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 .

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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

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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

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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 .

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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 .

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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 .

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 ) .

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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 ) .

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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 .

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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 ) .

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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 .

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,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 .

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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 .

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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 .

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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 .

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 .

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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 .

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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 .

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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 .

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

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 .

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

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 .

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

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 .

O O O O O O O O O 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

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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 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 O O O O O O O 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 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 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).


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

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 O O O O O O O O O O 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) 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 O 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 .

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

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

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

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

D-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 O O O 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O 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 .

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 .

O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O 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.


FOXA2 and FOXA1 expression in wild type and genome - edited high - grade PANC1 cells . Three different bulk populations were used for RNA - seq experiments .

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

FOXA2 and FOXA1 expression in wild type and genome-edited high-grade PANC1 cells. Three different bulk populations were used for RNA-seq experiments.


Heatmap showing row - normalized expression of differentially expressed genes in PANC1 cells deleted of FOXA2 ( FDR ≤ 0 . 05 and absolute log2 FC ≥ 1 ) . Blue and orange depict down - and up - regulation , respectively .

O O O O O O O O O O O O O O O O O O O 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 PANC1 cells deleted of FOXA2 (FDR ≤ 0.05 and absolute log2 FC ≥ 1). Blue and orange depict down- and up-regulation, respectively.


Representative Gene Ontology ( GO ) terms enriched in genes differentially expressed upon FOXA1 / 2 depletion in CFPAC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA1 / 2 depletion on cell morphology and adhesion to multiple substrates . Three replicates per condition were used : circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition .

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

Representative Gene Ontology (GO) terms enriched in genes differentially expressed upon FOXA1/2 depletion in CFPAC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA1/2 depletion on cell morphology and adhesion to multiple substrates. Three replicates per condition were used: circularity index was measured on 100 different cells per condition while adhesion is reported as mean of three different fields per replicate for a total of nine observations per condition.


Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells . Terms are ranked by the negative log10 p - value from the hypergeometric test . Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates . Three different bulk populations were used as biological replicates : circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition .

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Representative GO terms enriched in genes differentially expressed upon FOXA2 deletion in PANC1 cells. Terms are ranked by the negative log10 p-value from the hypergeometric test. Panels on the right show the effects of FOXA2 deletion on cell morphology and adhesion to multiple substrates. Three different bulk populations were used as biological replicates: circularity index was measured on 60 different cells per bulk population while adhesion is reported as mean of six different fields per bulk for a total of eighteen observations per condition.


Scatter plots showing the correlation between FOXA2 ChIP - seq biological duplicates in CFPAC1 ( top panel ) and PANC1 cells ( bottom panel ) . Pearson ' s correlation ( r ) between samples is indicated .

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Scatter plots showing the correlation between FOXA2 ChIP-seq biological duplicates in CFPAC1 (top panel) and PANC1 cells (bottom panel). Pearson's correlation (r) between samples is indicated.


Scatter plot showing the correlation between FOXA2 ChIP - seq peaks in CFPAC1 and PANC1 cells . Green and blue dots indicate FOXA2 peaks that are significantly ( FDR < 0 . 001 ) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind . All other peaks are indicated as grey dots . The boxplots on the right show the intensity of CFPAC1 - specific , PANC1 - specific and other FOXA2 peaks . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers .

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Scatter plot showing the correlation between FOXA2 ChIP-seq peaks in CFPAC1 and PANC1 cells. Green and blue dots indicate FOXA2 peaks that are significantly (FDR < 0.001) different between the two cell types and with log2 fold enrichment ≥ 2 as determined by DiffBind. All other peaks are indicated as grey dots. The boxplots on the right show the intensity of CFPAC1-specific, PANC1-specific and other FOXA2 peaks. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.


Representative FOXA2 ChIP - seq snapshot showing FOXA2 peaks classified as CFPAC1 - specific , shared or PANC1 - specific .

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Representative FOXA2 ChIP-seq snapshot showing FOXA2 peaks classified as CFPAC1-specific, shared or PANC1-specific.


The five top transcription factor motifs over - represented in TSS - distal CFPAC1 - specific FOXA2 peaks . TFs were grouped into families or subfamilies and ranked based on the best PWM q - value . The POU domain TF family includes HNF1β . Top scoring motifs are shown on the right .

