes are indicated.


B . Network analysis of significant immune mediators between symptomatic and asymptomatic COVID - 19 patients . Interactive relationships between the cytokines or chemokines were determined by STRING ( Search Tool for the Retrieval of Interacting Genes / Proteins ) analysis , with a confidence threshold of 0 . 5 .

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B. Network analysis of significant immune mediators between symptomatic and asymptomatic COVID-19 patients. Interactive relationships between the cytokines or chemokines were determined by STRING (Search Tool for the Retrieval of Interacting Genes/ Proteins) analysis, with a confidence threshold of 0.5.


C . SARS - CoV - 2 specific CD4 + non T follicular helper ( TFH ) cells from symptomatic ( n = 5 ) and asymptomatic COVID - 19 ( n = 5 ) patients were characterized with flow cytometry based on the expression of IL - 17a , IFN - γ , TNF - ⍺ , IL - 4 and IL - 10 upon SARS - CoV - 2 peptide stimulation . Representative plot overlay of unstimulated ( black ) on peptide stimulated ( red ) was performed on asymptomatic patient . Data are presented as Mean ± SD * * P < 0 . 01 ( Mann - Whitney U test ) .

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C. SARS-CoV-2 specific CD4+ non T follicular helper (TFH) cells from symptomatic (n=5) and asymptomatic COVID-19 (n=5) patients were characterized with flow cytometry based on the expression of IL-17a, IFN-γ, TNF-⍺, IL-4 and IL-10 upon SARS-CoV-2 peptide stimulation. Representative plot overlay of unstimulated (black) on peptide stimulated (red) was performed on asymptomatic patient. Data are presented as Mean ± SD **P<0.01 (Mann-Whitney U test).


A , B . ( A ) IgG and ( B ) IgM responses were analysed by screening plasma samples of asymptomatic ( n = 39 , median study day 29 ) and symptomatic ( n = 57 , median study day 31 ) COVID - 19 patients . Data are presented as Mean ± SD * * * P < 0 . 001 ( Mann - Whitney U test ) .

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A, B. (A) IgG and (B) IgM responses were analysed by screening plasma samples of asymptomatic (n=39, median study day 29) and symptomatic (n=57, median study day 31) COVID-19 patients. Data are presented as Mean ± SD ***P<0.001 (Mann-Whitney U test).


A . Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic ( n = 16 ) and symptomatic ( n = 52 ) COVID - 19 patients and healthy controls ( n = 17 ) . Data are presented as Mean ± SD * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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A. Flow cytometry acquisition of patient whole blood was performed and number of mature and immature neutrophils were compared between asymptomatic (n=16) and symptomatic (n=52) COVID-19 patients and healthy controls (n=17). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


B . Mass cytometry was performed on PBMCs obtained from acute asymptomatic ( n = 19 ) and acute symptomatic ( n = 37 ) COVID - 19 patients and healthy donors ( n = 10 ) . Monocytes were characterized based on their CD14 and CD16 expression . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) . C . Classical , intermediate and non - classical monocytes were further defined in acute asymptomatic ( n = 19 ) and acute symptomatic ( n = 37 ) COVID - 19 patients and healthy donors ( n = 10 ) with inflammatory marker CD169 . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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B. Mass cytometry was performed on PBMCs obtained from acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10). Monocytes were characterized based on their CD14 and CD16 expression. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison). C. Classical, intermediate and non-classical monocytes were further defined in acute asymptomatic (n=19) and acute symptomatic (n=37) COVID-19 patients and healthy donors (n=10) with inflammatory marker CD169. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


D . Expression data of genes associated with inflammatory monocytes , activated neutrophils and pro - inflammatory cytokines were compared between acute asymptomatic ( n = 30 ) and asymptomatic ( n = 26 ) COVID - 19 patients and were represented by ratio with respect to expression in asymptomatic patients . Bar graphs show DEGs with FDR adjusted P value < 0 . 05 and fold change values are represented in the log2 scale .

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D. Expression data of genes associated with inflammatory monocytes, activated neutrophils and pro-inflammatory cytokines were compared between acute asymptomatic (n=30) and asymptomatic (n=26) COVID-19 patients and were represented by ratio with respect to expression in asymptomatic patients. Bar graphs show DEGs with FDR adjusted P value < 0.05 and fold change values are represented in the log2 scale.


E . Pro - inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45 - plex microbead - based immunoassay . Immune mediator levels were compared between asymptomatic patients ( n = 48 ) and symptomatic patients stratified by COVID - 19 severity ( mild , n = 61 ; moderate , n = 43 ; severe , n = 68 ) . Immune mediator levels for healthy controls ( HC ) ( n = 23 ) are indicated by the black dotted line . Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification ( LOQ ) . Data are presented as Mean ± SD * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( One - way ANOVA with post hoc t test ) .

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E. Pro-inflammatory associated immune mediator levels in plasma fraction samples from first collection timepoint during hospital admission were quantified with 45-plex microbead-based immunoassay. Immune mediator levels were compared between asymptomatic patients (n=48) and symptomatic patients stratified by COVID-19 severity (mild, n=61; moderate, n=43; severe, n=68). Immune mediator levels for healthy controls (HC) (n=23) are indicated by the black dotted line. Patient samples with concentrations out of measurement range are presented as the value of Limit of Quantification (LOQ). Data are presented as Mean ± SD *P<0.05; **P<0.01; ***P<0.001 (One-way ANOVA with post hoc t test).


A . Growth factors in the plasma of asymptomatic ( n = 48 ) , symptomatic patients ( n = 173 ) and healthy controls ( n = 23 ) were analyzed by Luminex . A ratio of VEGF - A / VEGF - D was calculated and used to perform receiver operating characteristics analysis . The area under the curve was shown . Data are presented as Mean ± SD * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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A. Growth factors in the plasma of asymptomatic (n=48), symptomatic patients (n=173) and healthy controls (n=23) were analyzed by Luminex. A ratio of VEGF-A/VEGF-D was calculated and used to perform receiver operating characteristics analysis. The area under the curve was shown. Data are presented as Mean ± SD **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


B . RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown . Genes with false discovery rate less than 0 . 05 ( * ) , 0 . 01 ( * * ) or 0 . 001 ( * * * ) are indicated .

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B. RNAseq analysis was performed on whole blood and selected transcripts that are overexpressed in asymptomatic patients over symptomatic patients are shown. Genes with false discovery rate less than 0.05 (*), 0.01 (**) or 0.001 (***) are indicated.


