Live images show that xbp1 / atf6 ( RNAi ) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls . The remaining planarians in the starved condition are dead by the time point of regeneration shown . In the feeding condition , the remaining planarians at 8 - 10dR are either dead ( 13 / 145 ) or have a tiny blastema and died or regenerated later ( 40 / 145 ) while at 21 - 27dR are dead .

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Live images show that xbp1/atf6(RNAi) planarians in starved conditions form a minimal blastema when compared to controls while at the feeding condition most of them can regenerate as controls. The remaining planarians in the starved condition are dead by the time point of regeneration shown. In the feeding condition, the remaining planarians at 8-10dR are either dead (13/145) or have a tiny blastema and died or regenerated later (40/145) while at 21-27dR are dead.


Maximum projections of representative regenerating trunks after FISH for smedwi - 1 in starved conditions . The dashed line delimits the body of the planarian .

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Maximum projections of representative regenerating trunks after FISH for smedwi-1 in starved conditions. The dashed line delimits the body of the planarian.


TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions . The dashed line delimits the body of the planarian . The graph shows the number of TUNEL + cells per mm2 at the same time points in anterior ( A ) and posterior ( P ) blastemas . n ≥ 6 planarians per time point .

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TUNEL maximum projections of representative blastemas from regenerating trunks in starved conditions. The dashed line delimits the body of the planarian. The graph shows the number of TUNEL+ cells per mm2 at the same time points in anterior (A) and posterior (P) blastemas. n ≥ 6 planarians per time point.


Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1 / atf6 RNAi and controls in starved conditions after double immunostaining with anti - tyrosine - tubulin and anti - H3P ( * * * * P < 0 . 0001 , * * * P < 0 . 001 , * P < 0 . 05 , n . s . indicates not significant using two - sided Chi - square test ) ; n ≥ 5 planarians . Representative images are shown . Arrows indicate abnormal organization or number of spindle poles . j8 displays asymmetrical distribution of chromosome content .

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Quantification of the percentage of stem cells in different mitotic phases and the percentage of defective mitotic figures in 72hR anterior blastemas of xbp1/atf6 RNAi and controls in starved conditions after double immunostaining with anti-tyrosine-tubulin and anti-H3P (****P < 0.0001, ***P < 0.001, *P < 0.05, n.s. indicates not significant using two-sided Chi-square test); n ≥ 5 planarians. Representative images are shown. Arrows indicate abnormal organization or number of spindle poles. j8 displays asymmetrical distribution of chromosome content.


The Kaplan - Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions . Percentages indicate the number of survivals at the indicated time points . Three asterisks indicate p < 0 . 001 with log - rank ( Mantel - Cox ) test . Images are representative surviving animals for the different conditions at the displayed time point of regeneration . At the bottom are the number of planarians with the phenotype shown . The rest of planarians are dead at the displayed time point .

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The Kaplan-Meier curve demonstrates increased survival of cct3A RNAi animals when treated with DTT under starved conditions. Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate p < 0.001 with log-rank (Mantel-Cox) test. Images are representative surviving animals for the different conditions at the displayed time point of regeneration. At the bottom are the number of planarians with the phenotype shown. The rest of planarians are dead at the displayed time point.


Volcano plots displaying shRNA - Cct3 - and shRNA - luciferase - infected LSK cells cultured either in normal glucose ( upper graph ) or low glucose conditions ( lower graph ) . Dots represent all the NanoString analyzed transcripts . Red dots are UPR and ER stress related genes . The UPR and ER stress genes significantly down - regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph . For better visualization two dots with q ≤ 0 . 001 have been removed from the volcano plot ( see them in Dataset EV4 ) . Y - axis indicates the false discovery rate ( FDR ) ( q - value ) , and the X - axis indicates the log2 fold changes . The FDR - based method of p - value adjustment was conducted to calculate the q - values by nSolver software using Benjamini - Hochberg methods . Significance is established by q - value < 0 . 05 and indicated by the dotted horizontal line . n = 3 mice .

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Volcano plots displaying shRNA-Cct3- and shRNA-luciferase-infected LSK cells cultured either in normal glucose (upper graph) or low glucose conditions (lower graph). Dots represent all the NanoString analyzed transcripts. Red dots are UPR and ER stress related genes. The UPR and ER stress genes significantly down-regulated only under low glucose conditions in shCct3 cells compared to controls are indicated near the lower graph. For better visualization two dots with q ≤ 0.001 have been removed from the volcano plot (see them in Dataset EV4). Y-axis indicates the false discovery rate (FDR) (q- value), and the X-axis indicates the log2 fold changes. The FDR-based method of p-value adjustment was conducted to calculate the q-values by nSolver software using Benjamini-Hochberg methods. Significance is established by q-value < 0.05 and indicated by the dotted horizontal line. n= 3 mice.


The Kaplan - Meier curve demonstrates increased survival of starved cct3A ( RNAi ) animals when treated with palmitic acid / palmitoylcarnitine ( PA / PC ) . Percentages indicate the number of survivals at the indicated time points . Three asterisks indicate P < 0 . 001 with log - rank ( Mantel - Cox ) test .

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The Kaplan-Meier curve demonstrates increased survival of starved cct3A(RNAi) animals when treated with palmitic acid/palmitoylcarnitine (PA/PC). Percentages indicate the number of survivals at the indicated time points. Three asterisks indicate P < 0.001 with log-rank (Mantel-Cox) test.


( A ) Western blot of SMC3 - LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2 - phase . Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation ( asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments , presumably by repeated usage of the antibody samples ) . Note that ESCO1 levels were increased on chromatin after depletion of ESCO2 , raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin .

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(A) Western blot of SMC3-LAP expressing HeLa cells treated with siRNA as indicated and synchronized in G2-phase. Efficiency of RNA interference and SMC3 acetylation levels were analyzed from total extracts and after chromatin fractionation (asterisks indicate unspecific signals that were depleted from antibody dilutions used in later experiments, presumably by repeated usage of the antibody samples). Note that ESCO1 levels were increased on chromatin after depletion of ESCO2, raising the possibility that a decrease in chromatin bound ESCO2 might be compensated for by ESCO1 recruitment to chromatin.


