( D ) The endpoint analysis of NET - DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα - / - mice without ( control ) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point . The NET - DNA release index was reported in comparison to an assigned value of 100 % for the total DNA released by neutrophils lysed by 0 . 5 % ( v / v ) Triton - X - 100 .

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(D) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and PKCα-/- mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at the end of the 3 h time point. The NET-DNA release index was reported in comparison to an assigned value of 100% for the total DNA released by neutrophils lysed by 0.5% (v/v) Triton-X-100.


( E , F ) ( E ) Representative and ( F ) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα - / - mice stimulated without or with 10 µM PAF for 3 h , and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye , without fixation . Images were taken by Olympus confocal microscopy , followed by automated quantification of NETs on 5 - 6 non - overlapping area per well using ImageJ for calculation of % cells with NET formation . ( E ) Scale bars , 50 μm . % cells with NET formation by image analysis using ImageJ ( F )

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(E, F) (E) Representative and (F) summary analysis of NET formation of mouse peritoneal mPMNs from WT vs PKCα-/- mice stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red dye and cell impermeable Sytox Green dye, without fixation. Images were taken by Olympus confocal microscopy, followed by automated quantification of NETs on 5-6 non-overlapping area per well using ImageJ for calculation of % cells with NET formation. (E) Scale bars, 50 μm. % cells with NET formation by image analysis using ImageJ (F)


( A , B ) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT ( A ) vs Lmnb1TG ( B ) mice with UVB exposure , in which DNA was stained by DAPI , citrullinated histone H3 was probed by rabbit anti - mouse citrullinated Histone H3 , following stained by Alexa Fluor - 488 labeled donkey anti - rabbit secondary antibody , while neutrophil surface marker Ly6B was probed by rat anti - mouse Ly6B Ab following stained by Alexa Fluor - 647 conjugated goat anti - rat secondary antibody . The images of different staining and their overlays are shown ( A , B ) . The parental neutrophils ( stained by neutrophil membrane surface marker Ly6B ) and their released extracellular NET structures ( co - stained by anti - cit H3 and DAPI ) are indicated by red arrows and light blue arrows respectively . The white arrows indicated neutrophils without NET release . Scale bar , 50 µm .

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(A,B) representative analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.


( C ) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT ( A ) vs Lmnb1TG ( B ) mice with UVB exposure , in which DNA was stained by DAPI , citrullinated histone H3 was probed by rabbit anti - mouse citrullinated Histone H3 , following stained by Alexa Fluor - 488 labeled donkey anti - rabbit secondary antibody , while neutrophil surface marker Ly6B was probed by rat anti - mouse Ly6B Ab following stained by Alexa Fluor - 647 conjugated goat anti - rat secondary antibody . The images of different staining and their overlays are shown ( A , B ) . The parental neutrophils ( stained by neutrophil membrane surface marker Ly6B ) and their released extracellular NET structures ( co - stained by anti - cit H3 and DAPI ) are indicated by red arrows and light blue arrows respectively . The white arrows indicated neutrophils without NET release . Scale bar , 50 µm .

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(C) summary analysis of confocal fluorescent microscopy images of neutrophils with NET formation in the skin tissue of WT (A) vs Lmnb1TG (B) mice with UVB exposure, in which DNA was stained by DAPI, citrullinated histone H3 was probed by rabbit anti-mouse citrullinated Histone H3, following stained by Alexa Fluor-488 labeled donkey anti-rabbit secondary antibody, while neutrophil surface marker Ly6B was probed by rat anti-mouse Ly6B Ab following stained by Alexa Fluor-647 conjugated goat anti-rat secondary antibody. The images of different staining and their overlays are shown (A,B). The parental neutrophils (stained by neutrophil membrane surface marker Ly6B) and their released extracellular NET structures (co-stained by anti-cit H3 and DAPI) are indicated by red arrows and light blue arrows respectively. The white arrows indicated neutrophils without NET release. Scale bar, 50 µm.


( D , E ) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL - 17A ( D ) or TNFα ( E ) , in the skin tissue of WT vs Lmnb1TG mice without ( sham ) or with UVB exposure , by co - staining of Ly6B ( stained as described in panels A - C ) with IL - 17A ( D ) or TNFα ( E ) using FITC - conjugated rat anti - mouse IL - 17A , or FITC conjugated rat anti - mouse TNFα Abs .

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(D,E) Summary analysis for staining area of neutrophils with NET formation and their exhibition of IL-17A (D) or TNFα (E), in the skin tissue of WT vs Lmnb1TG mice without (sham) or with UVB exposure, by co-staining of Ly6B (stained as described in panels A-C) with IL-17A (D) or TNFα (E) using FITC-conjugated rat anti-mouse IL-17A, or FITC conjugated rat anti-mouse TNFα Abs.


A Macroscopic seedling phenotype of 12 - day - old b16b18CRISPR , quadriCRISPR F33 # 1 / # 6 , pentaCRISPR and amiR - 2572 , compared to WT ( Col - 0 ) .

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A Macroscopic seedling phenotype of 12-day-old b16b18CRISPR, quadriCRISPR F33#1/#6, pentaCRISPR and amiR-2572, compared to WT (Col-0).


E Macroscopic seedling phenotype of 12 - day - old quadriCRISPR B64 compared to WT ( Col - 0 ) and amiR - 2572 . Scale bars = 1 cm . F , G Boxplots showing the quantification of primary root length ( F ) and LR density ( G ) in seedlings depicted in E . n = 12 ( Col - 0 ) , 13 ( quadriCRISPR B64 ) , 11 ( amiR - 2572 ) .

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E Macroscopic seedling phenotype of 12-day-old quadriCRISPR B64 compared to WT (Col-0) and amiR-2572. Scale bars = 1 cm. F, G Boxplots showing the quantification of primary root length (F) and LR density (G) in seedlings depicted in E. n = 12 (Col-0), 13 (quadriCRISPR B64), 11 (amiR-2572). D


C Confocal images of root tips in 5 - day - old Col - 0 , amiR - 2572 and syn - tasi - 1522A # 1 seedlings . LRC cells were indicated by white asterisks . The distal LRC cells were indicated by white

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C Confocal images of root tips in 5-day-old Col-0, amiR-2572 and syn-tasi-1522A#1 seedlings. LRC cells were indicated by white asterisks. The distal LRC cells were indicated by white arrows


G Macroview stereo microscopic view of DR5 : VENUS expression in root tips of 3 - day - old WT , amiR - 2572 and syn - tasi - 1522A # 1 seedlings , red arrows indicate DR5 stripes in the lateral root cap .

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G Macroview stereo microscopic view of DR5:VENUS expression in root tips of 3-day-old WT, amiR-2572 and syn-tasi-1522A#1 seedlings, red arrows indicate DR5 stripes in the lateral root cap.


A Co - localisation of YFP - ABCB15 with Propidium Iodide ( PI ) in root epidermal cells of 3 - day - old seedlings . A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks .

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A Co-localisation of YFP-ABCB15 with Propidium Iodide (PI) in root epidermal cells of 3-day-old seedlings. A plot of the fluorescence intensity of YFP and PI along the dashed lines shows the colocalization of the YFP and PI intensity peaks.


C IAA export assay . Export of [ 3H ] - IAA , assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1 , ABCB15 - 22 and ABCB17P980G against vector control . mean ± SE ; n = 26 ( vector control ) , 44 ( ABCB1 ) , 5 ( ABCB15 ) , 4 ( ABCB16 ) , 9 ( ABCB17 ) , 5 ( ABCB18 ) , 9 ( ABCB22 ) and 8 ( ABCB17P980G ) , transport experiments generated from independent tobacco transfections .

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C IAA export assay. Export of [3H]-IAA, assayed in parallel from tobacco mesophyll protoplasts expressing ABCB1, ABCB15-22 and ABCB17P980G against vector control. mean ± SE; n = 26 (vector control), 44 (ABCB1), 5 (ABCB15), 4 (ABCB16), 9 (ABCB17), 5 (ABCB18), 9 (ABCB22) and 8 (ABCB17P980G),transport experiments generated from independent tobacco transfections.