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The five top transcription factor motifs over-represented in TSS-distal CFPAC1-specific FOXA2 peaks. TFs were grouped into families or subfamilies and ranked based on the best PWM q-value. The POU domain TF family includes HNF1β. Top scoring motifs are shown on the right.


Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP - seq peaks .

Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with HNF1β ChIP-seq peaks.


Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS ( left ) and JUNB ( right ) ChIP - seq peaks .

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Bar plot showing the percentage of different sets of FOXA2 peaks overlapping with FOS (left) and JUNB (right) ChIP-seq peaks.


Two representative ChIP - seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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Two representative ChIP-seq snapshots indicating the genomic distribution of FOXA2 and partner transcription factors in CFPAC1 peaks. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Representative areas from human PDAC tissue showing expression of HNF1β in G1 , G2 and G3 areas . The red dotted line indicates the borders of a G3 area . The percentage of HNF1β - positive cells in areas of different grade from a PDAC tissue microarray is shown .

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Representative areas from human PDAC tissue showing expression of HNF1β in G1, G2 and G3 areas. The red dotted line indicates the borders of a G3 area. The percentage of HNF1β-positive cells in areas of different grade from a PDAC tissue microarray is shown.


Colocalization of HNF1β and FOXA2 in human PDAC areas . PDAC sections from three different patients were stained with the anti - HNF1β antibody , stripped and re - stained with the anti - FOXA2 antibody . The red dotted areas on the left represent poorly differentiated ( G3 ) tumor regions .

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Colocalization of HNF1β and FOXA2 in human PDAC areas. PDAC sections from three different patients were stained with the anti-HNF1β antibody, stripped and re-stained with the anti-FOXA2 antibody. The red dotted areas on the left represent poorly differentiated (G3) tumor regions.


FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility ( determined by ATAC - seq ) . Clusters 1 - 4 were HNF1β - positive while cluster 5 was HNF1β - negative . The heat - maps on the left show FOXA2 , HNF1β , ATAC - seq and H3K27Ac profiles at FOXA2 - bound genomic regions . The three heat - maps on the right show JunB ChIP - seq data and ATAC - seq data in wild type or Jun / JunB double knockout ( DKO ) CFPAC1 cells . Data are shown in + / - 3 kb regions centered on the summit of FOXA2 peaks .

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FOXA2 peaks in CFPAC1 cells were divided into 5 clusters based on their association with HNF1β and the effects of HNF1β loss on FOXA2 binding and chromatin accessibility (determined by ATAC-seq). Clusters 1-4 were HNF1β- positive while cluster 5 was HNF1β-negative. The heat-maps on the left show FOXA2, HNF1β, ATAC-seq and H3K27Ac profiles at FOXA2-bound genomic regions. The three heat-maps on the right show JunB ChIP-seq data and ATAC-seq data in wild type or Jun/JunB double knockout (DKO) CFPAC1 cells. Data are shown in +/- 3 kb regions centered on the summit of FOXA2 peaks.


Characterization of clusters 1 - 3 . Left : quantification of the indicated signals at FOXA2 bound regions in wild - type ( WT ) and HNF1β knock - out ( KO ) cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers . Middle : distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks . Right : over - represented motifs in clusters 1 - 3 and inferred binding models .

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Characterization of clusters 1-3. Left: quantification of the indicated signals at FOXA2 bound regions in wild-type (WT) and HNF1β knock-out (KO) cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers. Middle: distribution of FOXA and HNF1β motifs relative to the summit of FOXA2 peaks. Right: over-represented motifs in clusters 1-3 and inferred binding models.


Quantification of JunB ChIP - seq signals in clusters 1 to 5 . ( Right ) Quantification of ATAC - seq signals in clusters 1 to 5 in wild type and Jun / JunB double KO ( DKO ) CFPAC1 cells . Central values represent the median , the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers .

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Quantification of JunB ChIP-seq signals in clusters 1 to 5. (Right) Quantification of ATAC-seq signals in clusters 1 to 5 in wild type and Jun/JunB double KO (DKO) CFPAC1 cells. Central values represent the median, the bars the 25th and 75th percentile and the vertical lines the lower and upper whiskers.