( B ) Still images of an inverse fluorescence recovery after photobleaching ( iFRAP ) experiment . Fluorescence intensities are false - colored as indicated . Size bar , 5 μm . ( C ) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics . Error bars denote standard error of the mean ( s . e . m . ) , n = 10 cells per condition . ( D ) Quantification of the relative abundance of SMC3 - LAP in the stable chromatin binding mode .

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(B) Still images of an inverse fluorescence recovery after photobleaching (iFRAP) experiment. Fluorescence intensities are false-colored as indicated. Size bar, 5 μm.(C) Graph depicting the normalized intensity after photobleaching from two biological replicates to quantify chromatin dissociation kinetics. Error bars denote standard error of the mean (s.e.m.), n = 10 cells per condition.(D) Quantification of the relative abundance of SMC3-LAP in the stable chromatin binding mode.


( A ) Quantification of iFRAP experiments using HeLa cells expressing SMC3 - LAP in the wild - type form ( wt ) or with lysines 105 - 106 mutated to glutamines ( QQ ) or arginines ( RR ) and synchronized in G1 - phase . Error bars denote s . e . m . , n > 25 cells per condition . ( B ) Quantification of chromatin residence time of SMC3 - LAP alleles in G1 - phase .

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(A) Quantification of iFRAP experiments using HeLa cells expressing SMC3-LAP in the wild-type form (wt) or with lysines 105-106 mutated to glutamines (QQ) or arginines (RR) and synchronized in G1-phase. Error bars denote s.e.m., n > 25 cells per condition.(B) Quantification of chromatin residence time of SMC3-LAP alleles in G1-phase.


( C ) Quantification of iFRAP experiments using cells synchronized in G2 - phase . Error bars denote s . e . m . , n > 26 cells per condition . ( D ) Quantification of stably bound SMC3 - LAP alleles in G2 - phase .

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(C) Quantification of iFRAP experiments using cells synchronized in G2-phase. Error bars denote s.e.m., n > 26 cells per condition.(D) Quantification of stably bound SMC3-LAP alleles in G2-phase.


( E ) Western blot of mouse fibroblasts expressing SMC3 - LAP in the presence or absence of endogenous Smc3 after gene deletion . Extracts were prepared after subculturing two times . Cells without SMC3 - LAP do not proliferate after deletion of Smc3 .

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(E) Western blot of mouse fibroblasts expressing SMC3-LAP in the presence or absence of endogenous Smc3 after gene deletion. Extracts were prepared after subculturing two times. Cells without SMC3-LAP do not proliferate after deletion of Smc3.


( F ) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2 - phase . Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells . We currently do not know if this is an effect of sororin depletion , an off - target effect or an artefact of unequal sample loading .

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(F) Western blot showing HeLa cell extracts after treatment with siRNA as indicated and synchronized in G2-phase. Note that ESCO1 immunoblot signals were also reduced in sororin depleted cells. We currently do not know if this is an effect of sororin depletion, an off-target effect or an artefact of unequal sample loading.


( G ) iFRAP quantification of stably chromatin bound SMC3 - LAP after siRNA treatment using cells synchronized in G2 - phase . Error bars denote s . e . m . , n > 11 cells per condition .

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(G) iFRAP quantification of stably chromatin bound SMC3-LAP after siRNA treatment using cells synchronized in G2-phase. Error bars denote s.e.m., n > 11 cells per condition.


( A ) FACS profile of cells transfected with nondegradable KEN - box mutant sororin - FLAG ( sorKBM - FLAG ) after synchronization in G1 - phase and propidium iodide staining .

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(A) FACS profile of cells transfected with nondegradable KEN-box mutant sororin-FLAG (sorKBM-FLAG) after synchronization in G1-phase and propidium iodide staining.


( B ) Immunofluorescence microscopy experiment to determine transfection efficiency .

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(B) Immunofluorescence microscopy experiment to determine transfection efficiency.


( C ) Western blot of fractionated lysates to detect sororinKBM - FLAG .

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(C) Western blot of fractionated lysates to detect sororinKBM-FLAG.


( D ) Quantification of SMC3 - LAP residence time on chromatin after transfecting cells with sorKBM - FLAG in G1 - phase . Error bars denote s . e . m . ; n > 15 cells per condition .

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(D) Quantification of SMC3-LAP residence time on chromatin after transfecting cells with sorKBM-FLAG in G1-phase. Error bars denote s.e.m.; n > 15 cells per condition.


( E - F ) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2 - phase . ( G ) Quantification of stably chromatin - bound SMC3 - LAP from cells expressing nondegradable sororin in G2 - phase . Cells were treated with siRNA as indicated .

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(E-F) Analysis of cell cycle distribution and transfection efficiency using cells synchronized in G2-phase.(G) Quantification of stably chromatin-bound SMC3-LAP from cells expressing nondegradable sororin in G2-phase. Cells were treated with siRNA as indicated.


( H ) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2 - phase after immunoprecipitation with FLAG antibodies .

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(H) Western blot showing fractionated and nuclease digested chromatin from cells synchronized in G1 and G2-phase after immunoprecipitation with FLAG antibodies.


( A ) Examples of sororin ( dotted arrows ) and SMC3ChIP - seq and BrdU DIP - seq data from early S - phase as compared to G2 - phase using the Integrated Genome Browser ( IGB ; Nicol et al , 2009 ) . ( B ) Alignment of sororin , BrdU and BrdU - negative control sequencing bins in early S - phase to humanchromosome 18 ( GRCh37 / hg19 ) . Bars correspond to called peaks , curves to read enrichments after smoothing ( k = 25 kbp ) . ( C ) Quantification of the co - occupancy of BrdU incorporation and sororin localization in early S - phase and sororin in G2 - phase depicted as p - value distributions calculated with IntervalStats ( Chikina Troyanskaya , 2012 ) and compared to randomized controls . Sororin peaks were identified as overlapping peaks of 4 samples in early S - phase and 2 samples in G2 - phase , using only the common peaks . ( D ) Quantification of the co - occupancy of SMC3 in early S - phase and in G2 - phase with BrdU incorporation in early S - phase .