B Analysis of DR5 : : VENUS expression in the root elongation zone of 4 - day - old pWOX5 : XVE > > YUC1 - 2A - TAA1 , in Control and amiR - 2572 treated with β - estradiol ( 5 µM ) for 0 , 7 . 5 and 9h . Images are composed of several tiles generated in a single snap with automatic assembly , PI in gray . The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5 : : VENUS signal in the elongation zone .

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B Analysis of DR5::VENUS expression in the root elongation zone of 4-day-old pWOX5:XVE>>YUC1-2A-TAA1, in Control and amiR-2572 treated with β-estradiol (5 µM) for 0, 7.5 and 9h. Images are composed of several tiles generated in a single snap with automatic assembly, PI in gray. The zoomed images of the yellow squares are presented below each root showing the accumulation of the DR5::VENUS signal in the elongation zone.


A , B Expression pattern of proABCB15 : NLS - GFP - GUS , proABCB16 : NLS - GFP - GUS , proABCB17 : NLS - GFP - GUS , proABCB18 : NLS - GFP - GUS , and proABCB22 : NLS - GFP - GUS expression in roots of 3 - day - old seedlings , using confocal microscopy showing longitudinal overview pictures ( Propidium iodide in magenta ) , and images zoomed on the epidermis and LRC corresponding to yellow squares ( A ) ; cytological sections of GUS stained roots of 3 - day - old seedlings , counterstained with ruthenium red ( B ) . Scale bars represent 20 µm for both graphs .

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A, B Expression pattern of proABCB15:NLS-GFP-GUS, proABCB16:NLS-GFP-GUS, proABCB17:NLS-GFP-GUS, proABCB18:NLS-GFP-GUS, and proABCB22:NLS-GFP-GUS expression in roots of 3-day-old seedlings, using confocal microscopy showing longitudinal overview pictures (Propidium iodide in magenta), and images zoomed on the epidermis and LRC corresponding to yellow squares (A); cytological sections of GUS stained roots of 3-day-old seedlings, counterstained with ruthenium red (B). Scale bars represent 20 µm for both graphs.


A Fluorescence of YFP - ABCB17 in the root meristem and leaf in 3 - day - old F1 crosses with WT , syn - tasi - 1522A # 1 and amiR - 2572 , propidium iodide ( PI ) in magenta . Red arrows highlight the position of the epidermis . Scale bars = 50 µm . B Schematic representation of root geometry with the indication of regions of interest for YFP - ABCB17 signal quantification . Red ( Stele ) , and black ( epidermis / LRC ) . C , D Quantification of YFP - ABCB17 fluorescence intensity in the stele ( C ) and epidermis / LRC ( D ) of A , measured at regions of interest corresponding

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A Fluorescence of YFP-ABCB17 in the root meristem and leaf in 3-day-old F1 crosses with WT, syn-tasi-1522A#1 and amiR-2572, propidium iodide (PI) in magenta. Red arrows highlight the position of the epidermis . Scale bars = 50 µm. B Schematic representation of root geometry with the indication of regions of interest for YFP-ABCB17 signal quantification. Red (Stele), and black (epidermis/LRC). C, D Quantification of YFP-ABCB17 fluorescence intensity in the stele (C) and epidermis/LRC (D) of A, measured at regions of interest corresponding t


G , H Quantification of the oscillation period ( G ) and amplitude ( H ) of DR5 : : LUC in 3 - day - old WT and syn - tasi - 1522A # 1 , n = 27 ( WT ) and 23 ( syn - tasi - 1522A # 1 ) .

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G,H Quantification of the oscillation period (G) and amplitude (H) of DR5::LUC in 3-day-old WT and syn-tasi-1522A#1, n= 27 (WT) and 23 (syn-tasi-1522A#1).


Kaplan - Meier analysis showing cases from the Australian cohort ( n = 73 ) delineated on the basis of expression of a set of hazardous genes , termed PC - 1 . Patients with the highest expression had a significantly poorer prognosis ( red line , median survival : 11 . 6 months ) compared with those patients with the lowest expression ( blue line , median survival : 34 . 4 months , P = 0 . 01 ) . The black line shows those with medium expression .

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Kaplan-Meier analysis showing cases from the Australian cohort (n = 73) delineated on the basis of expression of a set of hazardous genes, termed PC-1. Patients with the highest expression had a significantly poorer prognosis (red line, median survival: 11.6 months) compared with those patients with the lowest expression (blue line, median survival: 34.4 months, P = 0.01). The black line shows those with medium expression.


Kaplan - Meier analysis showing that cases in the Australian cohort that fell in the highest quartile of a hypoxia signature ( red line , median survival : 11 . 4 months ) have significantly decreased survival compared with those in the lowest quartile ( blue line , median survival : 25 . 2 months ; P = 0 . 03 ) .

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Kaplan-Meier analysis showing that cases in the Australian cohort that fell in the highest quartile of a hypoxia signature (red line, median survival: 11.4 months) have significantly decreased survival compared with those in the lowest quartile (blue line, median survival: 25.2 months; P = 0.03).


Kaplan - Meier analysis showing that cases in the APGI cohort ( n = 266 ) with high LOX expression above the 3rd quantile ( red line ) have significantly decreased survival compared to those with low expression below the 1st quantile ( blue line ) .

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Kaplan-Meier analysis showing that cases in the APGI cohort (n = 266) with high LOX expression above the 3rd quantile (red line) have significantly decreased survival compared to those with low expression below the 1st quantile (blue line).


Kaplan - Meier analysis showing that cases in the Glasgow cohort with high LOX expression ( red line , n = 24 ) have significantly decreased survival compared with those with low expression ( blue line , n = 23 ; P = 0 . 005 ) .

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Kaplan-Meier analysis showing that cases in the Glasgow cohort with high LOX expression (red line, n = 24) have significantly decreased survival compared with those with low expression (blue line, n = 23; P = 0.005).


Kaplan - Meier analysis showing that fibrillar collagen is significantly associated with reduced survival in human PDAC ( 20 months vs . 28 . 3 months , P = 0 . 033 ) .

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Kaplan-Meier analysis showing that fibrillar collagen is significantly associated with reduced survival in human PDAC (20 months vs. 28.3 months, P = 0.033).


Mean decay distance of the second harmonic generation ( SHG ) signal emitted by human PDAC - associated collagen . Mean decay distance is represented by boxplots showing the second and third quartile of the data with the whiskers indicating the maximum and minimum data points . Outliers are indicated by individual markers . Stage T3 tumors have a higher collagen mean decay distance score compared with Stage T2 tumors ( P = 0 . 02 ) . Lymph node - positive tumors have a higher collagen mean decay distance score vs . lymph node - negative tumors ( P = 0 . 047 ) . Tumors showing vascular invasion have a higher collagen mean decay distance score than non - vascular invasive tumors ( P = 0 . 037 ) .

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Mean decay distance of the second harmonic generation (SHG) signal emitted by human PDAC-associated collagen. Mean decay distance is represented by boxplots showing the second and third quartile of the data with the whiskers indicating the maximum and minimum data points. Outliers are indicated by individual markers. Stage T3 tumors have a higher collagen mean decay distance score compared with Stage T2 tumors (P = 0.02). Lymph node-positive tumors have a higher collagen mean decay distance score vs. lymph node-negative tumors (P = 0.047). Tumors showing vascular invasion have a higher collagen mean decay distance score than non-vascular invasive tumors (P = 0.037).


PC - 1 signature is predictive of metastatic disease in mouse models of pancreatic cancer . Log - transformed expressions of signature transcripts from mouse tumor microarrays were mean - centered across samples and scaled to unit variance . These values were then multiplied by the matching loading values from the PC - 1 signature and summarized for each sample across all transcripts to yield the risk score for that sample .

