H Drug treatment increased calcium transient frequency in 6 - week neurons . Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest ( one - way ANOVA , F4 , 52 = 20 . 28 , Dunnett ' s multiple comparisons test vs untreated KO : Nefiracetam , * * P < 0 . 01 and Z = 2 . 364 ; PHA 543613 , * * * P < 0 . 001 and Z = 3 . 391 ; Acamprosate , * * * P < 0 . 001 and Z = 3 . 153 ; WT83 / Q83X cell lines were used ; N = 10 - 14 neurons / condition ) and the percentage of active neurons ( one - way ANOVA , F4 , 52 = 23 . 11 ; Dunnett ' s multiple comparison test vs untreated KO : Nefiracetam , * * * P < 0 . 001 and Z = 3 . 144 ; PHA 543613 , * * P < 0 . 01 and Z = 2 . 567 ; * * Acamprosate , P < 0 . 01 and Z = 2 . 285 ; N = 10 - 14 neurons / condition ) .

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

H Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10-14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10-14 neurons/condition).


I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2 - KO neurons on MEA ( two - sided unpaired Student ' s t - test compared to KO untreated : Nefiracetam , * P = 0 . 016 and Z = 2 . 418 ; PHA 543613 , * P = 0 . 027 and Z = 2 . 147 ; Acamprosate , P = 0 . 39 and Z = 0 . 477 ; WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 4 - 11 MEA wells / condition ) .

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I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared to KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4-11 MEA wells/condition).


B , C Immunofluorescent staining ( B ) and quantitation ( C ) confirmed the graded decrease in MeCP2 expression from control to 50 % MECP2 mosaic to full MECP2 - KO neurospheres . Scale bar = 50 µm .

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B, C Immunofluorescent staining (B) and quantitation (C) confirmed the graded decrease in MeCP2 expression from control to 50% MECP2 mosaic to full MECP2-KO neurospheres. Scale bar = 50 µm.


D 8 - week MECP2 - KO neurospheres ( N = 44 ) exhibited decreased size ( diameter ; * * P < 0 . 01 ) , but 50 % MECP2 - mosaic neurospheres ( CTR / KO ; N = 44 ) were unchanged from controls ( one - way ANOVA , F2 , 150 = 6 . 03 , P = 0 . 003 ; Dunnett ' s multiple comparisons test ; N = 65 ) . Left : representative brightfield images of neurospheres ; Right : neurosphere size quantification . Scale bar = 200 µm .

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D 8-week MECP2-KO neurospheres (N = 44) exhibited decreased size (diameter; **P < 0.01), but 50% MECP2-mosaic neurospheres (CTR/KO; N = 44) were unchanged from controls (one-way ANOVA, F2,150 = 6.03, P = 0.003; Dunnett's multiple comparisons test; N = 65). Left: representative brightfield images of neurospheres; Right: neurosphere size quantification. Scale bar = 200 µm.


E Calcium imaging analysis showing calcium transients normalized by control ( one - way ANOVA , F2 , 72 = 15 . 62 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs control : MECP2 - mosaic , * * * P < 0 . 001 ; MECP2 - KO , * * * P < 0 . 001 ) . WT83 / Q83X cell lines were used ; N = 44 Q83X neurospheres and 65 WT83 neurospheres .

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E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres.


F 8 - week neurosphere size ( diameter ) remained unchanged by treatment with either drug ( one - way ANOVA , F6 , 344 = 0 . 52 , P = 0 . 79 ; N = 44 Q83X neurospheres and 65 WT83 neurospheres ) .

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F 8-week neurosphere size (diameter) remained unchanged by treatment with either drug (one-way ANOVA, F6,344 = 0.52, P = 0.79; N = 44 Q83X neurospheres and 65 WT83 neurospheres).


G Cell viability was improved or unharmed in treated 8 - week neurospheres ( one - way ANOVA , F9 , 104 = 6 . 81 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : Nefi + PHA 1 µM , * * P < 0 . 01 and Z = 2 . 974 ; Nefi + PHA 10 µM , * * * P < 0 . 001 and Z = 3 . 733 ; N = 8 - 24 neurospheres per condition ) .

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G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition).


H , I Calcium transient frequency ( number of peaks in 10 mins recording ) normalized by CTR / KO untreated ( H ; one - way ANOVA , F9 , 239 = 6 . 18 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : Nefi 1 µM , * P < 0 . 05 and Z = 0 . 780 ; Nefi + PHA 10 µM , * * * P < 0 . 001 and Z = 1 . 972 ; N = 15 - 61 neurospheres per condition ) and calcium transient amplitude normalized by CTR / KO untreated ( I ; one - way ANOVA , F9 , 241 = 1 . 27 , P = 0 . 255 ; N = 16 - 62 neurospheres per condition ) .

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 B-CONTROLLED_VAR 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 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

H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition).