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(A) Examples of sororin (dotted arrows) and SMC3ChIP-seq and BrdU DIP-seq data from early S-phase as compared to G2-phase using the Integrated Genome Browser (IGB; Nicol et al, 2009).(B) Alignment of sororin, BrdU and BrdU-negative control sequencing bins in early S-phase to humanchromosome 18 (GRCh37/hg19). Bars correspond to called peaks, curves to read enrichments after smoothing (k=25 kbp).(C) Quantification of the co-occupancy of BrdU incorporation and sororin localization in early S-phase and sororin in G2-phase depicted as p-value distributions calculated with IntervalStats (Chikina Troyanskaya, 2012) and compared to randomized controls. Sororin peaks were identified as overlapping peaks of 4 samples in early S-phase and 2 samples in G2-phase, using only the common peaks.(D) Quantification of the co-occupancy of SMC3 in early S-phase and in G2-phase with BrdU incorporation in early S-phase.


( A ) Schematic representation of wild - type ( wt ) , targeted ( flx ) and deleted ( Δ ) Cdca5 alleles ( left ) and Southern blot of mouse genomic DNA ( right ) . Red triangles , loxP sites .

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(A) Schematic representation of wild-type (wt), targeted (flx) and deleted (Δ) Cdca5 alleles (left) and Southern blot of mouse genomic DNA (right). Red triangles, loxP sites.


( B ) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ / + mice ( newborn mice , n = 136 ; E9 . 5 , n = 54 ) .

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(B) Observed and expected genotype distribution among offspring of heterozygous Cdca5 Δ/+ mice (newborn mice, n = 136; E9.5, n = 54).


( D ) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen - induced Cre expression . Extracts were prepared either one or two days after release from the arrest as indicated . Chr , fractionated chromatin ; sol , soluble proteins .

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(D) Western blot of cell extracts to analyze depletion of sororin protein from fibroblasts after tamoxifen-induced Cre expression. Extracts were prepared either one or two days after release from the arrest as indicated. Chr, fractionated chromatin; sol, soluble proteins.


( E ) Proliferation curve of cells with the indicated genotype after tamoxifen treatment .

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(E) Proliferation curve of cells with the indicated genotype after tamoxifen treatment.


( F ) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium .

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(F) Observed cell cycle distribution of fibroblasts treated with tamoxifen under starvation and subcultured in rich medium.


( G ) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours . Size bar , 10 µm ; error bars denote s . e . m . ; n > 500 cells per condition .

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(G) Quantification of mitotic cells with misaligned chromosomes after Cdca5 deletion and proliferation for 36 to 60 hours. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.


( H ) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days . Size bar , 10 µm ; error bars denote s . e . m . ; n > 500 cells per condition .

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(H) Phenotypic classification of nuclear morphology after Cdca5 deletion and proliferation for 5 to 7 days. Size bar, 10 µm; error bars denote s.e.m.; n > 500 cells per condition.


( I ) Western blot showing protein depletion efficiency . Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts .

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(I) Western blot showing protein depletion efficiency. Cre expression was induced with tamoxifen for 24 hours in proliferating Smc3 and Cdca5 conditional knockout immortalized fibroblasts.


( J ) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment . Representative images from every category are shown ; size bar , 10 µm ; error bars denote s . e . m . ; n > 500 per genotype .

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(J) Analysis of chromosome spreads after Smc3 or Cdca5 deletion and nocodazole treatment. Representative images from every category are shown; size bar, 10 µm; error bars denote s.e.m.; n > 500 per genotype.


( A ) Schematic illustration of the LAP - AID - tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus . ( B ) Immunofluorescence staining of cells expressing sor - LAP - AID and the F - box helper protein after treatment with auxin . Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2 / M - phase . Scale bar , 10 µm . ( C ) Quantification of immunofluorescence after treating cells with auxin . Error bars denote s . e . m . ; a . u . , arbitrary units . ( D ) Time course quantification of GFP immunofluorescence signal decrease after auxin addition .

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(A) Schematic illustration of the LAP-AID-tag integrated into the bacterial artificial chromosome containing the mouse Cdca5 locus.(B) Immunofluorescence staining of cells expressing sor-LAP-AID and the F-box helper protein after treatment with auxin. Antibodies against GFP mark the fusion protein and Aurora B antibody stains cells in G2/M-phase. Scale bar, 10 µm.(C) Quantification of immunofluorescence after treating cells with auxin. Error bars denote s.e.m.; a.u., arbitrary units.(D) Time course quantification of GFP immunofluorescence signal decrease after auxin addition.


( E ) Analysis of chromosome spreads after auxin treatment .

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(E) Analysis of chromosome spreads after auxin treatment.


( F ) Western blot showing cell extracts after treatment with auxin .

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(F) Western blot showing cell extracts after treatment with auxin.


( A ) Cell cycle distribution of immortalized Cdca5 Δ / Δ sor - LAP - AID fibroblasts after 24 - hour incubation with aphidicolin and release .

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(A) Cell cycle distribution of immortalized Cdca5 Δ/Δ sor-LAP-AID fibroblasts after 24-hour incubation with aphidicolin and release.


( B ) Western blot showing cell extracts after incubation with aphidicolin and release .

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(B) Western blot showing cell extracts after incubation with aphidicolin and release.


( C ) Still images of a fluorescence recovery after photobleaching experiment . Size bar , 5 μm . Bleach area radius , 2 µm . ( D ) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin ( n = 15 ) . ( E ) Quantification of the relative abundance of sororin - LAP - AID on chromatin . Error bars denote s . e . m . ( F ) Quantification of the chromatin residence time of sor - LAP - AID at different times . Error bars denote s . e . m .

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(C) Still images of a fluorescence recovery after photobleaching experiment. Size bar, 5 μm. Bleach area radius, 2 µm.(D) Graph depicting the normalized intensity after photobleaching to quantify turnover of sororin (n = 15).(E) Quantification of the relative abundance of sororin-LAP-AID on chromatin. Error bars denote s.e.m.(F) Quantification of the chromatin residence time of sor-LAP-AID at different times. Error bars denote s.e.m.


( B ) Quantification of EdU - labeled prometaphase cells after treating cells with auxin and EdU at indicated time points . Error bars denote s . e . m . ; n > 200 cells per condition .

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(B) Quantification of EdU-labeled prometaphase cells after treating cells with auxin and EdU at indicated time points. Error bars denote s.e.m.; n > 200 cells per condition.


( C ) Analysis of chromosome spreads after auxin and EdU treatment and Giemsa staining .

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(C) Analysis of chromosome spreads after auxin and EdU treatment and Giemsa staining.