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PC-1 signature is predictive of metastatic disease in mouse models of pancreatic cancer. Log-transformed expressions of signature transcripts from mouse tumor microarrays were mean-centered across samples and scaled to unit variance. These values were then multiplied by the matching loading values from the PC-1 signature and summarized for each sample across all transcripts to yield the risk score for that sample.


Inverted invasion assays were performed with PDAC tumor cell lines from KPC and KPflC mice . Tumor cell lines bearing mutant p53R172H ( KPC ) invade significantly further than tumor cells with deletion of 1 copy of p53 ( KPflC ) ( P ≤ 0 . 01 ) . Data are shown as the average of four wells + SEM .

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Inverted invasion assays were performed with PDAC tumor cell lines from KPC and KPflC mice. Tumor cell lines bearing mutant p53R172H (KPC) invade significantly further than tumor cells with deletion of 1 copy of p53 (KPflC) (P ≤ 0.01). Data are shown as the average of four wells + SEM.


Introduction of shRNA targeting Lox into KPC tumor cells significantly inhibits invasion ( P ≤ 0 . 01 by unpaired Student ' s t - test ) . Data are shown as the average of four wells + SEM .

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Introduction of shRNA targeting Lox into KPC tumor cells significantly inhibits invasion (P ≤ 0.01 by unpaired Student's t-test). Data are shown as the average of four wells + SEM.


Introduction of exogenous LOX into KPflC tumor cells significantly promotes invasion ( left panel , P ≤ 0 . 01 by unpaired Student ' s t - test ) . LOX expression was assessed by immunoblotting ( right panel ) . Columns indicate the mean of four well and error bars indicate SEM .

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Introduction of exogenous LOX into KPflC tumor cells significantly promotes invasion (left panel, P ≤ 0.01 by unpaired Student's t-test). LOX expression was assessed by immunoblotting (right panel). Columns indicate the mean of four well and error bars indicate SEM.


A Representative fluorescence images showing the SHG signal from cross - linked collagen in untreated , LOX antibody - treated or isotype control - treated organotypic cultures of primary fibroblasts and rat tail fibrillar collagen I . B Chart showing the percentage of cross - linked collagen by depth , based on SHG signal , in organotypic matrices treated as indicated . Error bars indicate SEM . C Barchart showing total cross - linked collagen in organotypic matrices treated as indicated . The experiment was performed in triplicate with three different zones imaged for 80 μm , at 2 . 5 - μm sections each . Data are shown as total pixels ± SEM .

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A Representative fluorescence images showing the SHG signal from cross-linked collagen in untreated, LOX antibody-treated or isotype control-treated organotypic cultures of primary fibroblasts and rat tail fibrillar collagen I.B Chart showing the percentage of cross-linked collagen by depth, based on SHG signal, in organotypic matrices treated as indicated. Error bars indicate SEM.C Barchart showing total cross-linked collagen in organotypic matrices treated as indicated. The experiment was performed in triplicate with three different zones imaged for 80 μm, at 2.5-μm sections each. Data are shown as total pixels ± SEM.


D Kaplan - Meier ( non - parametric ) survival curve showing significantly extended tumor - free survival of KPC mice treated with LOX antibody ( 1 mg / kg i . p . , twice weekly ) + gemcitabine ( 100 mg / kg i . p . , twice weekly ) ( green line , n = 17 ) , compared with gemcitabine ± isotype control antibody - treated mice ( blue line , n = 22 , P = 0 . 014 ) or LOX antibody alone ( red line , n = 9 ) . Treatment was initiated when mice were 70 days old ( randomization was not used when recruiting the mice ) and the mice were treated twice weekly . Censored mice did not develop PDAC .

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D Kaplan-Meier (non-parametric) survival curve showing significantly extended tumor-free survival of KPC mice treated with LOX antibody (1 mg/kg i.p., twice weekly) + gemcitabine (100 mg/kg i.p., twice weekly) (green line, n = 17), compared with gemcitabine ± isotype control antibody-treated mice (blue line, n = 22, P = 0.014) or LOX antibody alone (red line, n = 9). Treatment was initiated when mice were 70 days old (randomization was not used when recruiting the mice) and the mice were treated twice weekly. Censored mice did not develop PDAC.


E H & amp ; amp ; E - stained section of PDAC harvested from LOX + gemcitabine - treated KPC mouse . Note necrosis . F Boxplot showing quantification of necrosis in H & amp ; amp ; E sections of PDACs harvested from these mice , as indicated . At least 30 fields of view from at least five mice per cohort were scored .

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E H&amp;E-stained section of PDAC harvested from LOX + gemcitabine-treated KPC mouse. Note necrosis.F Boxplot showing quantification of necrosis in H&amp;E sections of PDACs harvested from these mice, as indicated. At least 30 fields of view from at least five mice per cohort were scored.


G - H H & amp ; amp ; E - stained sections of PDAC harvested from LOX + gemcitabine - treated KPC mice . Note stromal collapse ( G ) and marked dilation of blood vessels ( H ) . I Representative H & amp ; amp ; E image showing a section of PDAC harvested from a control - treated KPC mouse .

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G-H H&amp;E-stained sections of PDAC harvested from LOX + gemcitabine-treated KPC mice. Note stromal collapse (G) and marked dilation of blood vessels (H).I Representative H&amp;E image showing a section of PDAC harvested from a control-treated KPC mouse.


A Outer left panels : Immunohistochemistry analysis of macrophage infiltration ( by F4 / 80 staining ) in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . Inner left panels : Immunohistochemistry analysis of neutrophil infiltration ( by MPO staining ) in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . Inner right panels : Immunohistochemistry analysis of tumorvasculature ( by CD31 staining ) in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . Outer right panels : Immunohistochemistry analysis of tenascin C expression in tumors from isotype control + gemcitabine - treated KPC mice vs . LOX antibody + gemcitabine - treated KPC mice . B - D Plots showing quantification of macrophage infiltration ( B ) , neutrophil infiltration ( C ) and tumorvasculature ( D ) in tumors from KPC mice treated as indicated . At least 30 fields of view from at least four mice per cohort were scored , and scoring was conducted blind . P - values were calculated using Mann - Whitney U - test and median is indicated by horizontal lines . E Correlation of LOX protein with tenascin C expression in 47 cases of PDAC ( Spearman ' s rho correlation coefficient = 0 . 61 ; P < 0 . 0001 ) .

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A Outer left panels: Immunohistochemistry analysis of macrophage infiltration (by F4/80 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner left panels: Immunohistochemistry analysis of neutrophil infiltration (by MPO staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Inner right panels: Immunohistochemistry analysis of tumorvasculature (by CD31 staining) in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice. Outer right panels: Immunohistochemistry analysis of tenascin C expression in tumors from isotype control + gemcitabine-treated KPC mice vs. LOX antibody + gemcitabine-treated KPC mice.B-D Plots showing quantification of macrophage infiltration (B), neutrophil infiltration (C) and tumorvasculature (D) in tumors from KPC mice treated as indicated. At least 30 fields of view from at least four mice per cohort were scored, and scoring was conducted blind. P-values were calculated using Mann-Whitney U-test and median is indicated by horizontal lines.E Correlation of LOX protein with tenascin C expression in 47 cases of PDAC (Spearman's rho correlation coefficient = 0.61; P < 0.0001).


( C ) Western blot analysis of cells transfected with 10 nM siRNA against ERK1 , ERK2 or a 5 nM combination of each ( ERK1 + 2 ) .

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(C) Western blot analysis of cells transfected with 10 nM siRNA against ERK1, ERK2 or a 5 nM combination of each (ERK1+2).


( A ) Dynamic regulation of fluorescently tagged Beta - catenin / Armadillo ( Arm - GFP ) during mitosis in the growing fly wing epithelium . Notice down - regulation of Arm - GFP as cells round up for mitosis ( arrow ) . Scale bar ~ 2µm . n > 10 independent biological replicates .

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(A) Dynamic regulation of fluorescently tagged Beta-catenin/Armadillo (Arm-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of Arm-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.