J , K MEA spike frequency heatmap ( J ) and quantification ( K ) normalized by CTR / KO untreated ( one - way ANOVA , F5 , 38 = 1 . 21 , P = 0 . 321 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : PHA , P = 0 . 65 and Z = 1 . 134 ; Nefi , P = 0 . 98 and Z = 0 . 06 ; PHA + Nefi , P = 0 . 86 and Z = 0 . 94 ; N = 7 - 8 wells per condition ) . ' X ' on activity heatmap signifies absence of a neurosphere in that position .

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J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.


B 2 - month old cortical organoid diameter ( one - way ANOVA , F2 , 173 = 91 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs untreated : Nefiracetam , * * * P < 0 . 001 and Z = 5 . 414 ; PHA 543613 , * * * P < 0 . 001 and Z = 5 . 351 ; WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 28 - 90 organoids per condition ) . Left : representative brightfield images of cortical organoids ; Right : cortical organoid size quantification . Scale bar = 200 µm .

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B 2-month old cortical organoid diameter (one-way ANOVA, F2,173 = 91.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, ***P < 0.001 and Z = 5.414; PHA 543613, ***P < 0.001 and Z = 5.351; WT83/Q83X and WT82/K82fs cell lines were used; N = 28-90 organoids per condition). Left: representative brightfield images of cortical organoids; Right: cortical organoid size quantification. Scale bar = 200 µm.


C Ki67 + proliferative area ( Student ' s t - test , t10 = 0 . 36 , P = 0 . 73 , N = 6 each ) . Left : representative immunofluorescence images of progenitor , proliferative , and neuronal markers in cortical organoids ; Right : quantification of Ki67 + proliferation in cortical organoids . Scale bar = 50 µm .

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C Ki67+ proliferative area (Student's t-test, t10 = 0.36, P = 0.73, N = 6 each). Left: representative immunofluorescence images of progenitor, proliferative, and neuronal markers in cortical organoids; Right: quantification of Ki67+ proliferation in cortical organoids. Scale bar = 50 µm.


RNA sequencing ( D , left : heatmap ) and GO analysis ( D , right ) of untreated MECP2 - KO organoids vs Nefiracetam ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated genetic expression in synapse - relevant pathways .

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RNA sequencing (D, left: heatmap) and GO analysis (D, right) of untreated MECP2-KO organoids vs Nefiracetam (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated genetic expression in synapse-relevant pathways.


E , center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles ; whiskers are minimum and maximum log2 normalized expression values .

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E, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.


RNA sequencing ( F , left : heatmap ) and GO analysis ( F , right ) of untreated MECP2 - KO organoids vs PHA 543613 ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated expression of genes in synapse - relevant pathways .

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RNA sequencing (F, left: heatmap) and GO analysis (F, right) of untreated MECP2-KO organoids vs PHA 543613 (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated expression of genes in synapse-relevant pathways.


G , center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles ; whiskers are minimum and maximum log2 normalized expression values .

G, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.


H Synapsin1 + puncta quantitation ( one - way ANOVA , F3 , 34 = 10 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs untreated : Nefiracetam , * * P < 0 . 01 and Z = 2 . 998 ; PHA 543613 , P > 0 . 05 and Z = 1 . 266 ; WT83 / Q83X cell lines were used ; N = 8 - 10 each ) . Left : representative image of Synapsin1 + immunofluorescent staining ; Right : Synapsin1 + puncta quantification . Scale bar = 20 µm .

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H Synapsin1+ puncta quantitation (one-way ANOVA, F3,34 = 10.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, **P < 0.01 and Z = 2.998; PHA 543613, P > 0.05 and Z = 1.266; WT83/Q83X cell lines were used; N = 8-10 each). Left: representative image of Synapsin1+ immunofluorescent staining; Right: Synapsin1+ puncta quantification. Scale bar = 20 µm.


MECP2 - KO and control organoids had proportions of cells positive for the neuronal marker NeuN ( I ; Student ' s t - test , t14 = 0 . 26 , P = 0 . 80 , N = 8 each )

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MECP2-KO and control organoids had proportions of cells positive for the neuronal marker NeuN (I; Student's t-test, t14 = 0.26, P = 0.80, N = 8 each)


J MECP2 - KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 ( J ; one - way ANOVA , F3 , 40 = 2 . 489 , P = 0 . 074 and Z < 1 , WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 8 - 14 organoids each ) . J , left : representative image of CTIP2 + immunofluorescent staining in cortical organoids ; J , right : quantification of CTIP2 + neurons . Scale bar = 50 µm .

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J MECP2-KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 (J; one-way ANOVA, F3,40 = 2.489, P = 0.074 and Z < 1, WT83/Q83X and WT82/K82fs cell lines were used; N = 8-14 organoids each). J, left: representative image of CTIP2+ immunofluorescent staining in cortical organoids; J, right: quantification of CTIP2+ neurons. Scale bar = 50 µm.


L ; left and center : representative population spiking traces of control and MECP2 - KO organoids ; right : quantification of organoid population spiking activity , N = 6 replicates each ) .