C . Cell counts of DCs , CD4 T cells , CD8 T cells and B cells that express specific chemokine markers between asymptomatic ( n = 19 ) , symptomatic patients ( n = 37 ) and healthy controls ( n = 10 ) are analyzed by CyTOF and shown . Data are presented as Mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Kruskal - Wallis test with Dunn ' s multiple comparison ) .

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C. Cell counts of DCs, CD4 T cells, CD8 T cells and B cells that express specific chemokine markers between asymptomatic (n=19), symptomatic patients (n=37) and healthy controls (n=10) are analyzed by CyTOF and shown. Data are presented as Mean ± SD. *P<0.05; **P<0.01; ***P<0.001 (Kruskal-Wallis test with Dunn's multiple comparison).


a , Etoposide - treated Atg5 - / - MEFs were assessed by EM ( left ) and by Lamp2immunofluorescence ( IF ; middle ) . Right : reverse Lamp2 immunofluorescence image merged with the electron microscopy image . Arrows indicate Lamp2 - positive autolysosomes . Magnified photos are provided in Supplementary Fig .

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a, Etoposide-treated Atg5 -/- MEFs were assessed by EM (left) and by Lamp2immunofluorescence (IF; middle). Right: reverse Lamp2 immunofluorescence image merged with the electron microscopy image. Arrows indicate Lamp2-positive autolysosomes. Magnified photos are provided in Supplementary Fig. 5


b , The size of each autophagic vacuole ( AV ) ( n = 30 cells ) in MEFs treated with etoposide . The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5 - / - cells .

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b, The size of each autophagic vacuole (AV) (n = 30 cells) in MEFs treated with etoposide. The total number of AVs in 30 cells was 483 in WT cells and 494 in Atg5-/- cells.


c , Percentage autophagic area ( n ≥ 20 cells ) in MEFs treated with etoposide .

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c, Percentage autophagic area (n ≥ 20 cells) in MEFs treated with etoposide.


d , e , Representative macroautophagy in etoposide - treated Atg5 - / - MEFs . Conventional ( d ) and quick freezing and freeze - substitution ( e ) techniques were used . The autolysosomes ( arrows ) contain multilamellar bodies ( arrowheads ) .

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d, e, Representative macroautophagy in etoposide-treated Atg5-/- MEFs. Conventional (d) and quick freezing and freeze-substitution (e) techniques were used. The autolysosomes (arrows) contain multilamellar bodies (arrowheads).


f , The number of autophagosomes ( white columns ) and autolysosomes ( black columns ) in MEFs ( n = 10 cells ) treated with etoposide .

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f, The number of autophagosomes (white columns) and autolysosomes (black columns) in MEFs (n = 10 cells) treated with etoposide.


g , Reduction of percentage autophagic area by 3 - methyladenine ( 3 - MA ; n = 22 cells ) .

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g, Reduction of percentage autophagic area by 3-methyladenine (3-MA; n = 22 cells).


. h , Time - course analysis of long - lived protein degradation ( n = 4 ) .

. h, Time-course analysis of long-lived protein degradation (n = 4).


i , Inhibition of long - lived protein degradation by bafilomycin A1 , protease inhibitor ( PI ) cocktail and 3 - MA ( * P 0 . 05 ; n = 4 ) .

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i, Inhibition of long-lived protein degradation by bafilomycin A1, protease inhibitor (PI) cocktail and 3-MA (*P 0.05; n = 4).


j , k , Similar experiments to those in c and i were performed by starvation . Red and blue lines in b , c , g and j indicate means and s . e . m . , respectively . The dashed line in c , g and j indicates the autophagic cell border ( 6 % ) . Black error bars indicate s . d .

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j, k, Similar experiments to those in c and i were performed by starvation. Red and blue lines in b, c, g and j indicate means and s.e.m., respectively. The dashed line in c, g and j indicates the autophagic cell border (6%). Black error bars indicate s.d.


a , The production of LC3 - II . Gapdh was used as a loading control .

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a, The production of LC3-II. Gapdh was used as a loading control.


b , c , Punctate GFP - LC3 fluorescence in WT MEFs but not in Atg5 - / - MEFs . b , Representative photographs . c , Graphical presentation of the percentages of WT MEFs and Atg5 - / - MEFs with punctate GFP - LC3 fluorescence .

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b, c, Punctate GFP-LC3 fluorescence in WT MEFs but not in Atg5-/- MEFs. b, Representative photographs. c, Graphical presentation of the percentages of WT MEFs and Atg5-/- MEFs with punctate GFP-LC3 fluorescence.


d , The same GFP - LC3 - transfected Atg5 - / - MEFs were assessed by IF and by EM . The magnified photo is the area indicated by the squares . Arrows indicate AVs .

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d, The same GFP-LC3-transfected Atg5-/- MEFs were assessed by IF and by EM. The magnified photo is the area indicated by the squares. Arrows indicate AVs.


e , Atg5 - / - MEFs were incubated without ( NT ) or with etoposide and immunostained with an anti - Lamp2 antibody ( green ) . Nuclei were counterstained ( red ) . The percentages of cells with punctate Lamp2 immunostaining are shown ( means ± s . d . , n = 4 ) .

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e, Atg5-/- MEFs were incubated without (NT) or with etoposide and immunostained with an anti-Lamp2 antibody (green). Nuclei were counterstained (red). The percentages of cells with punctate Lamp2 immunostaining are shown (means ± s.d., n = 4).


f , Microarray analysis of the Atg5 - / - MEFs during treatment with etoposide .

f, Microarray analysis of the Atg5-/- MEFs during treatment with etoposide.


g , h , Induction of Ulk1 in the etoposide - treated Atg5 - / - MEFs was assessed by qPCR ( g ) and by immunoblotting ( h ) .

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g, h, Induction of Ulk1 in the etoposide-treated Atg5-/- MEFs was assessed by qPCR (g) and by immunoblotting (h).


i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the Ulk1 complex ( i , j ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by Lamp2 IF ( i , k )

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i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k)


i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the Ulk1 complex ( i , j ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by EM ( j , l ) .

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i-l, Atg5-/- MEFs were transfected with the siRNAs related to the Ulk1 complex (i, j) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).


i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the PI ( 3 ) K complex ( k , l ) and a beclin 1 plasmid ( k ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by Lamp2 IF ( i , k ) .