( B ) Dynamic regulation of fluorescently tagged E - cadherin ( E - cad - GFP ) during mitosis in the growing fly wing epithelium . Notice down - regulation of E - cad - GFP as cells round up for mitosis ( arrow ) . Scale bar ~ 2µm . n > 10 independent biological replicates .

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(B) Dynamic regulation of fluorescently tagged E-cadherin (E-cad-GFP) during mitosis in the growing fly wing epithelium. Notice down-regulation of E-cad-GFP as cells round up for mitosis (arrow). Scale bar ~2µm. n>10 independent biological replicates.


( C ) E - cad - GFP remains downregulated in cells arrested in mitosis with Colchicine . Scale bar ~ 2µm . n > 10 independent biological replicates .

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(C) E-cad-GFP remains downregulated in cells arrested in mitosis with Colchicine. Scale bar ~2µm. n>10 independent biological replicates.


( D ) Quantification of the degree of down - regulation of Arm - GFP and E - cad - GFP at the junction between mitotic cells and their interphase neighbours ( n > 10 independent samples per cell cycle stage ) . Mean + / - 1 S . D . shown .

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(D) Quantification of the degree of down-regulation of Arm-GFP and E-cad-GFP at the junction between mitotic cells and their interphase neighbours (n > 10 independent samples per cell cycle stage). Mean +/- 1 S.D. shown.


( E ) Two adjacent mitotic cells show no detectable Arm - GFP at the junction between them . Scale bar ~ 2µm .

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(E) Two adjacent mitotic cells show no detectable Arm-GFP at the junction between them. Scale bar ~2µm.


( F - I ) Electron microscopy cross - sections of wing epithelial cells at interphase , prophase , prometaphase and telophase . Notice adherens junctions ( dark staining , highlighted in insets ) are downregulated as cells round up for mitosis . Scale bars ~ 1µm ( low mag . ) and ~ 10nm ( high mag . ) . n > 10 independent biological replicates . Asterisks indicate a single mitotic cell . Green arrowheads indicate adherens junctions .

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(F-I) Electron microscopy cross-sections of wing epithelial cells at interphase, prophase, prometaphase and telophase. Notice adherens junctions (dark staining, highlighted in insets) are downregulated as cells round up for mitosis. Scale bars ~1µm (low mag.) and ~10nm (high mag.). n>10 independent biological replicates. Asterisks indicate a single mitotic cell. Green arrowheads indicate adherens junctions.


( A ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells in the anterior ( A ) compartment , but not in Rho - kinase ( Rok ) RNAi expressing cells of the posterior ( P ) compartment ( GFP - positive ; green ) . Scale bar ~ 10µm . n > 10 independent biological replicates .

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(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells in the anterior (A) compartment, but not in Rho-kinase (Rok) RNAi expressing cells of the posterior (P) compartment (GFP-positive; green). Scale bar ~10µm. n>10 independent biological replicates.


( B ) High - magnification view of single mitotic cell stained for p - MLC ( purple ) and E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Scale bar ~ 1µm . n > 10 independent biological replicates . ( C ) High - magnification view of single UAS . Rok - RNAi expressing mitotic cell stained for p - MLC ( purple ) and E - cadherin ( green ) , with metaphase plate chromosomes stained by DAPI ( blue ) . Note loss of cortical p - MLC staining ( purple ) . Scale bar ~ 1µm . n > 10 independent biological replicates .

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(B) High-magnification view of single mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Scale bar ~1µm. n>10 independent biological replicates. (C) High-magnification view of single UAS.Rok-RNAi expressing mitotic cell stained for p-MLC (purple) and E-cadherin (green), with metaphase plate chromosomes stained by DAPI (blue). Note loss of cortical p-MLC staining (purple). Scale bar ~1µm. n>10 independent biological replicates.


( D ) Expression of constitutively active Rho - kinase ( UAS . Rok - CA ) in the posterior compartment of the wing epithelium is sufficient to elevate p - MLC and reduce E - cadherin immunostaining . Zoom ( right ) shows high p - MLC and low E - cad in both a mitotic cell and its neighbours . Scale bar ~ 10µm . n > 10 independent biological replicates .

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(D) Expression of constitutively active Rho-kinase (UAS.Rok-CA) in the posterior compartment of the wing epithelium is sufficient to elevate p-MLC and reduce E-cadherin immunostaining. Zoom (right) shows high p-MLC and low E-cad in both a mitotic cell and its neighbours. Scale bar ~10µm. n>10 independent biological replicates.


( E ) MyoII RLC - GFP ( MyoII - GFP ) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis ( basal section , diagrammed left ) . Treatment with the Rho - kinase inhibitor Y - 27632 inhibits mitotic accumulation of MyoII - GFP . Quantification shown right ( n = 8 independent samples per genotype ) . Scale bar ~ 1µm . Mean + / - 1 S . D . shown .

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(E) MyoII RLC-GFP (MyoII-GFP) accumulates at the cortex as cells round up for mitosis and later appears at the cleavage furrow during cytokinesis (basal section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic accumulation of MyoII-GFP. Quantification shown right (n = 8 independent samples per genotype). Scale bar ~1µm. Mean +/- 1 S.D. shown.


( F ) E - cad - GFP is downregulated as cells round up for mitosis ( apical section , diagrammed left ) . Treatment with the Rho - kinase inhibitor Y - 27632 inhibits mitotic downregulation of E - cad - GFP . Extended treatment with Y - 27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E - cad - GFP is generally lost . Arrow indicates mitotic cell cortex at maximal rounding . Quantification shown right ( n = 6 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

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(F) E-cad-GFP is downregulated as cells round up for mitosis (apical section, diagrammed left). Treatment with the Rho-kinase inhibitor Y-27632 inhibits mitotic downregulation of E-cad-GFP. Extended treatment with Y-27632 leads to a general defect in all interphase and mitotic cells in which apical surface areas enlarge and E-cad-GFP is generally lost. Arrow indicates mitotic cell cortex at maximal rounding. Quantification shown right (n = 6 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


( A ) Nuclear localised Pbl - GFP becomes cytoplasmic in mitotic cells , co - incident with Rok - GFP relocalisation to the plasma membrane . Scale bar ~ 1µm . n > 5 independent biological replicates .

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(A) Nuclear localised Pbl-GFP becomes cytoplasmic in mitotic cells, co-incident with Rok-GFP relocalisation to the plasma membrane. Scale bar ~1µm. n > 5 independent biological replicates.


( B ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells , but not those expressing UAS . pbl - RNAi . Phospho - Histone H3 ( pH3 ) staining ( green ) marks mitotic cell chromosomes . Quantification shown right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bar ~ 1µm .

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(B) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those expressing UAS.pbl-RNAi. Phospho-Histone H3 (pH3) staining (green) marks mitotic cell chromosomes. Quantification shown right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bar ~1µm.


( C ) A stripe of expression of ptc . Gal4 UAS . pbl - RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis , marked by pyknotic nuclei and loss of aPKC / Dlg staining . Scale bars ~ 20µm . n > 6 independent biological replicates .

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(C) A stripe of expression of ptc.Gal4 UAS.pbl-RNAi in the wing disc leads to both enlarged cells and many basally extruded cells that undergo apoptosis, marked by pyknotic nuclei and loss of aPKC/Dlg staining. Scale bars ~20µm. n > 6 independent biological replicates.


( A ) Nuclear localised Tum - GFP becomes cytoplasmic in mitotic cells , including a distinct plasma membrane localisation ( arrows ) . Note also localisation to the cytokinetic ring ( asterisk ) and remnant mid - bodies / ring canals in interphase cells . Quantification shown on the right ( n > 7 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 2µm .

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(A) Nuclear localised Tum-GFP becomes cytoplasmic in mitotic cells, including a distinct plasma membrane localisation (arrows). Note also localisation to the cytokinetic ring (asterisk) and remnant mid-bodies/ring canals in interphase cells. Quantification shown on the right (n > 7 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~2µm.