( D ) Representative picture of the most prominent phenotype class upon auxin and EdU treatment and immunofluorescence microscopy . Scale bar , 10 µm . ( E ) Analysis of chromosome spreads after auxin treatment and EdU and DAPI labeling . Error bars denote s . e . m . ; n > 200 cells per condition .

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(D) Representative picture of the most prominent phenotype class upon auxin and EdU treatment and immunofluorescence microscopy. Scale bar, 10 µm.(E) Analysis of chromosome spreads after auxin treatment and EdU and DAPI labeling. Error bars denote s.e.m.; n > 200 cells per condition.


Phloroglucinol staining of wild - type seedlings inoculated with PTI - inducing Pst DC3000 hrcC - , virulent Pst DC3000 , and avirulent Pst DC3000 ( AvrRpm1 ) and Pst DC3000 ( AvrRpt2 ) . Data information : Twelve - day - old seedlings were flood - inoculated with P . syringae at 108 cfu / ml in ( A hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post -

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Phloroglucinol staining of wild-type seedlings inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml in (A hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation


B Phloroglucinol staining of wild - type adult leaves inoculated with PTI - inducing Pst DC3000 hrcC - , virulent Pst DC3000 , and avirulent Pst DC3000 ( AvrRpm1 ) and Pst DC3000 ( AvrRpt2 ) . The upper images are enlarged ones of the lower boxes at 2 dpi . Scale bars , 100 μm . Data information Six - week - old leaves were syringe - infiltrated with P . syringae at 108 cfu / ml in ( B hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculation ; IS , infected site ; UIS , uninfected site .

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

B Phloroglucinol staining of wild-type adult leaves inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). The upper images are enlarged ones of the lower boxes at 2 dpi. Scale bars, 100 μm. Data information Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml in (B hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation; IS, infected site; UIS, uninfected site.


C Quantification of lignin content in pathogen - treated seedlings Data are shown as means ± SD ( n = 4 ; 20 - 30 seedlings each ) . Data information : Twelve - day - old seedlings were flood - inoculated with P . syringae at 108 cfu / ml Significant differences are indicated by different letters ( Tukey ' s HSD test ; P < 0 . 05 ) Experiments were repeated 3 times with similar results . hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post -

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

C Quantification of lignin content in pathogen-treated seedlings Data are shown as means ± SD (n = 4; 20-30 seedlings each). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation


D Quantification of lignin content in pathogen - treated wild - type leaves Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Data information : Six - week - old leaves were syringe - infiltrated with P . syringae at 108 cfu / ml Significant differences are indicated by different letters ( Tukey ' s HSD test ; P < 0 . 05 ) Experiments were repeated 3 times with similar results . hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post -

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

D Quantification of lignin content in pathogen-treated wild-type leaves Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation


E Quantification of lignin content in wild - type leaves treated with different titers of Pst DC3000 ( AvrRpm1 ) . Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Six - week - old leaves were syringe - infiltrated with P . syringae at the indicated titers in ( E ) . Significant differences are indicated by asterisks ( t test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Experiments were repeated 3 times with similar results . dpi , days post -

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E Quantification of lignin content in wild-type leaves treated with different titers of Pst DC3000 (AvrRpm1). Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at the indicated titers in (E). Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. dpi, days post-inoculation


F Quantification of lignin content in wild - type and rpm1 - 3 rps2 - 101c leaves treated with avirulent bacteria . Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Six - week - old leaves were syringe - infiltrated with P . syringae at 108 cfu / ml Significant differences are indicated by asterisks ( t test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Experiments were repeated 3 times with similar results . AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post -

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F Quantification of lignin content in wild-type and rpm1-3 rps2-101c leaves treated with avirulent bacteria. Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation


B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection . Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 108 cfu / ml for experiments . Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; dpi , days post - inoculation .

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B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.


C Measurements of Pst DC3000 ( AvrRpm1 ) growth . Data are shown as means ± SD ( n = 3 ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 105 cfu / ml for growth assays Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; dpi , days post - inoculation .

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

C Measurements of Pst DC3000 (AvrRpm1) growth. Data are shown as means ± SD (n = 3). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.


D Cell death phenotypes of leaves inoculated with Pst DC3000 ( AvrRpm1 ) . E Quantification of leaves ( n ≥ 30 ) with spreading cell death as in ( D ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 105 cfu / ml for growth assays M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; dpi , days post - inoculation .

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D Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). E Quantification of leaves (n ≥ 30) with spreading cell death as in (D). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.


F Quantification of total and free SA in Col - 0 and palQ leaves after the indicated treatments and pathogen infection . Data are shown as means ± SD ( n = 5 ; 6 leaves each ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 108 cfu / ml for experiments . Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . M , mock ; PA , piperonylic acid dpi , days post - inoculation .

O O O O O O B-SMALL_MOLECULE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O B-TISSUE O O O B-ORGANISM I-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

F Quantification of total and free SA in Col-0 and palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 5; 6 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid dpi, days post-inoculation.


A Colonization patterns of GFP - Pst DC3000 and Pst DC3000 ( AvrRpt2 ) in wild - type plants . Data information : Leaves were inoculated with GFP - labeled bacteria at 108 cfu / ml for 2 days . IS , infected site ; UIS , uninfected site . White dash lines indicate the boundary between IS and UIS . Scale bars , 100

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

A Colonization patterns of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) in wild-type plants. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm


B Time - lapse images of GFP - Pst DC3000 and Pst DC3000 ( AvrRpt2 ) captured at 0 ( magenta ) , 15 , ( yellow ) , and 30 ( green ) sec Time in seconds in the movies is shown at the top of images . Blue color was used for chlorophyll autofluorescence . Data information : Leaves were inoculated with GFP - labeled bacteria at 108 cfu / ml for 2 days . Scale bars , 10

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

B Time-lapse images of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) captured at 0 (magenta), 15, (yellow), and 30 (green) sec Time in seconds in the movies is shown at the top of images. Blue color was used for chlorophyll autofluorescence. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. Scale bars, 10 μm


C Colonization patterns of GFP - Pst DC3000 ( AvrRpt2 ) in wild - type and palQ plants after PA and CA pretreatments . Data information : Leaves were inoculated with GFP - labeled bacteria at 108 cfu / ml for 2 days . M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; IS , infected site ; UIS , uninfected site . White dash lines indicate the boundary between IS and UIS . Scale bars , 100

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C Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in wild-type and palQ plants after PA and CA pretreatments. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm