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i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l) and a beclin 1 plasmid (k) for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by Lamp2 IF (i, k).


i - l , Atg5 - / - MEFs were transfected with the siRNAs related to the PI ( 3 ) K complex ( k , l ) for 24 h and treated with etoposide for 18 h . Induction of macroautophagy was assessed by EM ( j , l ) .

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i-l, Atg5-/- MEFs were transfected with the siRNAs related to the PI(3)K complex (k, l)for 24 h and treated with etoposide for 18 h. Induction of macroautophagy was assessed by EM (j, l).


a - d , Electron micrographs of etoposide - treated Atg5 - / - MEFs . a , AVs were observed near the Golgi apparatus ( G ) . Inset , the isolation membrane ( I ) was extended from the Golgi stack . b , Developing autophagosome . rER , rough endoplasmic reticulum . c , d , Isolation membrane fusing with vesicles containing thick membrane ( d ) and a complete autophagosome ( c ) is generated .

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a-d, Electron micrographs of etoposide-treated Atg5-/- MEFs. a, AVs were observed near the Golgi apparatus (G). Inset, the isolation membrane (I) was extended from the Golgi stack. b, Developing autophagosome. rER, rough endoplasmic reticulum. c, d, Isolation membrane fusing with vesicles containing thick membrane (d) and a complete autophagosome (c) is generated.


e , f , Co - localization of Lamp2 with GFP - M6PR , GFP - Stx7 or GFP - Rab9 in etoposide - treated Atg5 - / - MEFs . Arrows indicate co - localized dots . Original magnification , × 200 .

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e, f, Co-localization of Lamp2 with GFP-M6PR, GFP-Stx7 or GFP-Rab9 in etoposide-treated Atg5-/- MEFs. Arrows indicate co-localized dots. Original magnification, × 200.


g - i , Atg5 - / - MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide . Induction of macroautophagy was assessed by Lamp2 IF ( g ) and by EM ( h ) , and the number of isolation membranes was calculated ( i ) . In g , error bars indicate s . d . ( n = 4 ) . In h and i , red and blue lines indicate means and s . e . m . , respectively ; n = 35 cells each .

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g-i, Atg5-/- MEFs were transfected with the indicated siRNAs for 24 h and treated with etoposide. Induction of macroautophagy was assessed by Lamp2 IF (g) and by EM (h), and the number of isolation membranes was calculated (i). In g, error bars indicate s.d. (n = 4). In h and i, red and blue lines indicate means and s.e.m., respectively; n = 35 cells each.


j , No effect of silencing of Rab9 ( siRab9 ) on the production of LC3 - II in etoposide - treated WT MEFs .

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j, No effect of silencing of Rab9 (siRab9) on the production of LC3-II in etoposide-treated WT MEFs.


a - e , Typical autophagic structures in the tissues of an Atg5 - / - embryo ( E14 . 5 ) : a , midbrain ; b - d , liver ; c , d , developing autophagosomes ; e , heart . Arrows indicate the autophagic vacuoles .

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a-e, Typical autophagic structures in the tissues of an Atg5-/- embryo (E14.5): a, midbrain; b-d, liver; c, d, developing autophagosomes; e, heart. Arrows indicate the autophagic vacuoles.


f , g , k , l , Electron micrographs of wild - type ( WT ) and Atg5 - / - ( KO ) reticulocytes ( f , g ) and erythrocytes ( k , l ) . An isolation membrane ( asterisk ) , autophagosomes ( arrows ) and autolysosomes ( arrowheads ) are shown . Inset , engulfed mitochondria .

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f, g, k, l, Electron micrographs of wild-type (WT) and Atg5-/- (KO) reticulocytes (f, g) and erythrocytes (k, l). An isolation membrane (asterisk), autophagosomes (arrows) and autolysosomes (arrowheads) are shown. Inset, engulfed mitochondria.


h - j , m - o , The numbers of autophagic vacuoles ( h , m ) and autophagic vacuoles containing mitochondria ( i , n ) and persisting mitochondria ( j , o ) in reticulocytes ( h - j ) and erythrocytes ( m - o ) . Error bars indicate s . d . ( * P 0 . 05 ; n = 55 cells ) .

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h-j, m-o, The numbers of autophagic vacuoles (h, m) and autophagic vacuoles containing mitochondria (i, n) and persisting mitochondria (j, o) in reticulocytes (h-j) and erythrocytes (m-o). Error bars indicate s.d. (*P  0.05; n = 55 cells).


( A ) GAPC enzymatic activity was measured in wild - type crude extracts treated with H2O ( mock ) , and 100 µM of Tyr - Asp , Tyr , Asp , and Ile - Glu . ( B - D ) The enzymatic activities of GAPC ( B ) , GAPA / B ( C ) , and GAPN ( D ) were measured in the wild type and double k . o . mutant gapc1gapc2 treated with H2O ( mock ) , and 100 µM of Tyr - Asp . Data information : Data are mean ± SEM of n = 4 ( A - D ; four technical replicates ) , significance was assessed using unpaired two - tailed Student t - test .

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

(A) GAPC enzymatic activity was measured in wild-type crude extracts treated with H2O (mock), and 100 µM of Tyr-Asp, Tyr, Asp, and Ile-Glu. (B-D) The enzymatic activities of GAPC (B), GAPA/B (C), and GAPN (D) were measured in the wild type and double k.o. mutant gapc1gapc2 treated with H2O (mock), and 100 µM of Tyr-Asp. Data information: Data are mean ± SEM of n=4 (A-D; four technical replicates) , significance was assessed using unpaired two-tailed Student t-test.


( A ) Fresh weight quantification of Arabidopsis and tobacco seedlings . Seedlings were pretreated with mock ( water ) , Tyr - Asp ( 100 μM ) , Tyr and Asp ( 100 μM ) , Ser - Leu ( 100 μM ) , or Gly - Pro ( 100 μM ) for 1 h prior an oxidative stress ( catechin or H2O2 ) . Each treatment was compared to its respective control , corresponding to plants grown in one 24 - well plate ( represented by the adjoined bars ) . Data information : Data are mean ± SEM of n = 10 - 12 ( seedlings ) . Unpaired two - tailed Student t - test was performed to compare treatments with control . Scale bar : 10 cm .

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

(A) Fresh weight quantification of Arabidopsis and tobacco seedlings. Seedlings were pretreated with mock (water), Tyr-Asp (100 μM), Tyr and Asp (100 μM), Ser-Leu (100 μM), or Gly-Pro (100 μM) for 1 h prior an oxidative stress (catechin or H2O2). Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm.