( B ) A stripe of expression of ptc . Gal4 UAS . tum - RNAi in the wing disc leads to basally extruded cells that undergo apoptosis , marked by Dcp1 immunostaining , similar to UAS . pbl - RNAi . Quantification shown on the right ( n > 10 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

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(B) A stripe of expression of ptc.Gal4 UAS.tum-RNAi in the wing disc leads to basally extruded cells that undergo apoptosis, marked by Dcp1 immunostaining, similar to UAS.pbl-RNAi. Quantification shown on the right (n > 10 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


( C ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; red ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild - type wing epithelial cells , but not those cells homozygous mutant for tum347 , marked by expression of GFP in single - celled clones ( MARCM system ) . pH3 staining ( blue ) marks mitotic cell chromosomes . Arrow indicates a GFP - positive single cell tum347 mutant clone . Quantification shown on the right ( n > 3 independent samples per genotype ; mean + / - 1 S . D . shown ) . Scale bars ~ 20µm .

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(C) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; red) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells in wild-type wing epithelial cells, but not those cells homozygous mutant for tum347, marked by expression of GFP in single-celled clones (MARCM system). pH3 staining (blue) marks mitotic cell chromosomes. Arrow indicates a GFP-positive single cell tum347 mutant clone. Quantification shown on the right (n > 3 independent samples per genotype; mean +/- 1 S.D. shown). Scale bars ~20µm.


( A ) Phosphorylated Myosin - II Regulatory Light Chain ( p - MLC ; purple ) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells ( pH3 , red ) in wild - type wing discs , and aurA homozygous mutant wing discs , but not upon inhibition of both AurA and AurB by treatment with VX - 680 . Note increased numbers of mitotic cells in aurA homozygous mutant wing discs , due to delayed mitotic progression . Scale bars ~ 20µm ( low mag . ) and ~ 1µm ( high mag . ) . n > 15 independent biological replicates .

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(A) Phosphorylated Myosin-II Regulatory Light Chain (p-MLC; purple) detected by antibody staining reveals high levels around the cortex of rounded mitotic cells (pH3, red) in wild-type wing discs, and aurA homozygous mutant wing discs, but not upon inhibition of both AurA and AurB by treatment with VX-680. Note increased numbers of mitotic cells in aurA homozygous mutant wing discs, due to delayed mitotic progression. Scale bars ~20µm (low mag.) and ~1µm (high mag.). n > 15 independent biological replicates.


( B ) Live - imaging of MyoII - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , while inhibition of both AurA and AurB by treatment with VX - 680 leads to complete loss of mitotic rounding and MyoII - GFP membrane localisation . Asterisks indicate a single mitotic cell . Scale bars ~ 1µm . Quantification shown on the bottom right ( n = 5 independent samples per genotype ) .

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(B) Live-imaging of MyoII-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, while inhibition of both AurA and AurB by treatment with VX-680 leads to complete loss of mitotic rounding and MyoII-GFP membrane localisation. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 5 independent samples per genotype).


( C ) Live - imaging of E - cad - GFP reveals delayed mitosis in aurA homozygous mutant wing discs , cytokinesis failure in UAS . aurB - RNAi expressing wing discs , and complete failure of mitotic rounding and E - cad - GFP downregulation upon inhibition of both AurA and AurB by treatment with VX - 680 . Asterisks indicate a single mitotic cell . Scale bars ~ 1µm . Quantification shown on the bottom right ( n = 9 independent samples per genotype ) .

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(C) Live-imaging of E-cad-GFP reveals delayed mitosis in aurA homozygous mutant wing discs, cytokinesis failure in UAS.aurB-RNAi expressing wing discs, and complete failure of mitotic rounding and E-cad-GFP downregulation upon inhibition of both AurA and AurB by treatment with VX-680. Asterisks indicate a single mitotic cell. Scale bars ~1µm. Quantification shown on the bottom right (n = 9 independent samples per genotype).


( A ) Striped ptc . Gal4 - driven expression of UAS . GFP in a control wing imaginal disc . Scale bars ~ 20µm . n > 6 independent biological replicates . ( B ) Striped ptc . Gal4 - driven expression of UAS . GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells . n > 7 independent biological replicates . ( C ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . aurB - RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei ( marked by DAPI ; white arrowhead ) . n > 6 independent biological replicates . ( D ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . aurB - RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells ( marked by Dcp1 and pyknotic nuclei ) . n > 8 independent biological replicates . ( E ) Striped ptc . Gal4 - driven expression of UAS . GFP and UAS . pbl - RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells ( marked by Dcp1 and pyknotic nuclei ) . n > 8 independent biological replicates . ( F ) ptc . Gal4 - driven expression of UAS . aurB - RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence . n > 6 independent biological replicates .

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(A) Striped ptc.Gal4-driven expression of UAS.GFP in a control wing imaginal disc. Scale bars ~20µm. n > 6 independent biological replicates. (B) Striped ptc.Gal4-driven expression of UAS.GFP in an aurA homozygous mutant wing imaginal disc leads to increased numbers of pH3 positive mitotic cells. n > 7 independent biological replicates. (C) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB-RNAi in a control wing imaginal disc leads to enlarged cells with large nuclei (marked by DAPI; white arrowhead). n > 6 independent biological replicates. (D) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.aurB -RNAi in an aurA homozygous mutant wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (E) Striped ptc.Gal4-driven expression of UAS.GFP and UAS.pbl-RNAi in the wing imaginal disc leads to extrusion and apoptosis of cells (marked by Dcp1 and pyknotic nuclei). n > 8 independent biological replicates. (F) ptc.Gal4-driven expression of UAS.aurB-RNAi in an aurA homozygous mutant endoreplicating salivary gland has no phenotypic consequence. n > 6 independent biological replicates.


( G ) MARCM clones ( GFP - positive ) expressing UAS . aurB - RNAi in an aurA homozygous mutant cells result in single cell clones ( i . e . : no cell division ) , while individual loss of AurA or AurB allows some cell proliferation . Quantification shown on the right ( n > 7 independent samples per genotype ; Mean + / - 1 S . D . shown ) . Scale bar ~ 20µm .

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(G) MARCM clones (GFP-positive) expressing UAS.aurB-RNAi in an aurA homozygous mutant cells result in single cell clones (i.e.: no cell division), while individual loss of AurA or AurB allows some cell proliferation. Quantification shown on the right (n > 7 independent samples per genotype; Mean +/- 1 S.D. shown). Scale bar ~20µm.


( A ) Live - imaging of MyoII - GFP in a control wing disc epithelium . Asterisk indicates the location of a single mitotic cell about to initiate rounding . Scale bar ~ 1µm . n > 5 independent biological replicates . ( B ) Live - imaging of MyoII - GFP upon treatment with the dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding and prevents cytokinesis . Scale bar ~ 1µm . n > 9 independent biological replicates .

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(A) Live-imaging of MyoII-GFP in a control wing disc epithelium. Asterisk indicates the location of a single mitotic cell about to initiate rounding. Scale bar ~1µm. n > 5 independent biological replicates. (B) Live-imaging of MyoII-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 9 independent biological replicates.


( C ) Live - imaging of E - cad - GFP upon treatment with the dual AurA / B inhibitor VX - 680 after initiation of mitotic rounding , which rapidly arrests progression of rounding and prevents cytokinesis . Scale bar ~ 1µm . n > 6 independent biological replicates .

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(C) Live-imaging of E-cad-GFP upon treatment with the dual AurA/B inhibitor VX-680 after initiation of mitotic rounding, which rapidly arrests progression of rounding and prevents cytokinesis. Scale bar ~1µm. n > 6 independent biological replicates.


( A ) Early stage Drosophila egg chambers showing giant germ - line nurse cells surrounded by E - cadherin - positive follicle cell epithelium . A follicle cell undergoing mitotic rounding is highlighted with an asterisk ( * ) . Scale bar ~ 3µm . n > 3 independent biological replicates .

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(A) Early stage Drosophila egg chambers showing giant germ-line nurse cells surrounded by E-cadherin-positive follicle cell epithelium. A follicle cell undergoing mitotic rounding is highlighted with an asterisk (*). Scale bar ~3µm. n > 3 independent biological replicates.