A CANBD - and CADMAC - incorporated lignin polymerization . CANBD ( green ) and CADMAC ( blue ) fluorescence was observed in the lignified infection site at 2 dpi , as revealed by phloroglucinol staining . Data information : Bacterial inoculum was at 108 cfu / ml . IS , infected site ; UIS , uninfected site White dash lines indicate the boundary between IS and UIS . Scale bars , 500

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A CANBD- and CADMAC-incorporated lignin polymerization. CANBD (green) and CADMAC (blue) fluorescence was observed in the lignified infection site at 2 dpi, as revealed by phloroglucinol staining. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site White dash lines indicate the boundary between IS and UIS. Scale bars, 500 μm


B Cross - sections of CANBD - and Pst DC3000 ( AvrRpm1 ) - treated leaves . Data information : Bacterial inoculum was at 108 cfu / ml . hpi , hours post - inoculation Scale bars , 40

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B Cross-sections of CANBD- and Pst DC3000 (AvrRpm1)-treated leaves. Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 40 μm


C Cross - section of the entire leaf with one half leaf infected . Data information : Bacterial inoculum was at 108 cfu / ml . IS , infected site ; UIS , uninfected site ; hpi , hours post - inoculation Scale bars , 120

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C Cross-section of the entire leaf with one half leaf infected. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site; hpi, hours post-inoculation Scale bars, 120 μm


D CADMAC - incorporated lignin deposition in the GFP - Pst DC3000 ( AvrRpt2 ) - infected site at 2 dpi . Data information : Bacterial inoculum was at 108 cfu / ml . IS , infected site ; UIS , uninfected site DC3000 , Pst DC3000 ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; Chl , chlorophyll . White dash lines indicate the boundary between IS and UIS . Scale bars , 100

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D CADMAC-incorporated lignin deposition in the GFP-Pst DC3000 (AvrRpt2)-infected site at 2 dpi. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site DC3000, Pst DC3000; AvrRpt2, Pst DC3000 (AvrRpt2); Chl, chlorophyll. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm


E Cellular observation of CADMAC - incorporated lignin formation against infecting GFP - Pst DC3000 ( AvrRpt2 ) . Data information : Bacterial inoculum was at 108 cfu / ml . hpi , hours post - inoculation Scale bars , 20

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E Cellular observation of CADMAC-incorporated lignin formation against infecting GFP-Pst DC3000 (AvrRpt2). Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 20 μm


F 3D image of CANBD - polymerized structure . Images were taken at 24 hpi Data information : Bacterial inoculum was at 108 cfu / ml . Scale bars , 20

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F 3D image of CANBD-polymerized structure. Images were taken at 24 hpi Data information: Bacterial inoculum was at 108 cfu/ml. Scale bars, 20 μm


A Time course expression profiles of CASPLs in response to Pst DC3000 ( AvrRpm1 ) treatment . Analysis was based on the Genevestigator database ( http : / / www . genevestigator . ethz . ch / ) . The darker color corresponds to stronger expression . ND , not determined .

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

A Time course expression profiles of CASPLs in response to Pst DC3000 (AvrRpm1) treatment. Analysis was based on the Genevestigator database (http://www.genevestigator.ethz.ch/). The darker color corresponds to stronger expression. ND, not determined.


Quantification of lignin content in wild - type and caspl mutant leaves after Pst DC3000 ( AvrRpm1 ) inoculation . ata are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . ata information : Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; dpi , days post - inoculation .

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

Quantification of lignin content in wild-type and caspl mutant leaves after Pst DC3000 (AvrRpm1) inoculation. ata are shown as means ± SD (n = 4; 3-9 leaves each). ata information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); dpi, days post-inoculation.


C Cell death phenotypes of leaves inoculated with Pst DC3000 ( AvrRpm1 ) . Data information : AvrRpm1 , Pst DC3000 ( AvrRpm1 ) dpi , days post - inoculation .

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C Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). Data information: AvrRpm1, Pst DC3000 (AvrRpm1) dpi, days post-inoculation.


D Quantification of leaves ( n ≥ 30 ) with spreading cell death after Pst DC3000 ( AvrRpm1 ) inoculation . dpi , days post - inoculation .

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D Quantification of leaves (n ≥ 30) with spreading cell death after Pst DC3000 (AvrRpm1) inoculation. dpi, days post-inoculation.


E Measurements of Pst DC3000 ( AvrRpm1 ) and Pst DC3000 ( AvrRpt2 ) growth . Data are shown as means ± SD ( n = 3 ) . Data information : Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculation .

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E Measurements of Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) growth. Data are shown as means ± SD (n = 3). Data information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation.


F Colonization patterns of GFP - Pst DC3000 ( AvrRpt2 ) in caspl mutant plants . IS , infected site ; UIS , uninfected site . White dash lines indicate the boundary between IS and UIS . Scale bars , 100 μm .

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F Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in caspl mutant plants. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm.


A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence . Data information : pCASPL4D1 : : CASPL4D1 - mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 ( AvrRpm1 ) . mCh , mCherry ; Chl , chlorophyll ; hpi , hours post - inoculation . Scale bars , 20 μm ( A )

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A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars, 20 μm (A)


B Magnified views of CASPL4D1 and lignin localization . Images in boxes were enlarged in the 2nd to 4th columns . Arrows indicate lignin deposition between cells . Data information : pCASPL4D1 : : CASPL4D1 - mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 ( AvrRpm1 ) . mCh , mCherry ; Chl , chlorophyll ; hpi , hours post - inoculation . Scale bars 10 μm ( B ) .

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

B Magnified views of CASPL4D1 and lignin localization. Images in boxes were enlarged in the 2nd to 4th columns. Arrows indicate lignin deposition between cells. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars 10 μm (B).


C Relative fluorescence intensity profiles of CASPL1D1 ( mCherry ) and lignin ( CADMAC ) across transects indicated by dotted lines Data information : pCASPL4D1 : : CASPL4D1 - mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 ( AvrRpm1 ) .

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

C Relative fluorescence intensity profiles of CASPL1D1 (mCherry) and lignin (CADMAC) across transects indicated by dotted lines Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1).


D A model showing morphological changes of CASPL - and lignin - deposited cells during the HR response .

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

D A model showing morphological changes of CASPL- and lignin-deposited cells during the HR response.