( B ) Catechin - ( left upper panel ) and ( catechin - ) Tyr - Asp - treated tobacco plants ( right upper panel ) grown for four days in liquid media . H2O2 - ( left upper panel ) and ( H2O2 - ) Tyr - Asp - treated tobacco plants ( right upper panel ) grown for two days in liquid media . All plants were 2 - weeks - old before starting the stress treatment . Data information : Data are mean ± SEM of n = 10 - 12 ( seedlings ) .

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

(B) Catechin- (left upper panel) and (catechin-) Tyr-Asp-treated tobacco plants (right upper panel) grown for four days in liquid media. H2O2- (left upper panel) and (H2O2-) Tyr-Asp-treated tobacco plants (right upper panel) grown for two days in liquid media. All plants were 2-weeks-old before starting the stress treatment. Data information: Data are mean ± SEM of n=10-12 (seedlings).


( A - C ) Fresh weight measurements of Arabidopsis wild - type and gapc1 / gapc2 double k . o . plants subjected to the oxidative stress ( e . g . catechin ) and Tyr - Asp treatments . Each treatment was compared to its respective control , corresponding to plants grown in one 24 - well plate ( represented by the adjoined bars ) . ( D - F ) Representative Arabidopsis wild type and gapc1 / gapc2 double k . o . seedlings from the different treatments . All plants were 10 - day - old at the stress onset . The seedlings were exposed to catechin for three days . Data information : Data are mean ± SEM of n = 10 - 12 ( seedlings ) . Unpaired two - tailed Student t - test was performed to compare treatments with control . Scale bar : 10 cm .

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

(A-C) Fresh weight measurements of Arabidopsis wild-type and gapc1/gapc2 double k.o. plants subjected to the oxidative stress (e.g. catechin) and Tyr-Asp treatments. Each treatment was compared to its respective control, corresponding to plants grown in one 24-well plate (represented by the adjoined bars). (D-F) Representative Arabidopsis wild type and gapc1/gapc2 double k.o. seedlings from the different treatments. All plants were 10-day-old at the stress onset. The seedlings were exposed to catechin for three days. Data information: Data are mean ± SEM of n=10-12 (seedlings). Unpaired two-tailed Student t-test was performed to compare treatments with control. Scale bar: 10 cm.


( C ) PEPCK activity was measured in a protein lysate prepared from 3 - week - old Arabidopsis leaves in absence ( control , C ) or presence of 500 µM of the different dipeptides . Measurements were performed in the carboxylation direction , as described in the Methods section . Data are mean ± SE of 4 independent lysate preparations . Unpaired two - tailed Student t - tests were performed to compare dipeptide treatments with the control .

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 O O O O O O O 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) PEPCK activity was measured in a protein lysate prepared from 3-week-old Arabidopsis leaves in absence (control, C) or presence of 500 µM of the different dipeptides. Measurements were performed in the carboxylation direction, as described in the Methods section. Data are mean ± SE of 4 independent lysate preparations. Unpaired two-tailed Student t-tests were performed to compare dipeptide treatments with the control.


( A ) Ribbon diagram of the HsAtg4B - LC3 ( 1 - 120 ) complex . HsAtg4B is coloured salmon red , and LC3 is coloured green .

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(A) Ribbon diagram of the HsAtg4B-LC3(1-120) complex. HsAtg4B is coloured salmon red, and LC3 is coloured green.


( B ) Structural comparison between free and HsAtg4B ‐ bound LC3 . Crystal structure of free LC3 ( 1 - 120 ) ( PDB code 1UGM ) is superimposed on the HsAtg4B ‐ bound LC3 . HsAtg4B ‐ bound LC3 is coloured green , and free LC3 is coloured grey .

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(B) Structural comparison between free and HsAtg4B‐bound LC3. Crystal structure of free LC3(1-120) (PDB code 1UGM) is superimposed on the HsAtg4B‐bound LC3. HsAtg4B‐bound LC3 is coloured green, and free LC3 is coloured grey.


( C ) Structural comparison between free and LC3 ‐ bound HsAtg4B . Crystal structure of free HsAtg4B ( PDB code 2CY7 ) is superimposed on the LC3 ‐ bound HsAtg4B . LC3 ‐ bound HsAtg4B is coloured salmon red , and free HsAtg4B is coloured grey . The regions with large conformational differences between free and LC3 ‐ bound forms are coloured black ( free ) and red ( LC3 bound ) . All the figures representing molecular structures were generated with PyMOL ( DeLano , 2002 ) .

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(C) Structural comparison between free and LC3‐bound HsAtg4B. Crystal structure of free HsAtg4B (PDB code 2CY7) is superimposed on the LC3‐bound HsAtg4B. LC3‐bound HsAtg4B is coloured salmon red, and free HsAtg4B is coloured grey. The regions with large conformational differences between free and LC3‐bound forms are coloured black (free) and red (LC3 bound). All the figures representing molecular structures were generated with PyMOL ( DeLano, 2002).


( A ) Interaction between HsAtg4B and the ubiquitin core of LC3 . HsAtg4B is coloured salmon red , and LC3 is coloured green . The side chains of the residues involved in the HsAtg4B - LC3 interaction are shown as stick models . Possible hydrophilic interactions are shown as broken lines .

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(A) Interaction between HsAtg4B and the ubiquitin core of LC3. HsAtg4B is coloured salmon red, and LC3 is coloured green. The side chains of the residues involved in the HsAtg4B-LC3 interaction are shown as stick models. Possible hydrophilic interactions are shown as broken lines.


( B ) Interaction between HsAtg4B and the C ‐ terminal tail of LC3 . Trp142 and the regulatory loop of HsAtg4B are coloured blue , whereas the remainder is coloured as in ( A ) . As the side chain of Cys74 has two conformations , both conformations are shown . Possible hydrophilic interactions are shown as broken lines .

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(B) Interaction between HsAtg4B and the C‐terminal tail of LC3. Trp142 and the regulatory loop of HsAtg4B are coloured blue, whereas the remainder is coloured as in (A). As the side chain of Cys74 has two conformations, both conformations are shown. Possible hydrophilic interactions are shown as broken lines.


( C ) In vitro proteolysis assay . Left , LC3 mutants fused to GST were incubated with HsAtg4B . Right , LC3 - GST was incubated with HsAtg4B mutants . Procedures are described in detail in Materials and methods .