( B ) High resolution imaging of the F - actin cortex , E - cadherin , and phospho - MyoII in a mitotically rounded follicle cell . Note maintenance of adherens junctions including both E - cad and the contractile actomyosin ring during mitotic rounding . Asterisks or pH3 staining indicate a single mitotic cell . Scale bar ~ 3µm . n > 4 independent biological replicates .

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(B) High resolution imaging of the F-actin cortex, E-cadherin, and phospho-MyoII in a mitotically rounded follicle cell. Note maintenance of adherens junctions including both E-cad and the contractile actomyosin ring during mitotic rounding. Asterisks or pH3 staining indicate a single mitotic cell. Scale bar ~3µm. n > 4 independent biological replicates.


( A ) DNA damage induces H2AX ADP - ribosylation . 293T cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) . ADP - ribosylated proteins were IPed with anti - ADP - ribose ( anti - ADPR ) antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody .

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(A) DNA damage induces H2AX ADP-ribosylation. 293T cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes). ADP-ribosylated proteins were IPed with anti-ADP-ribose (anti-ADPR) antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.


( B ) H2AX is ADP - ribosylated at glutamate 141 ( E141 ) . shPARG HCT116 cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) and ADP - ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15 . 0109 Da , an increment that can be readily distinguished by mass spectrometry . Fragmentation of the NH2OH - derivatized peptides yielded typical b - and y - ion series , allowing easy localization of ADP - ribosylation sites . Glutamate 141 ( E141 ) is indicated by an asterisk .

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(B) H2AX is ADP-ribosylated at glutamate 141 (E141). shPARG HCT116 cells were treated with H2O2 (2 mM in PBS, 5 minutes) and ADP-ribosylated residues were tagged by a hydroxamic acid derivative with an addition of 15.0109 Da, an increment that can be readily distinguished by mass spectrometry. Fragmentation of the NH2OH-derivatized peptides yielded typical b- and y-ion series, allowing easy localization of ADP-ribosylation sites. Glutamate 141 (E141) is indicated by an asterisk.


( C ) The E141A mutation abolishes the ADP - ribosylation of H2AX . Empty vector ( EV ) , wild type ( WT ) or the E141A mutant ( E141A ) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2 . ADP - ribosylated proteins were IPed with anti - ADPR antibody . ADP - ribosylated H2AX was examined by Western blotting using anti - H2AX antibody .

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(C) The E141A mutation abolishes the ADP-ribosylation of H2AX. Empty vector (EV), wild type (WT) or the E141A mutant (E141A) of H2AX was expressed in U2OS H2AX knockout cells that were treated with H2O2. ADP-ribosylated proteins were IPed with anti-ADPR antibody. ADP-ribosylated H2AX was examined by Western blotting using anti-H2AX antibody.


( D ) ADP - ribosylation on H2AX is suppressed by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . The samples were examined by IP and Western blotting .

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(D) ADP-ribosylation on H2AX is suppressed by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. The samples were examined by IP and Western blotting.


( E ) PARP1 , but not PARP2 , mediates the ADP - ribosylation on H2AX . Wild type ( WT ) cells , PARP1 - null cells or PARP2 - null cells were treated with H2O2 .

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(E) PARP1, but not PARP2, mediates the ADP-ribosylation on H2AX. Wild type (WT) cells, PARP1-null cells or PARP2-null cells were treated with H2O2.


( A ) U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or the E141A mutant H2AX ( E141A ) were treated with H2O2 or MMS . Cell viability was measured after 24 hours using MTT assay . Values are mean ± SD of three assays .

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(A) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 or MMS. Cell viability was measured after 24 hours using MTT assay. Values are mean ± SD of three assays.


( B ) U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with empty vector ( H2AX KO + EV ) , wild type H2AX ( H2AX KO + WT ) , or the E141A mutant ( E141A ) H2AX were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) , subsequently ; the cells were cultured in fresh DMEM medium for 2 weeks . The numbers of colony formation were counted . Values are mean ± SD of three assays .

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(B) U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with empty vector (H2AX KO+EV), wild type H2AX (H2AX KO+WT), or the E141A mutant (E141A) H2AX were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes), subsequently; the cells were cultured in fresh DMEM medium for 2 weeks. The numbers of colony formation were counted. Values are mean ± SD of three assays.


( C ) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant ( E141A ) . U2OS cells ( WT ) and U2OS H2AX knockout cells reconstituted with wild type H2AX ( H2AX KO + WT ) , or the E141A mutant H2AX ( E141A ) were treated with H2O2 ( 2 mM in PBS , 5 minutes ) or MMS ( 1 mM in medium , 30 minutes ) and harvested at the indicated recovery time points . Then FPG - modified comet assay was performed . NT : no treatment . Representative comet tails were shown . The olive tail moments ( OTM ) were summarized from at least 50 cells in each experiment . Values are mean ± SD of three assays .

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(C) Damaged bases repair are suppressed in U2OS cells expressing the E141A mutant (E141A). U2OS cells (WT) and U2OS H2AX knockout cells reconstituted with wild type H2AX (H2AX KO+WT), or the E141A mutant H2AX (E141A) were treated with H2O2 (2 mM in PBS, 5 minutes) or MMS (1 mM in medium, 30 minutes) and harvested at the indicated recovery time points. Then FPG-modified comet assay was performed. NT: no treatment. Representative comet tails were shown. The olive tail moments (OTM) were summarized from at least 50 cells in each experiment. Values are mean ± SD of three assays.


( D ) DNA replication in U2OS cells expressing the E141A mutant ( E141A ) is suppressed . Cells were treated with H2O2 ( 2 mM in PBS , 5 minutes ) , MMS ( 1 mM in medium , 30 minutes ) or mock ( PBS , 30 minutes ) , followed by BrdU incorporation ( 30 μM in medium , 30 minutes ) . Representative images show BrdU - positive cells in cells expressing WT H2AX or E141A following genotoxic stress . Nuclei were stained with DAPI ( blue ) . The bottom panel histogram shows the percentage of BrdU incorporation in each sample , Values are mean ± SD of three assays .

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(D) DNA replication in U2OS cells expressing the E141A mutant (E141A) is suppressed. Cells were treated with H2O2 (2 mM in PBS, 5 minutes), MMS (1 mM in medium, 30 minutes) or mock (PBS, 30 minutes), followed by BrdU incorporation (30 μM in medium, 30 minutes). Representative images show BrdU-positive cells in cells expressing WT H2AX or E141A following genotoxic stress. Nuclei were stained with DAPI (blue). The bottom panel histogram shows the percentage of BrdU incorporation in each sample, Values are mean ± SD of three assays.


( E ) Generation of AP sites is suppressed in cells expressing E141A . Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes . AP sites were calculated according to the standard curve . Values are mean ± SD of three assays .

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(E) Generation of AP sites is suppressed in cells expressing E141A. Cells were treated with 2 mM H2O2 for 5 minutes or 1 mM MMS for 30 minutes. AP sites were calculated according to the standard curve. Values are mean ± SD of three assays.


( A ) ADP - ribosylation mediates the recruitment of Neil3 . GFP - Neil3 was expressed in U2OS cells , and cells were treated with olaparib ( 1 µM , 1 hour ) or mock ( 0 . 1 % DMSO in medium , 1 hour ) . Following laser microirradiation treatment , the recruitment of Neil3 was examined with live - cell imaging at the indicated time points ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(A) ADP-ribosylation mediates the recruitment of Neil3. GFP-Neil3 was expressed in U2OS cells, and cells were treated with olaparib (1 µM, 1 hour) or mock (0.1 % DMSO in medium, 1 hour). Following laser microirradiation treatment, the recruitment of Neil3 was examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( B ) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage . Truncated mutants of Neil3 were generated and fused with a GFP tag . Following laser microirradiation treatment , the recruitment of truncated mutants Neil3 were examined with live - cell imaging at the indicated time points ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(B) The GRFs of Neil3 alone is sufficient to be recruited to the sites of DNA damage. Truncated mutants of Neil3 were generated and fused with a GFP tag. Following laser microirradiation treatment, the recruitment of truncated mutants Neil3 were examined with live-cell imaging at the indicated time points (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( C ) PARP inhibitor treatment ( olaparib : 1 µM , 1 hour ) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represents mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(C) PARP inhibitor treatment (olaparib: 1 µM, 1 hour) or lack of PARP1 suppresses the relocation of the GRFs of Neil3 to the DNA lesions. The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represents mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( D ) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment ( 1 µM , 1 hour ) . 0 . 1 % DMSO in medium for 1 hour as mock . Oxidative damage was induced by KillerRed ( KR ) system with the exposure to a 15 - W SYLVANIA cool white fluorescent bulb for 10 minutes . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : the percentage of Neil3 foci co - localized with KR was quantified .