Representative snapshot showing changes in FOXA2 occupancy , chromatin accessibility and histone acetylation in HNF1β knock - out CFPAC1 cells . The FOXA2 ChIP - seq track in PANC1 cells is also shown for comparison .

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Representative snapshot showing changes in FOXA2 occupancy, chromatin accessibility and histone acetylation in HNF1β knock-out CFPAC1 cells. The FOXA2 ChIP-seq track in PANC1 cells is also shown for comparison.


Western blot showing HNF1β over - expression after lentiviral transduction of PANC1 cells . Expression of FOXA2 in wild type and over - expressing ( O . E . ) cells is shown . Vinculin : loading control .

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Western blot showing HNF1β over-expression after lentiviral transduction of PANC1 cells. Expression of FOXA2 in wild type and over-expressing (O.E.) cells is shown. Vinculin: loading control.


Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β - expressing lentivirus ( HNF1B O . E . ) and in their matched mock - infected control . The fraction of gained , unchanged and lost FOXA2 peaks overlapping HNF1β ChIP - seq peaks in transduced PANC1 cells is shown in the pie - charts on the right .

Scatter plot showing the genomic distribution of FOXA2 in PANC1 cells transduced with the HNF1β-expressing lentivirus (HNF1B O.E.) and in their matched mock-infected control. The fraction of gained, unchanged and lost FOXA2 peaks overlapping HNF1β ChIP-seq peaks in transduced PANC1 cells is shown in the pie-charts on the right.


TF motif over - represented in gained or lost FOXA2 - bound genomic regions .

TF motif over-represented in gained or lost FOXA2-bound genomic regions.


Representative snapshot from PANC1 cells transduced with the HNF1β - expressing lentivirus . HNF1B O . E . : HNF1B over - expression .

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Representative snapshot from PANC1 cells transduced with the HNF1β-expressing lentivirus. HNF1B O.E.: HNF1B over-expression.


Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP - seq peaks .

Percentage of different sets of FOXA2 peaks overlapping HOXB8 ChIP-seq peaks.


Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP - seq peaks in PANC1 cells . The FOXA2 ChIP - seq track in CFPAC1 cells is also shown for comparison .

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Representative snapshots showing the overlap between HOXB8 and FOXA2 ChIP-seq peaks in PANC1 cells. The FOXA2 ChIP-seq track in CFPAC1 cells is also shown for comparison.


FOXA2 ChIP - seq in CFPAC1 cells transduced with a HOXB8 - expressing lentivirus and in their matched mock - infected control . Gained ( brown ) and lost ( yellow ) FOXA2 peaks are shown . Pie - charts on the right show the fraction of gained , unchanged and lost peaks overlapping HOXB8 ChIP - seq peaks in transduced CFPAC1 cells .

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FOXA2 ChIP-seq in CFPAC1 cells transduced with a HOXB8-expressing lentivirus and in their matched mock-infected control. Gained (brown) and lost (yellow) FOXA2 peaks are shown. Pie-charts on the right show the fraction of gained, unchanged and lost peaks overlapping HOXB8 ChIP-seq peaks in transduced CFPAC1 cells.


Expression of HNF1β protein in wild - type and HOXB8 - expressing PANC1 cells .

Expression of HNF1β protein in wild-type and HOXB8-expressing PANC1 cells.


Representative snapshots from CFPAC1 cells transduced with the HOXB8 - expressing lentivirus .

Representative snapshots from CFPAC1 cells transduced with the HOXB8-expressing lentivirus.


( A ) Co - localization of c - terminally GFP - or mCherry - tagged Apl5 , Sec7 and Vps41 in a vam3 temperature sensitive ( ts ) background strain . Cells were grown over night at 23 ° C and where indicated shifted to 37 ° C for 3 h prior to imaging . White arrowheads indicate co - localization events . Scale bar , 5 µm . Scale bar in inset , 2 µm .