C , D , E Examples of embryos expressing the tFT - Bcd reporter with C : mCherry - sfGFP - Bcd , D : fmCherry - sfGFP - Bcd , and E : mCherry - sfGFP . For all , ( i ) Images of " shells " of embryos in early n . c . 14 . 3D images of the embryos were generated , and then the interior of the embryo was erased , leaving only the embryo cortex to improve clarity ( ii ) Mean AP intensity profile of each color for the embryo in i . Shade region represents ± 1 s . d . Inset shows same profiles after multiplication of red intensity by constant factor . Data binned into 10 µm bins ( n = 4 embryos ) . ( iii ) Ratio of green over red signal , reflecting protein age . The thin lines represent individual embryos , while the thick solid line is the mean . The solid dashed line depicts the mean green / red ratio for a line with the tFT - Bcd reporter but lacking endogenous Bcd ( BcdE1 mutant , n = 4 ) .

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

C,D,E Examples of embryos expressing the tFT-Bcd reporter with C: mCherry-sfGFP-Bcd, D: fmCherry-sfGFP-Bcd, and E: mCherry-sfGFP. For all, (i) Images of "shells" of embryos in early n.c. 14. 3D images of the embryos were generated, and then the interior of the embryo was erased, leaving only the embryo cortex to improve clarity (ii) Mean AP intensity profile of each color for the embryo in i. Shade region represents ±1 s.d. Inset shows same profiles after multiplication of red intensity by constant factor. Data binned into 10 µm bins (n=4 embryos). (iii) Ratio of green over red signal, reflecting protein age. The thin lines represent individual embryos, while the thick solid line is the mean. The solid dashed line depicts the mean green/red ratio for a line with the tFT-Bcd reporter but lacking endogenous Bcd (BcdE1 mutant, n=4).


, B , C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd ( gray , open squares , N = 10 ) . Control embryos were injected with DMSO ( black , filled circles , N = 9 ) . All embryos are y / w and were injected while in cycle 14 and imaged 60 min later .

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

,B,C Injection of the proteasome inhibitor MG132 in embryos increases both the amount and the average age of Bcd (gray, open squares, N=10). Control embryos were injected with DMSO (black, filled circles, N=9). All embryos are y/w and were injected while in cycle 14 and imaged 60 min later.


D The age of Bcd and its gradient are unaltered in fsd mutant . Comparison of a representative y / w embryo ( left ) and loss - of - function mutation fsdKG02393 ( right ) are shown . Both embryos have the tFT - Bcd construct in the same locus , are in early n . c . 14 , were imaged side - by - side under identical conditions , and are displayed with the same settings . Embryo " shells " are displayed .

O O O O B-GENEPROD O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O

D The age of Bcd and its gradient are unaltered in fsd mutant. Comparison of a representative y/w embryo (left) and loss-of-function mutation fsdKG02393 (right) are shown. Both embryos have the tFT-Bcd construct in the same locus, are in early n.c. 14, were imaged side-by-side under identical conditions, and are displayed with the same settings. Embryo "shells" are displayed.


E Quantification of embryos Fluorescence intensity from sfGFP ( green ) and mCherry ( magenta ) for wt ( N = 8 , filled circles ) and fsd ( N = 6 , open squares ) at early cycle 14 . Inset : tFT - Bcd ratio for wt ( black ) and FSD ( gray ) embryos .

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

E Quantification of embryos Fluorescence intensity from sfGFP (green) and mCherry (magenta) for wt (N=8, filled circles) and fsd (N=6, open squares) at early cycle 14. Inset: tFT-Bcd ratio for wt (black) and FSD (gray) embryos.


Time - course of a representative y / w embryo with the tFT - Bcd reporter from the division cycle 10 to the early gastrulation . A SPIM images of the embryo early development . Embryo " shells " are displayed . Left : Green fluorescence , center : Red fluorescence , right : Intensity - weighted ratiometric image of the mCherry / sfGFP ratio , reporting average protein age . The scale corresponds to 50 μm . B Quantification of the same embryo as is A . Each line represents the fluorescence profile along the anterior - posterior axis of the embryo for a time frame ( separated by 6 min ) . In the lower panel , where the mCherry / sfGFP ratio is shown , the posterior half of the embryo is not shown because the signal drops to the background levels ( due to softer imaging conditions for the long term time - course ) . C The same data is shown as a function of time for different embryo segments .

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

Time-course of a representative y/w embryo with the tFT-Bcd reporter from the division cycle 10 to the early gastrulation. A SPIM images of the embryo early development. Embryo "shells" are displayed. Left: Green fluorescence, center: Red fluorescence, right: Intensity-weighted ratiometric image of the mCherry/sfGFP ratio, reporting average protein age. The scale corresponds to 50 μm. B Quantification of the same embryo as is A. Each line represents the fluorescence profile along the anterior-posterior axis of the embryo for a time frame (separated by 6 min). In the lower panel, where the mCherry/sfGFP ratio is shown, the posterior half of the embryo is not shown because the signal drops to the background levels (due to softer imaging conditions for the long term time-course). C The same data is shown as a function of time for different embryo segments.


Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α - V5 tagged . Cells were fixed and stained with anti - calnexin ( green ) or anti - V5 ( red ) . An overlay of the cells is presented in the third panel . Bar = 10 µm .

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Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α-V5 tagged. Cells were fixed and stained with anti-calnexin (green) or anti-V5 (red). An overlay of the cells is presented in the third panel. Bar = 10 µm.


Whole cell lysates from HEK293T cells , with or without stable expression of ATF6α ( as indicated ) were immunoisolated with mouse anti - V5 . RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3 . Immunoisolated material was separated by SDS - PAGE under reducing conditions followed by western blotting to determine the co - isolation of ER proteins with ATF6α . Ig - HC ( anti - BiP blot ) indicates immunoglobulin heavy chains .

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

Whole cell lysates from HEK293T cells, with or without stable expression of ATF6α (as indicated) were immunoisolated with mouse anti-V5. RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3. Immunoisolated material was separated by SDS-PAGE under reducing conditions followed by western blotting to determine the co-isolation of ER proteins with ATF6α. Ig-HC (anti-BiP blot) indicates immunoglobulin heavy chains.