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(D) The recruitment of Neil3 to the oxidative lesions is inhibited by olaparib treatment (1 µM, 1 hour). 0.1 % DMSO in medium for 1 hour as mock. Oxidative damage was induced by KillerRed (KR) system with the exposure to a 15-W SYLVANIA cool white fluorescent bulb for 10 minutes. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of Neil3 foci co-localized with KR was quantified.


( A ) The GRFs of Neil3 recognizes PAR . The recombinant GST fusion proteins were incubated with PAR and the protein - PAR complex was pulled down by glutathione agarose beads . Samples were serially diluted three - fold , spotted onto the nitrocellulose membrane , and subjected to dot blotting using anti - PAR antibody ( top panel ) . FL represents full length Neil3 , N - ter represents N - terminal Neil3 ( 1 - 300 aa ) , Mid represents middle Neil3 ( 300 - 404 aa ) , and GRFs represents C - terminal Neil3 GRFs domain ( 480 - 606 aa ) . Recombinant GST protein was used as the negative control ( NC ) . The GST fusion proteins were also examined by the SDS - PAGE followed with Coomassie blue staining ( middle panel ) . The relative PAR - binding in the pull down assay was summarized in the graph ( bottom panel ) . Values are mean ± SD of three assays .

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(A) The GRFs of Neil3 recognizes PAR. The recombinant GST fusion proteins were incubated with PAR and the protein-PAR complex was pulled down by glutathione agarose beads. Samples were serially diluted three-fold, spotted onto the nitrocellulose membrane, and subjected to dot blotting using anti-PAR antibody (top panel). FL represents full length Neil3, N-ter represents N-terminal Neil3 (1-300 aa), Mid represents middle Neil3 (300-404 aa), and GRFs represents C-terminal Neil3 GRFs domain (480-606 aa). Recombinant GST protein was used as the negative control (NC). The GST fusion proteins were also examined by the SDS-PAGE followed with Coomassie blue staining (middle panel). The relative PAR-binding in the pull down assay was summarized in the graph (bottom panel). Values are mean ± SD of three assays.


( B ) Tandem co - immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP - ribosylated H2AX but not the E141A mutant H2AX .

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(B) Tandem co-immunoprecipitation and Western blotting assays demonstrate that Neil3 interacts with ADP-ribosylated H2AX but not the E141A mutant H2AX.


( C , D ) The GRFs of Neil3 is sufficient and required for the interaction with ADP - ribosylated H2AX . Tandem co - immunoprecipitation and Western blotting assays were

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(C,D) The GRFs of Neil3 is sufficient and required for the interaction with ADP-ribosylated H2AX. Tandem co-immunoprecipitation and Western blotting assays were used


( E , F ) The relocation kinetic of full - length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed . GFP - tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells , and relocation kinetics was monitored in a time course following laser microirradiation ( left panel ) . The laser stripe is indicated with yellow arrowheads . The relocation kinetics is shown in the right panel . Data represent mean ± SD from three biologically independent experiments ( right panel ) . At least 20 cells were included in each experiment .

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(E,F) The relocation kinetic of full-length Neil3 or the GRFs of Neil3 to DNA lesions was analyzed. GFP-tagged full length Neil3 or the GRFs of Neil3 was expressed in the indicated cells, and relocation kinetics was monitored in a time course following laser microirradiation (left panel). The laser stripe is indicated with yellow arrowheads. The relocation kinetics is shown in the right panel. Data represent mean ± SD from three biologically independent experiments (right panel). At least 20 cells were included in each experiment.


( G , H ) The recruitment of full - length Neil3 or the GRFs of Neil3 to the KR - induced oxidative lesions is mediated by H2AX ADP - ribosylation ( left panel ) . Scale bars , 2 μm . The foci were indicated with white arrowheads . Scale bars , 0 . 5 μm . Right panel : the percentage of Neil3 or GRFs Neil3 foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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(G,H) The recruitment of full-length Neil3 or the GRFs of Neil3 to the KR-induced oxidative lesions is mediated by H2AX ADP-ribosylation (left panel). Scale bars, 2 μm. The foci were indicated with white arrowheads. Scale bars, 0.5 μm. Right panel: the percentage of Neil3 or GRFs Neil3 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( A ) PARP inhibition facilitates γH2AX following oxidative stress . The cells were pre - treated with olaparib ( 1 µM , 1 hour ) prior to H2O2 stimulation . 0 . 1 % DMSO in medium for 1 hour as mock . γH2AX or H2AX was detected using Western blotting and immunostaining assays . The intensity of γH2AX levels were examined by Western blotting normalized by β - actin and averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - test . * * P < 0 . 01 ( left panel ) . The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8 .

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

(A) PARP inhibition facilitates γH2AX following oxidative stress. The cells were pre-treated with olaparib (1 µM, 1 hour) prior to H2O2 stimulation. 0.1 % DMSO in medium for 1 hour as mock. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.


( B ) The effect of H2AX E141 ADP - ribosylation on γH2AX following oxidative stress . γH2AX or H2AX was detected using Western blotting and immunostaining assays . The intensity of γH2AX levels were examined by Western blotting normalized by β - actin and averaged . Values are mean ± SD of three assays . P - values were calculated using Student ' s t - test . * * P < 0 . 01 ( left panel ) . The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8 .

O O O O O O B-CONTROLLED_VAR 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) The effect of H2AX E141 ADP-ribosylation on γH2AX following oxidative stress. γH2AX or H2AX was detected using Western blotting and immunostaining assays. The intensity of γH2AX levels were examined by Western blotting normalized by β-actin and averaged. Values are mean ± SD of three assays. P-values were calculated using Student's t-test. **P < 0.01 (left panel). The number of γH2AX foci examined in immunostaining assay was counted by Image J software and graphed by GraphPad Prism 8.


( C ) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed - induced oxidative DNA lesions ( left panel ) . Scale bar , 2 μm . The foci were indicated with white arrowheads . Scale bar , 0 . 5 μm . Right panel : the relative intensity of γH2AX at the site of tetR ( intensity of γH2AX at the site of tetR / intensity of γH2AX area away from tetR ) was quantified . Mean value with SD is the relative intensity of γH2AX in 20 cells .

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(C) The intensity of γH2AX is increased in cell expressing the E141A mutant following KillerRed-induced oxidative DNA lesions (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Scale bar, 0.5 μm. Right panel: the relative intensity of γH2AX at the site of tetR (intensity of γH2AX at the site of tetR/intensity of γH2AX area away from tetR) was quantified. Mean value with SD is the relative intensity of γH2AX in 20 cells.


( D ) The level of MDC1 at KillerRed - induced oxidative DNA lesions is increased in the cells expressing the E141A mutant ( left panel ) . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : the percentage of MDC1 foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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(D) The level of MDC1 at KillerRed-induced oxidative DNA lesions is increased in the cells expressing the E141A mutant (left panel). Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: the percentage of MDC1 foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( E ) The recruitment of Neil3 / MDC1 chimera but not wild type Neil3 to the KR - induced oxidative lesions in cells expressing the E141A mutant ( left panel ) . Neil3 / MDC1 chimera : replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1 . Scale bar , 2 μm . The foci were indicated with white arrowheads . Right panel : The percentage of detected protein foci co - localized with KR was quantified . Mean value with SD is from 20 cells .