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(C) In vitro proteolysis assay. Left, LC3 mutants fused to GST were incubated with HsAtg4B. Right, LC3-GST was incubated with HsAtg4B mutants. Procedures are described in detail in Materials and methods.


( A ) Annealed Fo − Fc electron density map for the C ‐ terminal region ( residues 119 - 122 ) of LC3 ( 1 - 124 ) . Left , LC3 ( 1 - 124 ) bound to the HsAtg4B H280A mutant ; right , LC3 ( 1 - 124 ) bound to the HsAtg4B C74S mutant . Structure of the C ‐ terminal region of LC3 is also shown as a stick model , in which carbon , nitrogen and oxygen atoms are coloured green , blue and red , respectively .

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(A) Annealed Fo−Fc electron density map for the C‐terminal region (residues 119-122) of LC3(1-124). Left, LC3(1-124) bound to the HsAtg4B H280A mutant; right, LC3(1-124) bound to the HsAtg4B C74S mutant. Structure of the C‐terminal region of LC3 is also shown as a stick model, in which carbon, nitrogen and oxygen atoms are coloured green, blue and red, respectively.


( B ) Structure of the C ‐ terminal tail of LC3 ( 1 - 124 ) and the catalytic site of HsAtg4B . Left , LC3 ( 1 - 124 ) bound to HsAtg4B H280A mutant , right , LC3 ( 1 - 124 ) bound to the HsAtg4B C74S mutant . HsAtg4B is shown as a ribbon model , whereas LC3 is shown as a stick model . The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model . Carbon , nitrogen , oxygen and sulphur atoms are coloured green , blue , red and yellow , respectively . Mutated catalytic residues are coloured cyan .

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(B) Structure of the C‐terminal tail of LC3(1-124) and the catalytic site of HsAtg4B. Left, LC3(1-124) bound to HsAtg4B H280A mutant, right, LC3(1-124) bound to the HsAtg4B C74S mutant. HsAtg4B is shown as a ribbon model, whereas LC3 is shown as a stick model. The side chains of the residues comprising the catalytic triad and Tyr54 of HsAtg4B are also shown as a stick model. Carbon, nitrogen, oxygen and sulphur atoms are coloured green, blue, red and yellow, respectively. Mutated catalytic residues are coloured cyan.


( C ) Structural comparison between the HsAtg4B - LC3 precursor and SENP2 - SUMO ‐ 3 precursor complexes . Left , structure of the HsAtg4B - LC3 precursor complex ; right , structure of the SENP2 - SUMO ‐ 3 precursor complex . HsAtg4B and SENP2 are shown as a ribbon model , whereas LC3 and SUMO ‐ 3 precursors are shown as a stick model . The side chains of the important residues at the catalytic site are also shown as a stick model . Atom colouring is the same as in ( A , B ) .

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(C) Structural comparison between the HsAtg4B-LC3 precursor and SENP2-SUMO‐3 precursor complexes. Left, structure of the HsAtg4B-LC3 precursor complex; right, structure of the SENP2-SUMO‐3 precursor complex. HsAtg4B and SENP2 are shown as a ribbon model, whereas LC3 and SUMO‐3 precursors are shown as a stick model. The side chains of the important residues at the catalytic site are also shown as a stick model. Atom colouring is the same as in (A, B).


( A ) Surface model of free ( left ) and LC3 ( 1 - 124 ) ‐ bound ( right ) HsAtg4B . W142 and the regulatory loop are coloured blue , and the N ‐ terminal tail is coloured cyan . The catalytic Cys74 is coloured red . LC3 ( 1 - 124 ) is shown as a ribbon model .

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(A) Surface model of free (left) and LC3(1-124)‐bound (right) HsAtg4B. W142 and the regulatory loop are coloured blue, and the N‐terminal tail is coloured cyan. The catalytic Cys74 is coloured red. LC3(1-124) is shown as a ribbon model.


( B ) In vitro proteolysis assay using N ‐ terminal tail ‐ deleted HsAtg4B . LC3 - GST was incubated with either wild ‐ type or N ‐ terminally truncated HsAtg4B and was subjected to SDS – PAGE analysis .

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(B) In vitro proteolysis assay using N‐terminal tail‐deleted HsAtg4B. LC3-GST was incubated with either wild‐type or N‐terminally truncated HsAtg4B and was subjected to SDS–PAGE analysis.


( C ) Structural comparison between free and LC3 ( 1 - 124 ) ‐ bound HsAtg4B and SENP2 - SUMO ‐ 3 precursor complexes . Left , free HsAtg4B ; middle , the HsAtg4B - LC3 precursor complex ; right , the SENP2 - SUMO ‐ 3 precursor complex . The three structures are shown in the same orientation . Model colouring is the same as in Figure 3 .

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(C) Structural comparison between free and LC3(1-124)‐bound HsAtg4B and SENP2-SUMO‐3 precursor complexes. Left, free HsAtg4B; middle, the HsAtg4B-LC3 precursor complex; right, the SENP2-SUMO‐3 precursor complex. The three structures are shown in the same orientation. Model colouring is the same as in Figure 3.


( D ) urface model of LC3 ( 1 - 120 ) bound to HsAtg4B . Atom colouring is the same as in ( A ) .

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(D) urface model of LC3(1-120) bound to HsAtg4B. Atom colouring is the same as in (A).


( A ) Overall structure of Hs bound to both substrate and non ‐ substrate LC3 . Substrate LC3 is designated as LC3 ( S ) and coloured green , whereas non ‐ substrate LC3 is designated as LC3 ( N ) and coloured blue .

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(A) Overall structure of Hs bound to both substrate and non‐substrate LC3. Substrate LC3 is designated as LC3(S) and coloured green, whereas non‐substrate LC3 is designated as LC3(N) and coloured blue.


( B ) Stereo ‐ view of the N ‐ terminal tail of HsAtg4B bound to the WXXL ‐ binding site of LC3 . The side chains of the residues involved in the interaction between HsAtg4B and LC3 are labelled and shown with a stick model . Model colouring is the same as in ( A ) .

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(B) Stereo‐view of the N‐terminal tail of HsAtg4B bound to the WXXL‐binding site of LC3. The side chains of the residues involved in the interaction between HsAtg4B and LC3 are labelled and shown with a stick model. Model colouring is the same as in (A).