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L; left and center: representative population spiking traces of control and MECP2-KO organoids; right: quantification of organoid population spiking activity, N = 6 replicates each).


Drug treatment increased spiking network activity of MECP2 - KO cortical organoids to a level not significantly different from that of control organoids ( M ; one - way ANOVA , F3 , 31 = 3 . 775 , Dunnett ' s multiple comparisons test vs control : untreated KO , * * P < 0 . 01 and Z = 3 . 192 ; Nefiracetam , P = 0 . 98 and Z = 2 . 117 ; PHA 543613 , P = 0 . 12 and Z = 1 . 101 , WT83 / Q83X cell lines were used ; N = 7 - 10 MEA wells per condition ) .

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Drug treatment increased spiking network activity of MECP2-KO cortical organoids to a level not significantly different from that of control organoids (M; one-way ANOVA, F3,31 = 3.775, Dunnett's multiple comparisons test vs control: untreated KO, **P < 0.01 and Z = 3.192; Nefiracetam, P = 0.98 and Z = 2.117; PHA 543613, P = 0.12 and Z = 1.101, WT83/Q83X cell lines were used; N = 7-10 MEA wells per condition).


Quantification of fibroblast density ( number of PDGFRαH2BEGFP + cells , right ; n = 3 biological replicates per time point ) and dermis volume ( left ) with age ( n = 7 E12 . 5 ; n = 8 P0 , P6 ; n = 9 E16 . 5 , E17 . 5 , P4 ; n = 10 P10 , P50 ; n = 11 E10 . 5 ; n = 12 E18 . 5 , P2 , P21 ; n = 20 P12 biological replicates ) .

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Quantification of fibroblast density (number of PDGFRαH2BEGFP+ cells, right; n=3 biological replicates per time point) and dermis volume (left) with age (n=7 E12.5; n=8 P0, P6; n=9 E16.5, E17.5, P4; n=10 P10, P50; n=11 E10.5; n=12 E18.5, P2, P21; n=20 P12 biological replicates).


Changes in fibroblast proliferation and collagen deposition during dermal development in mice . Immunofluorescence staining for Ki67 ( red ) of PDGFRαH2BEGFP ( green ) back skin at indicated developmental time points ( upper panel ) . Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points ( lower panel ) .

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Changes in fibroblast proliferation and collagen deposition during dermal development in mice. Immunofluorescence staining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin at indicated developmental time points (upper panel). Polarised light images of Picrosirius red stained back skin sections shown in binary images at indicated time points (lower panel).


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Percentage of PDGFRαH2BEGFP+ cells in G1 cell cycle phase with age (n=1 E12.5; n=2 P2, P10; n=3 E16.5, E17, P0; n=4 E10.5; n=5 E17.5, E18.5, P50; n=7 P4 biological replicates) fitted using an exponential model.


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Quantification of fibroblast proliferation (left; n= 4 E16.5, P10; n= 3 E10.5, E18.5, P2, P21, P44, P50; n= 2 E17.5, P0 biological replicates) and collagen density (right; n= 4 E16.5, P2, P6, P10, P14, P16, P21, P50; n= 3 E10.5, E18.5, P2, P21, P44, P50 biological replicates) with age.


Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus - hGem ( green ) expressing cells are in S / G2 / M phase and mCherry - hCdt1 ( red ) positive cells are arrested in G1 phase . Percentage of labelled cells in S / G2 / M cell cycle phase in the upper and lower dermis at P0 ( n = 3 biological replicates ) and P2 ( n = 4 biological replicates ) .

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Immunofluorescence image of R26Fucci2a x Dermo1Cre back skin at P2 where mVenus-hGem (green) expressing cells are in S/G2/M phase and mCherry-hCdt1 (red) positive cells are arrested in G1 phase. Percentage of labelled cells in S/G2/M cell cycle phase in the upper and lower dermis at P0 (n=3 biological replicates) and P2 (n=4 biological replicates).


Changes in dermal cell proliferation and collagen deposition in human skin . ( G ) Immunofluorescence staining for Vimentin ( red ) and Ki67 ( green ) of back skin at indicated time points ( upper panel ) . Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points ( lower panel ) . ( H ) Quantification of proliferating cells in the upper and lower dermis ( n = 3 biological replicates per time point ) . ( I ) Quantification of dermal cell proliferation ( Ki67 + ) ( left ) and collagen density ( right ) with age ( n = 3 biological replicates per time point ) . Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent , non - proliferative state before collagen was efficiently deposited . Data shown are means ± s . d . Nuclei were labelled with DAPI ( blue in B , E , G ) . Scale bars , 100µm . wk , week .