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(A) Co-localization of c-terminally GFP- or mCherry-tagged Apl5, Sec7 and Vps41 in a vam3 temperature sensitive (ts) background strain. Cells were grown over night at 23°C and where indicated shifted to 37°C for 3 h prior to imaging. White arrowheads indicate co-localization events. Scale bar, 5 µm. Scale bar in inset, 2 µm.


( B ) Quantification of co - localization events Single events were counted and the percentage of co - localization normalized to the number of Apl5 - punctae was plotted ( n ≥ 187 cells ) . Bars show the mean percentage of co - localization ± standard deviation .

(B) Quantification of co-localization events Single events were counted and the percentage of co-localization normalized to the number of Apl5-punctae was plotted (n ≥ 187 cells). Bars show the mean percentage of co-localization ± standard deviation.


( C ) Re - localization of Apl5 upon Vps41 re - localization . Vps41 was tagged with a C - terminal FRBGFP tag in a strain , where Apl5 carries a C - terminal mCherry and Pma1 a FKBP - tag Cells were grown in either the absence ( control ) or presence of 10 μM rapamycin , and localization of Vps41 and Apl5 was analysed by fluorescence microscopy . Scale bar , 5 μm .

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(C) Re-localization of Apl5 upon Vps41 re-localization. Vps41 was tagged with a C-terminal FRBGFP tag in a strain, where Apl5 carries a C-terminal mCherry and Pma1 a FKBP-tag Cells were grown in either the absence (control) or presence of 10 μM rapamycin, and localization of Vps41 and Apl5 was analysed by fluorescence microscopy. Scale bar, 5 μm.


( D ) Effect of clathrin deletion on the AP - 3 pathway . GFP - tagged Nyv1 with a C - terminal Snc1 transmembrane domain ( GNS ) was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Labeling of the cell surface by GFP indicates a defect in the AP - 3 pathway ( E ) Quantification of AP - 3 defect from ( D ) . Linear intensity plots were laid over the cells , the AP - 3 defect was calculated by dividing GFP - intensities on the vacuolar membrane by the sum of the intensity of vacuolar , and plasma membrane signal ( n ≥ 30 cells ) . Bars show the mean vacuolar signal over total signal ± standard deviation .

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(D) Effect of clathrin deletion on the AP-3 pathway. GFP-tagged Nyv1 with a C-terminal Snc1 transmembrane domain (GNS) was localized relative to FM4-64 stained vacuoles in the indicated strains. Labeling of the cell surface by GFP indicates a defect in the AP-3 pathway (E) Quantification of AP-3 defect from (D). Linear intensity plots were laid over the cells, the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 30 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.


( C ) Tethering of AP - 3 vesicles to the mitochondrial surface . Cells were grown at 30 ° C in YPG and kept in logarithmic phase . Just prior to imaging , the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy . First column shows DIC ( difference interference contrast ) image of cells . Scale bar , 5 µm . The inset shows the indicated magnification . Scale bar in inset , 2 µm .

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(C) Tethering of AP-3 vesicles to the mitochondrial surface. Cells were grown at 30°C in YPG and kept in logarithmic phase. Just prior to imaging, the mitochondrial DNA was stained with DAPI Localization of Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC (difference interference contrast) image of cells. Scale bar, 5 µm. The inset shows the indicated magnification. Scale bar in inset, 2 µm.


Apl5 - dots co - localizing with GFP signal were counted and plotted in percent of the total number of Apl5 - dots . Bars show the mean percentage of co - localization ± standard deviation ( n ≥ 60 cells ) .

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Apl5-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5-dots. Bars show the mean percentage of co-localization ± standard deviation (n ≥ 60 cells).


( E , F ) Purification of mitochondria from selected strains . Mitochondria were purified from the indicated strains as described in the methods . The amount corresponding to 1 % of the total volume was loaded of the ' load ' ' sample , 5 % were used for ' crude ' and ' pure ' mitochondria fractions . Samples were loaded on a 10 % SDS PAGE gel and analysed by Western Blot . White asterisks indicate unspecific bands . ( G ) Quantification of relative Nyv1 signal intensity from ( F ) . Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted . Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments .

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(E, F) Purification of mitochondria from selected strains. Mitochondria were purified from the indicated strains as described in the methods. The amount corresponding to 1 % of the total volume was loaded of the 'load'' sample, 5% were used for 'crude' and 'pure' mitochondria fractions. Samples were loaded on a 10% SDS PAGE gel and analysed by Western Blot. White asterisks indicate unspecific bands. (G) Quantification of relative Nyv1 signal intensity from (F). Signal intensity was determined using ImageJ and a ratio of pure over crude signal intensity normalized to Tom40 signal intensity was plotted. Bars show the relative Nvy1 signal intensity ± standard deviation from n = 3 independent experiments.


( A - H ) Indicated strains were grown in YPG for expression of Vps41 - GFP - Fis constructs . Two representative images are shown for each strain . Mitochondria ( M ) are highlighted in red and vesicular structures in teal ( unlabeled for VPS41 - GFP - Fis ) or green ( unlabeled for VPS41 - GFP - Fis ) . Cell wall ( CW ) , plasma membrane ( PM ) , and vacuoles ( V ) are indicated . Scale bars , 250 nm .

(A-H) Indicated strains were grown in YPG for expression of Vps41-GFP-Fis constructs. Two representative images are shown for each strain. Mitochondria (M) are highlighted in red and vesicular structures in teal (unlabeled for VPS41-GFP-Fis) or green (unlabeled for VPS41-GFP-Fis). Cell wall (CW), plasma membrane (PM), and vacuoles (V) are indicated. Scale bars, 250 nm.


( A ) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects . First column shows DIC image of cells . Scale bar , 5 µm . ( B ) Quantification of the AP - 3 defect from ( A ) . Linear intensity plots were laid over cells grown in medium containing glucose ( D ) or galactose ( G ) , the AP - 3 defect was calculated by dividing GFP - intensities on the vacuolar membrane by the sum of the intensity of vacuolar , and plasma membrane signal ( n ≥ 15 cells ) . Bars show the mean vacuolar signal over total signal ± standard deviation .

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(A) GNS was localized by fluorescence microscopy in the indicated strains grown in medium containing glucose or galactose Images were inverted to visualize possible defects. First column shows DIC image of cells. Scale bar, 5 µm. (B) Quantification of the AP-3 defect from (A). Linear intensity plots were laid over cells grown in medium containing glucose (D) or galactose (G), the AP-3 defect was calculated by dividing GFP-intensities on the vacuolar membrane by the sum of the intensity of vacuolar, and plasma membrane signal (n ≥ 15 cells). Bars show the mean vacuolar signal over total signal ± standard deviation.


( C ) Effect of different truncations and deletions on AP - 3 targeting to mitochondria . Indicated strains were grown at 30 ° C in YPG and kept in logarithmic phase . Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy . First column shows DIC image of cells . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( D ) Quantification of ( C ) . ( - ) Control corresponds to Vps41 ( ΔPEST ) - GFP - Fis1 Apl6 - mCherry ( CUY12558 ) and ( + ) corresponds to Vps41 ( S - D ) - GFP - Fis1 Apl6 - mCherry ( CUY12560 ) . Apl5 - / Apl6 - dots co - localizing with GFP signal were counted and plotted in percent of the total number of Apl5 - / Apl6 - dots ( n ≥ 63 ) . Bars show the mean percentage of co - localization ± standard deviation .

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(C) Effect of different truncations and deletions on AP-3 targeting to mitochondria. Indicated strains were grown at 30°C in YPG and kept in logarithmic phase. Localization of Apl5 or Apl6 and Vps41 was analysed by fluorescence microscopy. First column shows DIC image of cells. Scale bar, 5 µm. Scale bar in inset, 2 µm. (D) Quantification of (C). (-) Control corresponds to Vps41(ΔPEST)-GFP-Fis1 Apl6-mCherry (CUY12558) and (+) corresponds to Vps41(S-D)-GFP-Fis1 Apl6-mCherry (CUY12560). Apl5- /Apl6-dots co-localizing with GFP signal were counted and plotted in percent of the total number of Apl5- /Apl6-dots (n ≥ 63). Bars show the mean percentage of co-localization ± standard deviation.