Whole cell lysates from HEK293T cells , with or without stable expression of

Whole cell lysates from HEK293T cells, with or without stable expression of ATF6α


Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated ( - ) or treated ( + ) with 5 µM thapsigargin ( TG ) for 1 h . THBS4 was transiently transfected into cells in the experiment depicted in panel 2 . ATF6α was either immunoisolated with anti - V5 ( panel 1 and 3 ) or anti - HA ( panel 2 ) . For the experiment in panel 3 the cells were cross - linked prior to immunoisolation . Samples were separated by SDS - PAGE under reducing conditions followed by western blots to determine co - isolation of endogenous PDIR , ERp18 or exogenously expressed THBS4 with ATF6α . HC indicates immunoglobulin heavy chain .

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

Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated (-) or treated (+) with 5 µM thapsigargin (TG) for 1 h. THBS4 was transiently transfected into cells in the experiment depicted in panel 2. ATF6α was either immunoisolated with anti-V5 (panel 1 and 3) or anti-HA (panel 2). For the experiment in panel 3 the cells were cross-linked prior to immunoisolation. Samples were separated by SDS-PAGE under reducing conditions followed by western blots to determine co-isolation of endogenous PDIR, ERp18 or exogenously expressed THBS4 with ATF6α. HC indicates immunoglobulin heavy chain.


HEK293T cells stably expressing ATF6α were either left untransfected ( UT ) or transfected with substrate - trapping mutants of PDI oxidoreductases . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots confirm mixed - disulfide complexes between ATF6α and ERp18 , ERp57 , and PDIR indicated with arrows ( lanes 2 , 4 , 5 ) . M , D and O refer to ATF6α monomer , dimer and oligomer respectively .

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

HEK293T cells stably expressing ATF6α were either left untransfected (UT) or transfected with substrate-trapping mutants of PDI oxidoreductases. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots confirm mixed-disulfide complexes between ATF6α and ERp18, ERp57, and PDIR indicated with arrows (lanes 2, 4, 5). M, D and O refer to ATF6α monomer, dimer and oligomer respectively.


Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain ( ATF6 DM ) were transfected with PDI substrate - trapping mutants . ATF6α was immunoisolated with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions , and ATF6α detected with rabbit anti - HA .

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

Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain (ATF6 DM) were transfected with PDI substrate-trapping mutants. ATF6α was immunoisolated with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions, and ATF6α detected with rabbit anti-HA.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µg / ml tunicamycin to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . * * indicates hypoglycosylated ATF6α . Data shown are representative of three independent experiments .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µg/ml tunicamycin to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 20 µM MG132 to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . * * indicates hypoglycosylated ATF6α . Data shown are representative of three independent experiments .

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

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 20 µM MG132 to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments.


HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . Samples from ( A ) were rerun and probed with anti - sera raised against respective PDI enzymes as indicated ( D ) . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes .

B-CELL_LINE O O O O O O B-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. Samples from (A) were rerun and probed with anti-sera raised against respective PDI enzymes as indicated (D). *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.


HEK293T cells were co - transfected with the ERp18 substrate - trapping mutant and the indicated ATF6α constructs . Cell were allowed to recover for 24 h post - transfection before being either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress . M , D and O refer to ATF6 monomer , dimer and oligomer respectively . * indicates positions of the ATF6 - ERp18 mixed - disulfide complexes .

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

HEK293T cells were co-transfected with the ERp18 substrate-trapping mutant and the indicated ATF6α constructs. Cell were allowed to recover for 24 h post-transfection before being either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress. M, D and O refer to ATF6 monomer, dimer and oligomer respectively. *indicates positions of the ATF6-ERp18 mixed-disulfide complexes.


HEK293T cells were co - transfected with GFP - ATF6α and ERp18 trapping mutant constructs as indicated . GFP - ATF6α complexes were immunoisolated using GFP - trap . Immunoisolated material was separated by SDS - PAGE under non - reducing conditions and silver stained to detect ATF6α disulfide - linked complexes . MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18 , whilst bands marked as monomer ( M ) and oligomer ( O ) contain ATF6α only .

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

HEK293T cells were co-transfected with GFP-ATF6α and ERp18 trapping mutant constructs as indicated. GFP-ATF6α complexes were immunoisolated using GFP-trap. Immunoisolated material was separated by SDS-PAGE under non-reducing conditions and silver stained to detect ATF6α disulfide-linked complexes. MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18, whilst bands marked as monomer (M) and oligomer (O) contain ATF6α only.


CRISPR / Cas9 - based knockout of ERp18 in HEK293T cells expressing ATF6α . ERp18 levels in control ( WT ) and knockout ( KO ) cells were detected with an antibody to ERp18 .

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

CRISPR/Cas9-based knockout of ERp18 in HEK293T cells expressing ATF6α. ERp18 levels in control (WT) and knockout (KO) cells were detected with an antibody to ERp18.


qPCR analysis of UPR target genes in the absence of ER stress . Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type . Error bars represent ± standard deviation for three independent experiments . qPCR analysis of UPR target genes following ER stress . Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls . Error bars represent ± standard deviation for three independent experiments .

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

qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.   qPCR analysis of UPR target genes following ER stress. Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls. Error bars represent ± standard deviation for three independent experiments.


qPCR analysis of UPR target genes in the absence of ER stress . Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type . Error bars represent ± standard deviation for three independent experiments .

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

qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.


Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) . Normalised BiP levels from ( D ) were quantified . Error bars represent ± standard deviation for three independent experiments .

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

Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised BiP levels from (D ) were quantified. Error bars represent ± standard deviation for three independent experiments.


Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) .

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

Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).


Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) . Normalised ERp72 levels from ( F ) were quantified . Error bars represent ± standard deviation for three independent experiments .

Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised ERp72 levels from (F) were quantified. Error bars represent ± standard deviation for three independent experiments.


Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) .

Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).


A , B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times . Cells were then subject to differential centrifugation to obtain membrane ( A ) and nuclear fractions ( B ) which were separated by SDS - PAGE under reducing conditions and analysed by western blotting for ATF6α . The positions of full - length ATF6α ( ATF6 - FL ) , cleaved membrane - associated ATF6α ( ATF6 - P ) and nuclear translocated S2P cleaved ATF6α ( ATF6 - N ) are as indicated . HDAC2 was used as a nuclear marker .

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

A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times. Cells were then subject to differential centrifugation to obtain membrane (A) and nuclear fractions (B) which were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of full-length ATF6α (ATF6-FL), cleaved membrane-associated ATF6α (ATF6-P) and nuclear translocated S2P cleaved ATF6α (ATF6-N) are as indicated. HDAC2 was used as a nuclear marker.