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Changes in dermal cell proliferation and collagen deposition in human skin. (G) Immunofluorescence staining for Vimentin (red) and Ki67 (green) of back skin at indicated time points (upper panel). Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points (lower panel). (H) Quantification of proliferating cells in the upper and lower dermis (n=3 biological replicates per time point). (I) Quantification of dermal cell proliferation (Ki67+) (left) and collagen density (right) with age (n= 3 biological replicates per time point). Note that cells in upper human dermis were more proliferative than cells in the lower dermis at all developmental time points analysed and that fibroblasts entered a quiescent, non-proliferative state before collagen was efficiently deposited. Data shown are means ±s.d. Nuclei were labelled with DAPI (blue in B,E,G). Scale bars, 100µm. wk, week.


Two independent primary mouse fibroblast isolations FB1 ( black ) and FB2 ( grey ) were cultured in ( triangle ) and outside ( circle ) collagen gels for up to 5 days . Cell number was measured by CellTiterGlo assay at indicated time points ( n = 3 technical replicates of one representative experiment repeated 3 times ) .

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Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in (triangle) and outside (circle) collagen gels for up to 5 days. Cell number was measured by CellTiterGlo assay at indicated time points (n= 3 technical replicates of one representative experiment repeated 3 times).


Two independent primary mouse fibroblast isolations FB1 ( black ) and FB2 ( grey ) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days ( triangle ) . Controls were kept on TCP for 3 days before plating ( circle ) . Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown ( n = 3 technical replicates ) .

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

Two independent primary mouse fibroblast isolations FB1 (black) and FB2 (grey) were cultured in collagen gels for 3 days before being released by collagenase treatment and plated on TCP for 4 days (triangle). Controls were kept on TCP for 3 days before plating (circle). Fold increase in confluency 24 hours after plating of collagen gel released cells and cells kept on TCP is shown (n= 3 technical replicates).


Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis . Immunofluorescence staining for Krt14 ( green ) , Ki67 ( red ) and Vimentin ( white ) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding . Quantification of Ki67 positive fibroblasts at indicated time point after seeding ( n = 8 section per condition of one representative experiment repeated two times ) .

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

Repopulation of DED organotypic cultures recapitulates adult dermal homeostasis. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures with and without fibroblasts at indicated time points after seeding. Quantification of Ki67 positive fibroblasts at indicated time point after seeding (n= 8 section per condition of one representative experiment repeated two times).


ECM degradation in DED organotypic cultures by collagenase treatment . Immunofluorescence staining for Krt14 ( green ) , Ki67 ( red ) and Vimentin ( white ) of DED organotypic cultures at indicated time points after collagenase treatment . Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment ( n = 8 section per condition of one representative experiment repeated two times ) . Note that fibroblasts around the injection sides start to proliferate after 48 hours . Nuclei were labelled with DAPI ( blue ) ( D , F ) . Scale bars , 100µm ( D , F ) . Data shown are means ± s . d . Fb , fibroblasts ; PD , post digestion ; TCP , tissue culture plastic ; h , hours .

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

ECM degradation in DED organotypic cultures by collagenase treatment. Immunofluorescence staining for Krt14 (green), Ki67 (red) and Vimentin (white) of DED organotypic cultures at indicated time points after collagenase treatment. Quantification of Ki67 positive fibroblasts at indicated time point after collagenase treatment (n= 8 section per condition of one representative experiment repeated two times). Note that fibroblasts around the injection sides start to proliferate after 48 hours. Nuclei were labelled with DAPI (blue) (D,F). Scale bars, 100µm (D,F). Data shown are means ± s.d. Fb, fibroblasts; PD, post digestion; TCP, tissue culture plastic; h, hours.


Resulting simulation of the system of equations


Live imaging of adult PDGFRαH2BEGFP mouse back skin . Representative time - lapse images of adult PDGFRαH2BEGFP ( green ) dermis with collagen shown as second harmonic generation ( SHG ) in purple at indicated imaging time points . Scale bar , 100 µm .

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

Live imaging of adult PDGFRαH2BEGFP mouse back skin. Representative time-lapse images of adult PDGFRαH2BEGFP (green) dermis with collagen shown as second harmonic generation (SHG) in purple at indicated imaging time points. Scale bar, 100 µm.


In silico lineage tracing of fibroblasts . ( E ) Labelling of fibroblasts close to the epidermis at indicated MCS . ( F ) Labelling of fibroblasts in the middle of the dermis at indicated MCS . ( G ) Labelling of fibroblasts close to the inside of the body at indicated MCS .

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

In silico lineage tracing of fibroblasts. (E) Labelling of fibroblasts close to the epidermis at indicated MCS. (F) Labelling of fibroblasts in the middle of the dermis at indicated MCS. (G) Labelling of fibroblasts close to the inside of the body at indicated MCS.