( C ) Structural comparison of the N ‐ terminal tail - LC3 interaction with the p62 - LC3 interaction . The structure of LC3 complexed with a p62 peptide is superimposed on that complexed with the N ‐ terminal tail peptide . As the structure of LC3 is almost identical to each other , only that bound to the N ‐ terminal tail peptide of Hs is shown .

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(C) Structural comparison of the N‐terminal tail-LC3 interaction with the p62-LC3 interaction. The structure of LC3 complexed with a p62 peptide is superimposed on that complexed with the N‐terminal tail peptide. As the structure of LC3 is almost identical to each other, only that bound to the N‐terminal tail peptide of Hs is shown.


( D ) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N ‐ terminal ‐ tail peptide . The combined 1H and 15N chemical shift differences , calculated using the equation Δp . p . m . = [ ( ΔδHN ) 2 + ( ΔδN / 5 ) 2 ] 1 / 2 were plotted against residue numbers .

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(D) Chemical shift perturbations of the LC3 backbone amide groups upon complex formation with the N‐terminal‐tail peptide. The combined 1H and 15N chemical shift differences, calculated using the equation Δp.p.m.=[(ΔδHN)2+(ΔδN/5)2]1/2 were plotted against residue numbers.


( E ) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B . The residues with Δp . p . m . > 0 . 4 are shown in blue , 0 . 4 > Δp . p . m . > 0 . 3 in cyan and 0 . 3 > Δp . p . m . > 0 . 2 in green .

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(E) Mapping of chemical shift perturbation results on the crystal structure of LC3 bound to HsAtg4B. The residues with Δp.p.m. >0.4 are shown in blue, 0.4>Δp.p.m.>0.3 in cyan and 0.3>Δp.p.m. >0.2 in green.


B . Immunoblot shows GST - GGA - GAT domain bound Arf1 ( active ; top ) and total Arf1 ( input lysates ; bottom ) from equal aliquots of lysates of HeLa cells that were stimulated with EGF for the indicated time points prior to lysis .

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B. Immunoblot shows GST-GGA-GAT domain bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of HeLa cells that were stimulated with EGF for the indicated time points prior to lysis.


C . Graphs display the model fitting for Arf1 activation dynamics . The experimentally determined Arf1 activation ( in B ) dynamics are displayed as black dots with error bars , representing mean ± S . E . M ( n = 3 biological replicates ) , and numerical simulation is shown by the blue continuous line .

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C. Graphs display the model fitting for Arf1 activation dynamics. The experimentally determined Arf1 activation (in B) dynamics are displayed as black dots with error bars, representing mean ± S.E.M (n=3 biological replicates), and numerical simulation is shown by the blue continuous line.


E . HeLa cells expressing Arf1 - HA were serum starved overnight ( E , top ) and subsequently stimulated with EGF for 5 min ( E , bottom ) prior to fixation with PFA . Fixed cells were stained for Arf1 ( HA ; green ) and GIV ( red ) and nuclei ( DAPI ; blue ) . Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1 - stained pixels . Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels . Scale bar = 10 µm .

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E. HeLa cells expressing Arf1-HA were serum starved overnight (E, top) and subsequently stimulated with EGF for 5 min (E, bottom) prior to fixation with PFA. Fixed cells were stained for Arf1 (HA; green) and GIV (red) and nuclei (DAPI; blue). Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1-stained pixels. Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels. Scale bar = 10 µm.


C . Equal aliquots ( ~ 45 µg ) of whole cell lysates of control ( shControl ; top ) and GIV - GEM depleted ( shGIV ; bottom ) HeLa cells were analyzed for GIV and tubulin ( loading control ) by immunoblotting ( IB ) .

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C. Equal aliquots (~45 µg) of whole cell lysates of control (shControl; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were analyzed for GIV and tubulin (loading control) by immunoblotting (IB).


D . Control ( sh Control ; top ) and GIV - GEM depleted ( shGIV ; bottom ) HeLa cells were co - transfected with Gαi1 - YFP , Gβ1 - CFP and Gγ2 ( untagged ) and live cells were analyzed by FRET imaging at steady - state , after being serum starved in 0 . 2 % FBS overnight and then after stimulation with 50 nM EGF . Representative freeze - frame FRET images are shown . FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel . FRET scale is shown in the inset . Golgi and PM regions of interest are indicated with arrows . Scale bar = 10 µm . See also Figure EV2A - B for free - frame images for additional time points in control HeLa cells .

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D. Control (sh Control; top) and GIV-GEM depleted (shGIV; bottom) HeLa cells were co-transfected with Gαi1-YFP, Gβ1-CFP and Gγ2 (untagged) and live cells were analyzed by FRET imaging at steady-state, after being serum starved in 0.2% FBS overnight and then after stimulation with 50 nM EGF. Representative freeze-frame FRET images are shown. FRET image panels display intensities of acceptor emission due to efficient energy transfer in each pixel. FRET scale is shown in the inset. Golgi and PM regions of interest are indicated with arrows. Scale bar = 10 µm. See also Figure EV2A-B for free-frame images for additional time points in control HeLa cells.


F . HeLa cells starved with 0 . 2 % FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi ( green ; anti - Gαi : GTP mAb ) and Man II ( red ) and analyzed by confocal microscopy . Activation of Gαi was detected exclusively after EGF stimulation . When detected , active Gαi colocalizes with Man II ( yellow pixels in merge panel ) . See also Figure EV2C - D for additional time points and stimulus . Scale bar = 7 . 5 µm .

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F. HeLa cells starved with 0.2% FBS overnight or stimulated subsequently with 50 nM EGF were fixed and stained for active Gαi (green; anti-Gαi:GTP mAb) and Man II (red) and analyzed by confocal microscopy. Activation of Gαi was detected exclusively after EGF stimulation. When detected, active Gαi colocalizes with Man II (yellow pixels in merge panel). See also Figure EV2C-D for additional time points and stimulus. Scale bar = 7.5 µm.


G . Model fit for the fold change of active tGTPase ( denoted as tG * ) . Experiment data is the fold change of ∆ FRET / CFP in D and is shown as mean ± S . E . M ( n = 8 ; 3 - 5 cells from 2 independent biological experiments ) . Continuous lines display the model simulation results after parameter

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G. Model fit for the fold change of active tGTPase (denoted as tG*). Experiment data is the fold change of ∆FRET/CFP in D and is shown as mean ± S.E.M (n=8; 3-5 cells from 2 independent biological experiments). Continuous lines display the model simulation results after parameter fitting


I . Immunoblot shows bound Arf1 ( active ; top ) and total Arf1 ( input lysates ; bottom ) from equal aliquots of lysates of control ( sh Control ) and GIV - depleted ( shGIV ) HeLa cells . Cells were stimulated with EGF for the indicated time points prior to lysis . Bar graphs in Figure EV2E display the fold change in Arf1 activity normalized to t0 min . ' Low ' and ' high ' indicate exposures .