( C ) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α . Samples were separated by SDS - PAGE and ATF6α was detected by western blotting . HC indicates immunoglobulin heavy chains .

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

(C) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α. Samples were separated by SDS-PAGE and ATF6α was detected by western blotting. HC indicates immunoglobulin heavy chains.


( D , E ) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α , either untreated ( - ) or treated ( + ) with 30 µM PF429242 ( S1P inhibitor ) prior to and following induction of ER stress with 10 mM DTT . The positions of ATF6 - P , ATF6 - M and ATF6 - N are as indicated . The unprocessed ER - localised ATF6α ( designated ER ) and the O - linked glycan modified Golgi - translocated ATF6α ( designated G ) are indicated . HDAC2 was included as a nuclear marker . The blots in ( D , E ) confirm that ATF6 - P is produced independently of S1P and also the absence of ATF6α processing to ATF6 - N in the presence of the S1P inhibitor .

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

(D, E) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α, either untreated (-) or treated (+) with 30 µM PF429242 (S1P inhibitor) prior to and following induction of ER stress with 10 mM DTT. The positions of ATF6-P, ATF6-M and ATF6-N are as indicated. The unprocessed ER-localised ATF6α (designated ER) and the O-linked glycan modified Golgi-translocated ATF6α (designated G) are indicated. HDAC2 was included as a nuclear marker. The blots in (D, E) confirm that ATF6-P is produced independently of S1P and also the absence of ATF6α processing to ATF6-N in the presence of the S1P inhibitor.


HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor . Cell lysates were prepared , separated by SDS - PAGE and then probed with anti - ATF6α . Blot reveals the presence of O - linked glycan modified Golgi - translocated ATF6 ( designated G ) , which is absent for the D564G mutant , confirming a lack of ER to Golgi trafficking .

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

HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor. Cell lysates were prepared, separated by SDS-PAGE and then probed with anti-ATF6α. Blot reveals the presence of O-linked glycan modified Golgi-translocated ATF6 (designated G), which is absent for the D564G mutant, confirming a lack of ER to Golgi trafficking.


Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated ( - ) or treated ( + ) with 5 µg / ml brefeldin A for1 h prior to cell lysis . The anti - ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6 - M , only in the brefeldin A treated cells . Nuclear fractions confirm the presence of S2P cleavage product , ATF6 - N following brefeldin A treatment . Anti - HDAC2 was used as a loading control for the nuclear fraction .

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

Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated (-) or treated (+) with 5 µg/ml brefeldin A for1 h prior to cell lysis. The anti-ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6-M, only in the brefeldin A treated cells. Nuclear fractions confirm the presence of S2P cleavage product, ATF6-N following brefeldin A treatment. Anti-HDAC2 was used as a loading control for the nuclear fraction.


A - C Wild type cells stably expressing ATF6α ( A ) or ERp18 KO cells stably expressing ATF6α ( B ) , and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 ( RV cells ) ( C ) , were pre - treated with 30 µM PF429242 ( S1P inhibitor ) for 1 h prior to induction of ER stress with 10 mM DTT as indicated .

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

A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated.


D - F Wild type cells transiently expressing ATF6 C467A ( D ) , ATF6 C618A ( E ) , or ATF6 DM ( F ) were treated as above . Whole cell lysates were separated by SDS - PAGE under reducing conditions and analysed by western blotting for ATF6α . The positions of unprocessed ER - localised ATF6α ( ER ) and the O - linked glycan modified Golgi - translocated ATF6α ( Gol ) are indicated as is ATF6 - P .

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

D-F Wild type cells transiently expressing ATF6 C467A (D), ATF6 C618A (E), or ATF6 DM (F) were treated as above. Whole cell lysates were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of unprocessed ER-localised ATF6α (ER) and the O-linked glycan modified Golgi-translocated ATF6α (Gol) are indicated as is ATF6-P.


G The percentage of Golgi - ATF6α was quantified , the error bars represent ± standard deviation for at least three independent experiments .

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

G The percentage of Golgi-ATF6α was quantified, the error bars represent ± standard deviation for at least three independent experiments.


FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade . Immunohistochemistry analysis of FOXA1 ( top ) and FOXA2 ( bottom ) in human PDAC . Representative G1 , G2 and G3 areas from one patient are shown . Red dotted lines indicate the borders of poorly differentiated ( G3 ) tumor areas .

B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-ORGANISM B-DISEASE O O O O O O B-EXP_ASSAY O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM B-DISEASE O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

FOXA1 and FOXA2 expression in human PDAC areas with distinct differentiation grade. Immunohistochemistry analysis of FOXA1 (top) and FOXA2 (bottom) in human PDAC. Representative G1, G2 and G3 areas from one patient are shown. Red dotted lines indicate the borders of poorly differentiated (G3) tumor areas.


Percent of FOXA1 - and FOXA2 - positive nuclei in PDAC cells from areas of different grade . Tissue microarrays were automatically acquired and quantified using QuPath . The number ( n ) of analyzed areas of each grade is indicated .

O O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O B-DISEASE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O

Percent of FOXA1- and FOXA2-positive nuclei in PDAC cells from areas of different grade. Tissue microarrays were automatically acquired and quantified using QuPath. The number (n) of analyzed areas of each grade is indicated.


Expression of FOXA1 and FOXA2 in low - grade and high - grade PDAC cell lines . Vinculin is shown as loading control .

B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O B-DISEASE O O O B-GENEPROD O O O O O O

Expression of FOXA1 and FOXA2 in low-grade and high-grade PDAC cell lines. Vinculin is shown as loading control.


Western blot showing siRNA - mediated depletion of FOXA1 and / or FOXA2 in the low - grade CFPAC1 cell line . The three biological replicates used in the subsequent experiments are shown .

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

Western blot showing siRNA-mediated depletion of FOXA1 and/or FOXA2 in the low-grade CFPAC1 cell line. The three biological replicates used in the subsequent experiments are shown.


Heatmap showing row - normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1 / 2 ( FDR ≤ 0 . 05 and absolute log2 FC ≥ 1 ) . Blue and orange depict down - and up - regulation , respectively .

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

Heatmap showing row-normalized expression of differentially expressed genes in CFPAC1 cells depleted of FOXA1/2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.