( C ) Volcano plots of proteins in close proximity to Apl5 - TurboID versus control cells , from a label - free proteomics analysis of streptavidin - biotin pulldowns . The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student ' s t - test , equal variance , performed from n = 3 independent experiments . The red dashed line ( significance , 0 . 05 ) separates specifically identified proteins ( top right portion of plot ) from background . Selected top hits are indicated with black dots ,

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(C) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells, from a label-free proteomics analysis of streptavidin-biotin pulldowns. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.05) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots,


( E ) Volcano plots of proteins in close proximity to Apl5 - TurboID versus control cells in a GAL1pr - Vps41 - GFP - Fis1 background , from a label - free proteomics analysis of streptavidin - biotin pulldowns with cells grown in YPG . The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student ' s t - test , equal variance , performed from n = 3 independent experiments . The red dashed line ( significance , 0 . 001 ) separates specifically identified proteins ( top right portion of plot ) from background . Selected top hits are indicated with black

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(E) Volcano plots of proteins in close proximity to Apl5-TurboID versus control cells in a GAL1pr-Vps41-GFP-Fis1 background, from a label-free proteomics analysis of streptavidin-biotin pulldowns with cells grown in YPG. The logarithmic ratios of protein intensities are plotted against negative logarithmic P values of two tailed Student's t-test, equal variance, performed from n = 3 independent experiments. The red dashed line (significance, 0.001) separates specifically identified proteins (top right portion of plot) from background. Selected top hits are indicated with black dots


( B ) AP - 3 sorting defects in GAP deletions . The AP - 3 reporter was localized relative to FM4 - 64 stained vacuoles in the indicated strains . First column shows the DIC image of cells . Scale bar , 5 µm .

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(B) AP-3 sorting defects in GAP deletions. The AP-3 reporter was localized relative to FM4-64 stained vacuoles in the indicated strains. First column shows the DIC image of cells. Scale bar, 5 µm.


( D ) Localization of AP - 3 in ArfGAP deletion strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . First column shows DIC image of cells . Scale bar , 5 µm . ( E ) Quantification of Apl5 - dots in ( D ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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(D) Localization of AP-3 in ArfGAP deletion strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. First column shows DIC image of cells. Scale bar, 5 µm. (E) Quantification of Apl5-dots in (D). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


( F ) Co - localization of C - terminally GFP - tagged Apl5 and C - terminally mCherry - tagged Sec7 in wt and age2Δ background . Cells were grown at 30 ° C and kept in logarithmic phase . Scale bar , 5 µm . Scale bar in inset , 2 µm . ( G ) Quantification of co - localization from ( F ) normalized per cell . Number of co - localizing dots was determined and normalized to the number of co - localization events per cell . Bars show the mean number of co - localizing dots per cell ± standard deviation ( n ≥ 60 ) .

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(F) Co-localization of C-terminally GFP-tagged Apl5 and C-terminally mCherry-tagged Sec7 in wt and age2Δ background. Cells were grown at 30°C and kept in logarithmic phase. Scale bar, 5 µm. Scale bar in inset, 2 µm. (G) Quantification of co-localization from (F) normalized per cell. Number of co-localizing dots was determined and normalized to the number of co-localization events per cell. Bars show the mean number of co-localizing dots per cell ± standard deviation (n ≥ 60).


( A , B ) AP - 3 sorting defects in strains overexpressing Gcs1 ( A ) or Age2 ( B ) . The AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm .

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(A, B) AP-3 sorting defects in strains overexpressing Gcs1 (A) or Age2 (B). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.


( C ) Localization of AP - 3 in ArfGAP overexpression strains . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( D ) Quantification of Apl5 dots in ( C ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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(C) Localization of AP-3 in ArfGAP overexpression strains. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (D) Quantification of Apl5 dots in (C). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


( E , F ) AP - 3 sorting defects in GAP - dead mutants of Gcs1 ( E ) and Age2 ( F ) . The AP - 3 reporter GNS was localized relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm .

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

(E, F) AP-3 sorting defects in GAP-dead mutants of Gcs1 (E) and Age2 (F). The AP-3 reporter GNS was localized relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm.