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I. Immunoblot shows bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of control (sh Control) and GIV-depleted (shGIV) HeLa cells. Cells were stimulated with EGF for the indicated time points prior to lysis. Bar graphs in Figure EV2E display the fold change in Arf1 activity normalized to t0 min. 'Low' and 'high' indicate exposures.


D - E . The secretion ( D ) or the cell number ( E ) as a function of stimulus in coupled and uncoupled switches . The stimulus = 0 , 0 . 00046 , 0 . 0046 , 0 . 046 , 0 . 115 , 0 . 23 and 0 . 46 correspond to varying doses of EGF in simulations , ranging from 0 , 0 . 1 nM , 1 nM , 10 nM , 25 nM , 50 nM , and 100 nM , respectively . The error bar denotes the S . D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections .

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D-E. The secretion (D) or the cell number (E) as a function of stimulus in coupled and uncoupled switches. The stimulus =0, 0.00046, 0.0046, 0.046, 0.115, 0.23 and 0.46 correspond to varying doses of EGF in simulations, ranging from 0, 0.1 nM, 1 nM, 10 nM, 25 nM, 50 nM, and 100 nM, respectively. The error bar denotes the S.D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections.


F . The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches , at different levels of stimulus . For the first two bars , the height and error bars are the mean and S . D . of cell number when stimulus = 0 . 046 in E , respectively . For the last two bars , the height and error bars are the mean and S . D . of cell number when stimulus = 0 in E , respectively .

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F. The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches, at different levels of stimulus. For the first two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0.046 in E, respectively. For the last two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0 in E, respectively.


B . Immunoblots showing intracellular ( left ) and secreted ( in the media ; right ) GFP - MMP9 at 24 h after stimulation with varying doses of EGF . Tubulin , used as a loading control , confirms the presence of a similar number of plated cells in the assay .

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B. Immunoblots showing intracellular (left) and secreted (in the media; right) GFP-MMP9 at 24 h after stimulation with varying doses of EGF. Tubulin, used as a loading control, confirms the presence of a similar number of plated cells in the assay.


C . Left : Graph displays experimentally determined secretion of GFP - MMP9 in response to varying doses of EGF in control ( shControl ) and GIV - depleted ( shGIV ) HeLa cells ( as in B ) , and quantified by band densitometry . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . p values were determined by two - sided unpaired t - test . Right : Schematic diagram of dose responses ( mG * and secretion ) for the single switch and coupled switches . Coupled switches stretch the range of proportionate responses . Single mG switch results in misaligned responses . DoRA , dose response alignment .

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C. Left: Graph displays experimentally determined secretion of GFP-MMP9 in response to varying doses of EGF in control (shControl) and GIV-depleted (shGIV) HeLa cells (as in B), and quantified by band densitometry. Results are expressed as mean ± S.E.M; n = 3 biological replicates. p values were determined by two-sided unpaired t-test. Right: Schematic diagram of dose responses (mG* and secretion) for the single switch and coupled switches. Coupled switches stretch the range of proportionate responses. Single mG switch results in misaligned responses. DoRA, dose response alignment.


G - H . Control ( parental ) and GIV - depleted ( GIV KO ) HeLa cells grown in different concentrations of serum ( FBS % ) were treated or not with varying concentrations of BFA ( µM ) as indicated . Line graphs in 3D ( G ) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested . Bar graphs ( H ) depict the cell number in serum - free growth conditions that are supported exclusively by autocrine secrete - and - sense loop ( without BFA ; BFA = 0 . 0 µM ) or when such loop is interrupted ( BFA = 0 . 1 µM ) . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . Statistical significance was determined by one way ANOVA .

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G-H. Control (parental) and GIV-depleted (GIV KO) HeLa cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as indicated. Line graphs in 3D (G) depict the formazan absorbance expressed as a measure of cell viability from the HeLa cells in various conditions tested. Bar graphs (H) depict the cell number in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA.


I - K . Control ( parental ) and GIV - depleted ( GIV KO ) MDA MB - 231 cells grown in different concentrations of serum ( FBS % ) were treated or not with varying concentrations of BFA ( µM ) as in G - H . Line graphs in 3D ( I ) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB - 231 cells in various conditions tested . Bar graphs ( J ) depict the viability of the MDA MB - 231 cells in serum - free growth conditions that are supported exclusively by autocrine secrete - and - sense loop ( without BFA ; BFA = 0 . 0 µM ) or when such loop is interrupted ( BFA = 0 . 1 µM ) . Results are expressed as mean ± S . E . M ; n = 3 biological replicates . Statistical significance was determined by one way ANOVA . Immunoblots ( K ) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin ( loading control ) . See also Appendix Figure S5D - H for dot plots and early and late apoptotic fractions . Results are expressed as mean ± S . E . M ; n = 3 biological replicates .

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I-K. Control (parental) and GIV-depleted (GIV KO) MDA MB-231 cells grown in different concentrations of serum (FBS%) were treated or not with varying concentrations of BFA (µM) as in G-H. Line graphs in 3D (I) depict the formazan absorbance expressed as a measure of cell viability from the MDA MB-231 cells in various conditions tested. Bar graphs (J) depict the viability of the MDA MB-231 cells in serum-free growth conditions that are supported exclusively by autocrine secrete-and-sense loop (without BFA; BFA = 0.0 µM) or when such loop is interrupted (BFA = 0.1 µM). Results are expressed as mean ± S.E.M; n = 3 biological replicates. Statistical significance was determined by one way ANOVA. Immunoblots (K) of equal aliquots of whole cell lysates confirm the depletion of GIV compared to tubulin (loading control). See also Appendix Figure S5D-H for dot plots and early and late apoptotic fractions. Results are expressed as mean ± S.E.M; n = 3 biological replicates.