Fibroblast activation during wound repair . Immunofluorescence staining for α − sma ( red ) of PDGFRαH2BEGFP ( green ) wound sites at indicated post wounding ( PW ) times Nuclei were labelled with DAPI ( blue )

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

Fibroblast activation during wound repair. Immunofluorescence staining for α−sma (red) of PDGFRαH2BEGFP (green) wound sites at indicated post wounding (PW) times Nuclei were labelled with DAPI (blue)


Fibroblast activation during wound repair . quantification of α − sma fluorescence intensity in pixel per area inside the upper and lower wound bed ( n = 4 PW0 , PW2 , PW7 , PW21 ; n = 3 PW10 , PW14 ; n = 2 PW4 biological replicates )

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

Fibroblast activation during wound repair. quantification of α−sma fluorescence intensity in pixel per area inside the upper and lower wound bed (n= 4 PW0, PW2, PW7, PW21; n= 3 PW10, PW14; n= 2 PW4 biological replicates)


Changes in fibroblast density , proliferation and collagen deposition during wound repair . Immunostaining for Ki67 ( red ) of PDGFRαH2BEGFP ( green ) back skin sections at indicated wound healing time points . Nuclei were labelled with

Changes in fibroblast density, proliferation and collagen deposition during wound repair. Immunostaining for Ki67 (red) of PDGFRαH2BEGFP (green) back skin sections at indicated wound healing time points. Nuclei were labelled with DAPI


Changes in fibroblast density , proliferation and collagen deposition during wound repair . Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) .

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

Changes in fibroblast density, proliferation and collagen deposition during wound repair. Quantification of fibroblast proliferation in the upper and lower dermis inside the wound bed over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Quantification of fibroblast density during wound repair inside and outside the wound bed ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) .

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

Quantification of fibroblast density during wound repair inside and outside the wound bed (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding .

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

Polarised light image of Picrosirius red stained back skin section shown as binary image at indicated time points after wounding.


Quantification of proliferating ( Ki67 positive ) keratinocytes and fibroblasts during wound repair over time ( n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) .

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

Quantification of proliferating (Ki67 positive) keratinocytes and fibroblasts during wound repair over time (n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates).


Quantification of fibroblast proliferation ( left ; n = 4 PW0 , PW2 , PW21 ; n = 3 PW4 , PW10 ; n = 2 PW7 , PW14 biological replicates ) and collagen density ( right ; n = 4 PW0 , PW2 ; n = 3 PW4 , PW7 , PW10 , PW14 ; n = 2 PW21 biological replicates ) inside ( A ) and outside ( B ) the wound bed over time .

O O O O O O O O O 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

Quantification of fibroblast proliferation (left; n= 4 PW0, PW2, PW21; n= 3 PW4, PW10; n= 2 PW7, PW14 biological replicates) and collagen density (right; n= 4 PW0, PW2; n= 3 PW4, PW7, PW10, PW14; n= 2 PW21 biological replicates) inside (A) and outside (B) the wound bed over time.


Temporal evolution of proliferating fibroblasts ( PF ) and dermal ECM density over time .

Temporal evolution of proliferating fibroblasts (PF) and dermal ECM density over time.


Live imaging of adult PDGFRαH2BEGFP back skin during wound healing . Representative wound bed Z - stack stitched maximum projections of the entire wound bed ( upper panel ) showing fibroblasts ( green ) and collagen with second harmonic generation ( SHG ) in purple at indicated time points after wounding and before live imaging . Dotted line indicates wound edge and boxed area indicates the position of the magnified area below .

O O O O O O O O O O O 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

Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative wound bed Z-stack stitched maximum projections of the entire wound bed (upper panel) showing fibroblasts (green) and collagen with second harmonic generation (SHG) in purple at indicated time points after wounding and before live imaging. Dotted line indicates wound edge and boxed area indicates the position of the magnified area below.


Live imaging of adult PDGFRαH2BEGFP back skin during wound healing . Representative time - lapse images of adult wound bed at PW4 , where Z - directional movement is visualized by pseudo colouring the fibroblast nuclei ( red , movement towards the basement membrane ; blue , movement away from the basement membrane ) .

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

Live imaging of adult PDGFRαH2BEGFP back skin during wound healing. Representative time-lapse images of adult wound bed at PW4, where Z-directional movement is visualized by pseudo colouring the fibroblast nuclei (red, movement towards the basement membrane; blue, movement away from the basement membrane).


In vivo lineage tracing of upper ( Blimp1Cre ) and lower ( Dlk1CreER ) dermal fibroblasts during wound healing . Immunofluorescence image of Blimp1Cre x Confetti ( upper panels ) and Dlk1CreER x Confetti ( lower panel ) back skin inside and outside the wound bed at 10 days post wounding . Colours show YFP ( green ) , RFP ( red ) and CFP ( blue ) with bright field image overlaid . ( G ) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding ( n = 400 clones of 4 biological replicates ) . ( H ) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding ( n = 300 clones of 2 biological replicates ) .

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

In vivo lineage tracing of upper (Blimp1Cre) and lower (Dlk1CreER) dermal fibroblasts during wound healing. Immunofluorescence image of Blimp1Cre x Confetti (upper panels) and Dlk1CreER x Confetti (lower panel) back skin inside and outside the wound bed at 10 days post wounding. Colours show YFP (green), RFP (red) and CFP (blue) with bright field image overlaid. (G) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n= 400 clones of 4 biological replicates). (H) Clone size quantification of Blimp1Cre labelled fibroblasts inside and outside the wound bed at 10 days post wounding (n=300 clones of 2 biological replicates).