( G ) Localization of AP - 3 in GAP - dead mutants of Gcs1 and Age2 . Localization of C - terminally GFP - tagged Apl5 relative to FM4 - 64 stained vacuoles in the indicated strains . Scale bar , 5 µm . ( H ) Quantification of Apl5 dots in ( G ) . Apl5 - positve dots were counted and averaged to the number of dots per cell . Box boundaries indicate 25 % and 75 % of the dataset . The middle line indicates the median and the small square the mean .

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(G) Localization of AP-3 in GAP-dead mutants of Gcs1 and Age2. Localization of C-terminally GFP-tagged Apl5 relative to FM4-64 stained vacuoles in the indicated strains. Scale bar, 5 µm. (H) Quantification of Apl5 dots in (G). Apl5-positve dots were counted and averaged to the number of dots per cell. Box boundaries indicate 25% and 75% of the dataset. The middle line indicates the median and the small square the mean.


B . Analysis of cell cycle arrest ( in G2 ) in response to DSB induction assayed by flow cytometry .

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B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.


C . Rad53 phosphorylation ( Rad53 - P ) in response to DSB assayed by western blotting .

C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.


D . Cell survival upon DSB induction . Average ± SD ( n = 4 ) is shown for each genotype .

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D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.


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B. Srs2 requirement in de novo telomere addition in cells with and without functional HR. All strains are pif1-m2. Strains used: NK1264; NK2375, NK2376; NK2014, NK2015; NK2451, NK2452; NK2012, NK2013; NK2457, NK2458; NK2363, NK2364; NK2469; 2369, NK2370; NK2473-2475.


D . Dynamics of ( TG1 - 3 ) n addition monitored by qPCR through a time - course experiment ( asynchronous populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 ; NK4112 , NK4113 ; NK4114 , NK4115 ; NK3292 est2 ∆ , NK3293 est2 ∆ ; NK4232 , NK4233 .

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D. Dynamics of (TG1-3)n addition monitored by qPCR through a time-course experiment (asynchronous populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Strains used: NK3292, NK3293; NK4670, NK4671; NK4112, NK4113; NK4114, NK4115; NK3292 est2∆, NK3293 est2∆; NK4232, NK4233.


F . Comparative analysis of ssDNA / dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time - course experiment ( synchronised populations ) . The Y axis shows a fold increase in de novo telomere - specific PCR product relative to the background levels at 0 h and normalised against an internal control ( ARO1 locus ) . Average ± SD ( n = 3 ) is shown for each time - point of each genotype . Top set of error bars represents SD in relative increase of the de novo telomere - specific PCR product ( as in panel D ) while the lower set of error bars corresponds to quantifications of ss / dsDNA fractions . Strains used : NK3292 , NK3293 ; NK4670 , NK4671 .

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

F. Comparative analysis of ssDNA/dsDNA at de novo telomere addition loci in SRS2 and srs2Δ during a time-course experiment (synchronised populations). The Y axis shows a fold increase in de novo telomere-specific PCR product relative to the background levels at 0 h and normalised against an internal control (ARO1 locus). Average ± SD (n=3) is shown for each time-point of each genotype. Top set of error bars represents SD in relative increase of the de novo telomere-specific PCR product (as in panel D) while the lower set of error bars corresponds to quantifications of ss/dsDNA fractions. Strains used: NK3292, NK3293; NK4670, NK4671.


B . Southern blot analysis of re - synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three RS probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C . Re - synthesis of resected DNA on chr . VIIL in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 15 . 2 ( RS15 . 2 ) , 6 . 8 ( RS6 . 8 ) and 2 . 6 ( RS2 . 6 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

O O 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 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 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

B. Southern blot analysis of re-synthesis of resected DNA during BIR in SRS2 and srs2Δ corresponding to the data quantifications in C. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three RS probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown.C. Re-synthesis of resected DNA on chr.VIIL in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 15.2 (RS15.2), 6.8 (RS6.8) and 2.6 (RS2.6) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


D . Southern blot analysis of BIR - dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D . DNA was digested with EcoRI and BamHI , resolved on 0 . 7 % agarose gels , transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes ( three BIR probes and a reference probe , REF , hybridizing to an ARS522 - containing fragment on chr . V which is not involved in the repair ) . A representative image of one of the three repeats is shown . C - control strain NK3980 . E . BIR - dependent DNA synthesis in SRS2 and srs2Δ cells ( solid and dashed lines , respectively ) at the distance of 6 ( BIR6 ) , 36 ( BIR36 ) and 77 ( BIR77 ) kb away from the homology region . Average ± SD ( n = 3 ) is shown for each time point .