( A ) SA - β - gal staining of U2 - OS cells , harvested at indicated times post irradiation ( IR ) ; h , hours ; D , days ; UT , untreated . n = 3 . Scale bar : 50 μm . Bar Graph : Significance was determined by Brown Forsythe ANOVA with Dunnett ' s 3T test , using n = 3 biological replicates . SD shown . * * * p < 0 . 001 , n . s . = not significant .

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(A) SA-β-gal staining of U2-OS cells, harvested at indicated times post irradiation (IR); h, hours; D, days; UT, untreated. n = 3. Scale bar: 50 μm. Bar Graph: Significance was determined by Brown Forsythe ANOVA with Dunnett's 3T test, using n = 3 biological replicates. SD shown. *** p < 0.001, n.s. = not significant.


( B ) Nuclear fractions from U2 - OS cells were harvested at indicated time points post IR ( 20 Gy ) as in ( A ) . Top panel : NF - κB DNA binding was analyzed by EMSA . Lower panels : Western blot of indicated NF - κB subunits and PARP1 as loading control . The two phases of nuclear

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(B) Nuclear fractions from U2-OS cells were harvested at indicated time points post IR (20 Gy) as in (A). Top panel: NF-κB DNA binding was analyzed by EMSA. Lower panels: Western blot of indicated NF-κB subunits and PARP1 as loading control. The two phases of nuclear translocation


( C ) Gene expression analysis of two NF - κB phases following damage . RNA - Seq analysis ( n = 5 biological replicates per group ) of U2 - OS cells , either untreated or analyzed 1 . 5 hours or 7 days after irradiation ( 20 Gy ) , as indicated . The heatmap shows significantly regulated genes ( log2 value > 0 . 5 and p value < 0 . 05 ) . Black arrow points to NFKBIA . Significance determined by ANOVA with Bonferroni correction for multiple testing . See also Dataset EV1 .

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(C) Gene expression analysis of two NF-κB phases following damage. RNA-Seq analysis (n = 5 biological replicates per group) of U2-OS cells, either untreated or analyzed 1.5 hours or 7 days after irradiation (20 Gy), as indicated. The heatmap shows significantly regulated genes (log2 value > 0.5 and p value < 0.05). Black arrow points to NFKBIA. Significance determined by ANOVA with Bonferroni correction for multiple testing. See also Dataset EV1.


( D ) Female mice 12 - 16 weeks old ( n = 6 mice per condition ( with three technical repeats per mouse ) , were irradiated ( 5 Gy ) and sacrificed after 1 hour ( first phase ) , 7 days ( second phase ) or left untreated . Skin or renal tissue RNA was analyzed by RT - qPCR , shown as a mean + / - SD . Significance was confirmed by ANOVA with Tukey multiple comparisons test . SD shown : * = p < 0 . 05 , * * = p < 0 . 01 .

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(D) Female mice 12-16 weeks old (n = 6 mice per condition (with three technical repeats per mouse), were irradiated (5 Gy) and sacrificed after 1 hour (first phase), 7 days (second phase) or left untreated. Skin or renal tissue RNA was analyzed by RT-qPCR, shown as a mean +/- SD. Significance was confirmed by ANOVA with Tukey multiple comparisons test. SD shown: * = p < 0.05, ** = p < 0.01.


( E ) In situ hybridization using an anti - sense IκBα / Nfkbia riboprobe on longitudinal skin sections from male and female mice ( 5 - 8 weeks of age ) treated as in ( D ) for the time indicated . Representative sections shown from n = 6 mice per condition . Bu , bulge region ( dashed lines ) , HS , hair shaft , IFE , interfollicular epidermis . Scale bar : 50 μm . ( F ) Quantitation of ( E ) from n = 6 mice per condition and 3 - 4 sections per mouse . Bulge regions positive for Nfkbia were counted and presented as percentage of total . One way ANOVA analysis with Tukey multiple comparisons test was performed . SD shown . * * = p < 0 . 01 .

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(E) In situ hybridization using an anti-sense IκBα/Nfkbia riboprobe on longitudinal skin sections from male and female mice (5-8 weeks of age) treated as in (D) for the time indicated. Representative sections shown from n = 6 mice per condition. Bu, bulge region (dashed lines), HS, hair shaft, IFE, interfollicular epidermis. Scale bar: 50 μm. (F) Quantitation of (E) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for Nfkbia were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. ** = p < 0.01.


( G ) Skin sections as in ( E ) analyzed by immunofluorescence with IκBα ( red ) or IL - 6 antibody ( green ) and nuclear DAPI staining ( blue ) . Dotted lines delineate hair follicles , Bu , bulge region , HS , hair shaft , IFE , interfollicular epidermis , SG , sebaceous gland . Scale bar : 50 μm . ( H ) Quantitation of ( G ) from n = 6 mice per condition and 3 - 4 sections per mouse . Bulge regions positive for IκBα or IL - 6 were counted and presented as percentage of total . One way ANOVA analysis with Tukey multiple comparisons test was performed . SD shown . * = p < 0 . 05 , * * * = p < 0 . 001 .

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(G) Skin sections as in (E) analyzed by immunofluorescence with IκBα (red) or IL-6 antibody (green) and nuclear DAPI staining (blue). Dotted lines delineate hair follicles, Bu, bulge region, HS, hair shaft, IFE, interfollicular epidermis, SG, sebaceous gland. Scale bar: 50 μm. (H) Quantitation of (G) from n = 6 mice per condition and 3-4 sections per mouse. Bulge regions positive for IκBα or IL-6 were counted and presented as percentage of total. One way ANOVA analysis with Tukey multiple comparisons test was performed. SD shown. * = p < 0.05, *** = p < 0.001.


( A ) Left panel : IκBα western blot of U2 - OS cells . Lane 1 , untreated ( ut ) ; Lanes 2 - 4 , single IR ( 20 Gy ) at indicated time points prior to harvest ; Lane 5 , IR at 5 days prior to harvest , plus 1 . 5 hours prior to harvest . Right panel : Samples treated as in the left panel , analyzed by RT - qPCR . Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test . SD shown . * * * = p < 0 . 001 .

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(A) Left panel: IκBα western blot of U2-OS cells. Lane 1, untreated (ut); Lanes 2-4, single IR (20 Gy) at indicated time points prior to harvest; Lane 5, IR at 5 days prior to harvest, plus 1.5 hours prior to harvest. Right panel: Samples treated as in the left panel, analyzed by RT-qPCR. Significance from n = 3 replicates per condition determined by ANOVA analysis with Tukey multiple comparisons test. SD shown. *** = p < 0.001.