B . Scatter plot illustrates individual mouse TSPO - PET values derived from a whole brain volume of interest . 8 - 15 female mice per group at an average age of 11 . 1 ± 1 . 6 months ( Grn - / - ( n = 8 ) , Trem2 - / - ( n = 9 ) , Double - / - ( n = 10 ) , WT ( n = 15 ) ) . Data represent mean ± SD . For statistical analysis one - way ANOVA with Tukey post hoc test was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatter plot illustrates individual mouse TSPO-PET values derived from a whole brain volume of interest. 8-15 female mice per group at an average age of 11.1 ± 1.6 months (Grn-/- (n = 8), Trem2-/- (n = 9), Double-/- (n = 10), WT (n= 15)). Data represent mean ± SD. For statistical analysis one-way ANOVA with Tukey post hoc test was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ns, not significant.


B . Volcano plot presentation of the differently expressed transcripts in FCRLS - and CD11b - positive Grn - / - ( n = 6 ) in comparison to WT ( n = 6 ) microglia isolated from 6 - month - old male mice . 35 out of 65 analyzed genes are significantly changed more than 20 % , with 22 genes upregulated ( purple ) and 13 genes downregulated ( blue ) . Data information : For statistical analysis the unpaired , two - tailed student ` s t - test was performed Statistical significance was set at * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 000 ; ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Volcano plot presentation of the differently expressed transcripts in FCRLS- and CD11b-positive Grn-/- (n=6) in comparison to WT (n=6) microglia isolated from 6-month-old male mice. 35 out of 65 analyzed genes are significantly changed more than 20%, with 22 genes upregulated (purple) and 13 genes downregulated (blue). Data information: For statistical analysis the unpaired, two-tailed student`s t-test was performed Statistical significance was set at **, p < 0.01; ***, p < 0.001; ****, p < 0.000; ns, not significant.


B . AlphaLISA - mediated quantification of p - Syk in human macrophages with a dose titration treatment of Ab1 and Ab2 with or without liposomes for 5 min . IC50 and maximal inhibition ( max ) are indicated by a dotted line . Data represent the mean ± SEM ( n = 3 independent experiments ) . Data information : Data represent mean ± SEM . For statistical analysis of patient samples in comparison to HC , the unpaired , two - tailed student ` s t - test was performed . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; ns , not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. AlphaLISA-mediated quantification of p-Syk in human macrophages with a dose titration treatment of Ab1 and Ab2 with or without liposomes for 5 min. IC50 and maximal inhibition (max) are indicated by a dotted line. Data represent the mean ± SEM (n=3 independent experiments). Data information: Data represent mean ± SEM. For statistical analysis of patient samples in comparison to HC, the unpaired, two-tailed student`s t-test was performed. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant


G . Western blot of TREM2 in lysates and media of cultured human macrophages isolated from GRN - FTLD # 1 and HC # 1 upon treatment with Ab1 and Ab2 . An isotype antibody was used as a negative control . ADAM protease inhibition ( GM ) does not further increase mTREM2 levels in GRN - FTLD patients . Equal amounts of protein were loaded . GAPDH was used as loading control .

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

G. Western blot of TREM2 in lysates and media of cultured human macrophages isolated from GRN-FTLD #1 and HC #1 upon treatment with Ab1 and Ab2. An isotype antibody was used as a negative control. ADAM protease inhibition (GM) does not further increase mTREM2 levels in GRN-FTLD patients. Equal amounts of protein were loaded. GAPDH was used as loading control.


A . Western blot of PGRN in whole - cell lysates of WT and GRN - / - hiMGL . Actin was used as loading control . B . Western blot of TREM2 in whole - cell lysates of WT and GRN - / - hiMGL . Mature ( mTREM ) and immature TREM2 ( imTREM2 ) are indicated . Actin was used as loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot of PGRN in whole-cell lysates of WT and GRN-/- hiMGL. Actin was used as loading control. B. Western blot of TREM2 in whole-cell lysates of WT and GRN-/- hiMGL. Mature (mTREM) and immature TREM2 (imTREM2) are indicated. Actin was used as loading control.