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

(E) The recruitment of Neil3/MDC1 chimera but not wild type Neil3 to the KR-induced oxidative lesions in cells expressing the E141A mutant (left panel). Neil3/MDC1 chimera: replaced the GRFs of Neil3 with the tandem BRCT domains of MDC1. Scale bar, 2 μm. The foci were indicated with white arrowheads. Right panel: The percentage of detected protein foci co-localized with KR was quantified. Mean value with SD is from 20 cells.


( F ) AP sites were measured in cells expressing the E141A mutant or the Neil3 / MDC1 chimera , according to the standard curve . Values are mean ± SD of three assays .

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(F) AP sites were measured in cells expressing the E141A mutant or the Neil3/MDC1 chimera, according to the standard curve. Values are mean ± SD of three assays.


( G ) DNA replication of U2OS cells expression with individual wild type ( WT ) , the H2AX KO + WT , the E141A mutant ( E141A ) or the Neil3 / MDC1 Chimera ( Chimera ) were analyzed by BrdU incorporation following treatment with H2O2 , MMS or mock ( PBS ) . Represented images are shown in the left panel . The graph shows percentages of BrdU - positive cells expressing WT , E141A or Chimera ( right panel ) .

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

(G) DNA replication of U2OS cells expression with individual wild type (WT), the H2AX KO + WT, the E141A mutant (E141A) or the Neil3/MDC1 Chimera (Chimera) were analyzed by BrdU incorporation following treatment with H2O2, MMS or mock (PBS). Represented images are shown in the left panel. The graph shows percentages of BrdU-positive cells expressing WT, E141A or Chimera (right panel).


A ) HFFs were pre - stimulated with IFNγ ( 10U / mL ) for 24 h . Plaque assays were performed for each strain and each condition . Plaque number and area loss were calculated 4 days p . i . for RH and 6 days p . i . for Pru . Assays were performed with RH ( n = 4 ) , Pru ( n = 4 ) .

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A) HFFs were pre-stimulated with IFNγ (10U/mL) for 24 h. Plaque assays were performed for each strain and each condition. Plaque number and area loss were calculated 4 days p.i. for RH and 6 days p.i. for Pru. Assays were performed with RH (n=4), Pru (n=4).


B ) Relative parasite growth was measured 24 h p . i . in IFNγ - stimulated and unstimulated HFFs by luciferase assay . Growth of each strain in IFNγ - stimulated HFFs is expressed relative to growth in unstimulated HFFs . Experiments were performed with RH ( n = 4 ) , Pru ( n = 4 ) .

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B) Relative parasite growth was measured 24 h p.i. in IFNγ-stimulated and unstimulated HFFs by luciferase assay. Growth of each strain in IFNγ-stimulated HFFs is expressed relative to growth in unstimulated HFFs. Experiments were performed with RH (n=4), Pru (n=4).


C ) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U / mL IFNγ for 24 h . Following stimulation , HFFs were infected with either RH ( MOI = 1 ) or Pru ( MOI = 3 ) for another 24 h . Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei . The number of PVs in 5 - 6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field . Experiments were performed with RH ( n = 3 ) , Pru ( n = 3 ) . Representative images of percentage infected cells for each strain and condition are provided . Scale bar is 100 µm .

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C) Number of PVs was calculated using HFFs grown on coverslips and stimulated with 10 U/mL IFNγ for 24 h. Following stimulation, HFFs were infected with either RH (MOI=1) or Pru (MOI=3) for another 24 h. Coverslips were fixed and stained with GRA7 for parasite PVM and Hoechst 33258 for nuclei. The number of PVs in 5-6 fields of the coverslips were counted for each condition and normalized with the number of host cells in each field. Experiments were performed with RH (n=3), Pru (n=3). Representative images of percentage infected cells for each strain and condition are provided. Scale bar is 100 µm.


D ) Parasites per vacuole were determined 24 h p . i . with similar conditions and staining as described in ( C ) except MOIs used were 0 . 5 and 1 for RH ( n = 3 ) and Pru ( n = 3 ) , respectively .

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D) Parasites per vacuole were determined 24 h p.i. with similar conditions and staining as described in (C) except MOIs used were 0.5 and 1 for RH (n=3) and Pru (n=3), respectively.


E ) Plaque assays were performed similarly as described for ( a ) with Pru WT ( n = 20 for plaque number and n = 7 for plaque area ) , PruΔgra15 ( n = 20 for plaque number and n = 7 for plaque area ) , PruΔgra15 + GRA15 complemented ( n = 12 for plaque number and n = 7 for plaque area ) .

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E) Plaque assays were performed similarly as described for (a) with Pru WT (n=20 for plaque number and n=7 for plaque area), PruΔgra15 (n=20 for plaque number and n=7 for plaque area), PruΔgra15+GRA15 complemented (n=12 for plaque number and n=7 for plaque area).


F ) Plaque assays were performed similarly as described for ( A ) with RH ( n = 5 for plaque number and n = 3 for plaque area ) and RH + GRA15II ( n = 5 for plaque number and n = 3 for plaque area ) .

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F) Plaque assays were performed similarly as described for (A) with RH (n=5 for plaque number and n=3 for plaque area) and RH+GRA15II (n=5 for plaque number and n=3 for plaque area).


G ) Relative parasite growth was measured as described in ( B ) with Pru WT ( n = 12 ) , PruΔgra15 ( n = 12 ) , PruΔgra15 + GRA15 complemented ( n = 8 ) .

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G) Relative parasite growth was measured as described in (B) with Pru WT (n=12), PruΔgra15 (n=12), PruΔgra15+GRA15 complemented (n=8).


H ) Number of PVs was calculated as described in ( C ) with Pru WT ( n = 3 ) , PruΔgra15 ( n = 3 ) , RH ( n = 3 ) and RH + GRA15II ( n = 3 ) and normalized with the number of host cells in each field .

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H) Number of PVs was calculated as described in (C) with Pru WT (n=3), PruΔgra15 (n=3), RH (n=3) and RH+GRA15II (n=3) and normalized with the number of host cells in each field.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( A ) Total ubiquitin ( n = 3 for RH , n = 5 for Pru and n = 3 for PruΔgra15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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 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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (A) Total ubiquitin (n=3 for RH, n=5 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( B ) K63 - linked ubiquitin ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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 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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (B) K63-linked ubiquitin (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( C ) p62 ( n = 3 for RH , n = 10 for Pru and n = 7 for PruΔgra15 , n = 4 for Pru Δgra15 + GRA15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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 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 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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (C) p62 (n=3 for RH, n=10 for Pru and n=7 for PruΔgra15, n=4 for Pru Δgra15+GRA15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.


HFFs were stimulated with for 24 h with 10U / mL IFNγ or left unstimulated and subsequently infected with RH , Pru or PruΔgra15 parasites for 3 h . The percentage of vacuoles that stained positive for ( D ) NDP52 ( n = 3 for RH , n = 3 for Pru and n = 3 for PruΔgra15 ) , is shown in the left bar diagram . On the right - hand side , a representative fluorescent image is shown for the Toxoplasma Pru strain , which expresses GFP . DNA was stained with Hoechst 33258 . Scale bar is 10 µm . The yellow box inside each representative image is shown as inset pictures with magnification .

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 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

HFFs were stimulated with for 24 h with 10U/mL IFNγ or left unstimulated and subsequently infected with RH, Pru or PruΔgra15 parasites for 3 h. The percentage of vacuoles that stained positive for (D) NDP52 (n=3 for RH, n=3 for Pru and n=3 for PruΔgra15), is shown in the left bar diagram. On the right-hand side, a representative fluorescent image is shown for the Toxoplasma Pru strain, which expresses GFP. DNA was stained with Hoechst 33258. Scale bar is 10 µm. The yellow box inside each representative image is shown as inset pictures with magnification.