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 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 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

D. Southern blot analysis of BIR-dependent DNA synthesis in SRS2 and srs2Δ corresponding to the data quantifications in D. DNA was digested with EcoRI and BamHI, resolved on 0.7% agarose gels, transferred onto charged Nylon membrane and hybridised to the mixture of 4 probes (three BIR probes and a reference probe, REF, hybridizing to an ARS522-containing fragment on chr. V which is not involved in the repair). A representative image of one of the three repeats is shown. C - control strain NK3980.E. BIR-dependent DNA synthesis in SRS2 and srs2Δ cells (solid and dashed lines, respectively) at the distance of 6 (BIR6), 36 (BIR36) and 77 (BIR77) kb away from the homology region. Average ± SD (n=3) is shown for each time point.


B . Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A . Average ± SD ( n = 4 ) is shown for each genotype . Strains used in B and D : NK4691 - 4693 ; NK4805 - 4808 ; NK5081 - 5084 ; NK5085 - 5091 .

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B. Frequency of DSB repair via SSA in SRS2 and srs2Δ cells with and without functional HR in the assay based on the system shown in panel A. Average ± SD (n=4) is shown for each genotype. Strains used in B and D: NK4691-4693; NK4805-4808; NK5081-5084; NK5085-5091.


D . Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51 . Also see Figure EV3 for blot images . Average ± SD ( n = 4 ) is shown for each time point .

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D. Fragment L formation in SRS2 and srs2Δ cells in the presence and absence of Rad51. Also see Figure EV3 for blot images. Average ± SD (n=4) is shown for each time point.


( D ) Absolute cell numbers of the indicated cell types were determined by flow - cytometric analysis of the bone marrow from 3 - week - old Pax5 + / + and Pax5Jak2 / + mice . Average cell numbers are shown with SEM and were statistically analyzed by multiple t - tests ( unpaired two - tailed with Holm - Šídák ' s correction ) ; ns , not significant ( P > 0 . 05 ) . One of 3 independent experiments is shown .

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(D) Absolute cell numbers of the indicated cell types were determined by flow-cytometric analysis of the bone marrow from 3-week-old Pax5+/+ and Pax5Jak2/+ mice. Average cell numbers are shown with SEM and were statistically analyzed by multiple t-tests (unpaired two-tailed with Holm-Šídák's correction); ns, not significant (P > 0.05). One of 3 independent experiments is shown.


( F ) Kaplan - Meier survival analysis of Pax5Jak2 / + ( black ) and control Pax5 + / + ( grey ) mice . n , number of mice analyzed . A P value of < 0 . 0001 was determined for the survival curves by statistical analysis with the log - rank ( Mantel - Cox ) test .

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(F) Kaplan-Meier survival analysis of Pax5Jak2/+ (black) and control Pax5+/+ (grey) mice. n, number of mice analyzed. A P value of < 0.0001 was determined for the survival curves by statistical analysis with the log-rank (Mantel-Cox) test.


( H ) Eosin - hematoxylin - stained sections of the lung and liver of a Pax5Jak2 / + tumor mouse . Infiltrating and blasting tumor cells are indicated by an arrow .

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(H) Eosin-hematoxylin-stained sections of the lung and liver of a Pax5Jak2/+ tumor mouse. Infiltrating and blasting tumor cells are indicated by an arrow.


( I ) Flow - cytometric analysis of lymph node cells from a control Pax5 + / + mouse and a 10 - week - old Pax5Jak2 / + tumor mouse . ( J ) Flow - cytometric analysis of B220lowCD19 + B cells from the bone marrow of a 4 - week - old Pax5Jak2 / + mouse .

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(I) Flow-cytometric analysis of lymph node cells from a control Pax5+/+ mouse and a 10-week-old Pax5Jak2/+ tumor mouse. (J) Flow-cytometric analysis of B220lowCD19+ B cells from the bone marrow of a 4-week-old Pax5Jak2/+ mouse.