E . ELISA - mediated quantification of sTREM2 in conditioned media of GRN - / - hiMGL upon treatment with Ab1 and Ab2 ( 20 μg / ml , 40 μg / ml ) ( n = 3 , biological replicates ) . An isotype antibody ( 10 μg / ml ) was used as a negative control . F . AlphaLISA - mediated quantification of p - Syk levels in GRN - / - hiMGL upon treatment with Ab1 and Ab2 ( 5 μg / ml , 10 μg / ml ) with liposomes ( 1 mg / ml ) for 5 min . ( n = 8 , biological replicates ) . Data represent mean ± SEM . For statistical analysis in E and F one - way ANOVA with Dunnett ` s post hoc test , was used to compare untreated , Ab1 , and Ab2 ( 20 µg / ml and 40µg / ml ) conditions to the isotype treated 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. ELISA-mediated quantification of sTREM2 in conditioned media of GRN-/- hiMGL upon treatment with Ab1 and Ab2 (20 μg/ml, 40 μg/ml) (n = 3, biological replicates). An isotype antibody (10 μg/ml) was used as a negative control. F. AlphaLISA-mediated quantification of p-Syk levels in GRN-/- hiMGL upon treatment with Ab1 and Ab2 (5 μg/ml, 10 μg/ml) with liposomes (1 mg/ml) for 5 min. (n = 8, biological replicates). Data represent mean ± SEM. For statistical analysis in E and F one-way ANOVA with Dunnett`s post hoc test, was used to compare untreated, Ab1, and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition.


H . Uptake assay for fluorescent myelin . GRN - / - hiMGL phagocytose significantly more myelin as compare to WT hiMGL . This is reversed upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 ( n = 4 , biological replicates ) .

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

H. Uptake assay for fluorescent myelin. GRN-/- hiMGL phagocytose significantly more myelin as compare to WT hiMGL. This is reversed upon treatment with TREM2 antagonistic antibodies Ab1 and Ab2 (n=4, biological replicates).


E . Transcript levels of DAM gene transcripts significantly altered in GRN - / - hiMGL , treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A , normalized to the mean of the WT hiMGL samples ( n = 4 , biological replicates ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Dunnett ` s post hoc test was used to compare Ab1 and Ab2 ( 20 µg / ml and 40µg / ml ) conditions to the isotype treated condition , Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , * * * * * , p < 0 . 00001 , and ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transcript levels of DAM gene transcripts significantly altered in GRN-/- hiMGL, treated with Ab1 or Ab2 in comparison to isotype treatment from the data set in A, normalized to the mean of the WT hiMGL samples (n=4, biological replicates). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Dunnett`s post hoc test was used to compare Ab1 and Ab2 (20 µg/ml and 40µg/ml) conditions to the isotype treated condition, Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, *****, p < 0.00001, and ns, not significant.


A . Western blot of CatD in total brain lysates from 14 - month - old female WT , Grn - / - , Trem2 - / - , and Double - / - mice . CatD maturation variants are indicated ( sc : single chain ; hc : heavy chain ; n = 3 ) . B . , C . Quantification of CatD variants in A normalized to WT ( n = 3 per genotype ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ' s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Western blot of CatD in total brain lysates from 14-month-old female WT, Grn-/-, Trem2-/-, and Double-/- mice. CatD maturation variants are indicated (sc: single chain; hc: heavy chain; n=3). B., C. Quantification of CatD variants in A normalized to WT (n=3 per genotype). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


D . , E . Catalytic activity of CatD in brain lysates from female 6 - month - old ( n = 4 per genotype ) ( D ) or 14 - month - old ( n = 3 per genotype ) ( E ) Grn - / - , Trem2 - / - , Double - / - mice normalized to WT . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ' s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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., E. Catalytic activity of CatD in brain lysates from female 6-month-old (n=4 per genotype) (D) or 14-month-old (n=3 per genotype) (E) Grn-/-, Trem2-/-, Double-/- mice normalized to WT. Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


F . Immunohistochemical analysis of lipofuscin ( green ) in coronal brain sections . Representative images of thalamus are shown . Scalebars = 50 μm . G . Quantification of lipofuscin autofluorescence . Five images per mouse were taken , means were normalized to WT samples ( n = 3 per genotype , female ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ' s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

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 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 O O O

F. Immunohistochemical analysis of lipofuscin (green) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm. G. Quantification of lipofuscin autofluorescence. Five images per mouse were taken, means were normalized to WT samples (n=3 per genotype, female). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey's post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


A - D . Volcano plot presentation of lipids and metabolites upregulated ( purple ) or downregulated ( blue ) in total brain homogenates from 6 - month - old male Grn - / - ( A , n = 5 ) , Trem2 - / - ( B , n = 5 ) , Double - / - ( C , n = 5 ) mice in comparison to WT ( n = 4 ) , and Double - / - in comparison to Grn - / - mice ( D ) . Counts for each sample were normalized to the mean value of WT followed by a log2 transformation ( n = 4 - 5 per genotype ) . Analyte values were adjusted with an FDR < 10 % to exclude type I errors in null hypothesis testing . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ` s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Volcano plot presentation of lipids and metabolites upregulated (purple) or downregulated (blue) in total brain homogenates from 6-month-old male Grn-/- (A, n=5), Trem2-/- (B, n=5), Double-/- (C, n=5) mice in comparison to WT (n=4), and Double-/- in comparison to Grn-/- mice (D). Counts for each sample were normalized to the mean value of WT followed by a log2 transformation (n=4-5 per genotype). Analyte values were adjusted with an FDR <10% to exclude type I errors in null hypothesis testing. Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey`s post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


E - G . Abundance of BMP species and glucosylsphingosine ( GlcSph ) in total brain of 6 - month - old Grn - / - , Trem2 - / - , Double - / - and WT mice ( n = 4 - 5 per genotype ) . H . Glucocerebrosidase ( GCase ) activity in whole brain lysates from 6 - month - old male Grn - / - , Trem2 - / - , Double - / - and WT mice . The linear increase of fluorescence signal was measured and then normalized to WT mice ( n = 3 - 6 per genotype ) . Data information : Data represent mean ± SEM . For statistical analysis one - way ANOVA with Tukey ` s post hoc test of Grn - / - , Trem2 - / - and Double - / - was used . Statistical significance was set at * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 , and ns , not significant .

O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G. Abundance of BMP species and glucosylsphingosine (GlcSph) in total brain of 6-month-old Grn-/-, Trem2-/-, Double-/- and WT mice (n=4-5 per genotype). H. Glucocerebrosidase (GCase) activity in whole brain lysates from 6-month-old male Grn-/-, Trem2-/-, Double-/- and WT mice. The linear increase of fluorescence signal was measured and then normalized to WT mice (n=3-6 per genotype). Data information: Data represent mean ± SEM. For statistical analysis one-way ANOVA with Tukey`s post hoc test of Grn-/-, Trem2-/- and Double-/- was used. Statistical significance was set at ***, p < 0.001; ****, p < 0.0001, and ns, not significant.


A . Immunohistochemical analysis of synaptophysin ( SPH , pink ) and VGAT ( yellow ) in coronal brain sections . Representative images of thalamus are shown . Scalebars = 50 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

A. Immunohistochemical analysis of synaptophysin (SPH, pink) and VGAT (yellow) in coronal brain sections. Representative images of thalamus are shown. Scalebars = 50 μm.


F . Morphological analysis of cortical microglia . Representative maximum intensity projections of confocal z - stack images showing IBA1 + microglial cells of female WT , Grn - / - , Trem2 - / - and Double - / - mice ( scalebar = 50 µm ) . Arrows point to individual microglia , which are shown as three - dimensional reconstruction , scalebar = 10 µm .

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

F. Morphological analysis of cortical microglia. Representative maximum intensity projections of confocal z-stack images showing IBA1+ microglial cells of female WT, Grn-/-, Trem2-/- and Double-/- mice (scalebar = 50 µm). Arrows point to individual microglia, which are shown as three-dimensional reconstruction, scalebar = 10 µm.


A . Immunoassay - based quantification of neurofilament - light chain ( NfL ) protein levels in CSF of 6 - month - old Grn - / - ( n = 4 ) , Trem2 - / - ( n = 4 ) , Double - / - ( n = 4 ) and WT ( n = 5 ) male mice . B . Immunoassay - based quantification of NfL levels in CSF of 14 - month - old Grn - / - , Trem2 - / - , Double - / - and WT female mice ( n = 3 per genotype ) . Data information : Data in A , B represent mean ± SEM . For statistical analysis in A - B two - way ANOVA with Dunnett ` s post hoc test was used the

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Immunoassay-based quantification of neurofilament-light chain (NfL) protein levels in CSF of 6-month-old Grn-/- (n=4), Trem2-/-(n=4), Double-/- (n=4) and WT (n=5) male mice. B. Immunoassay-based quantification of NfL levels in CSF of 14-month-old Grn-/-, Trem2-/-, Double-/- and WT female mice (n=3 per genotype). Data information: Data in A, B represent mean ± SEM. For statistical analysis in A-B two-way ANOVA with Dunnett`s post hoc test was used the unpaired


D . Transcript levels of all significantly changed genes in Double - / - vs . Grn - / - brain mRNA of 6 - month - old and 14 - month - old mice analyzed in C . Grin3b was under detection limit in the 14 - month - old cohort . Transcript expression is normalized to the mean of the WT cohort . Data in D represent mean ± SEM . in D two - tailed student ` s t - test was performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Transcript levels of all significantly changed genes in Double-/- vs. Grn-/- brain mRNA of 6-month-old and 14-month-old mice analyzed in C. Grin3b was under detection limit in the 14-month-old cohort. Transcript expression is normalized to the mean of the WT cohort. Data in D represent mean ± SEM. in D two-tailed student`s t-test was performed.


F . Bar graph illustrates individual FDG - PET values derived from a whole brain volume of interest . Data represent mean ± SD . 8 - 15 female mice per group at an average age of 10 . 7 ± 1 . 5 months ( Grn - / - n = 8 , Trem2 - / - n = 10 , Double - / - n = 10 , WT n = 15 ) were used . Data in F two - tailed student ` s t - test was performed .

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

F. Bar graph illustrates individual FDG-PET values derived from a whole brain volume of interest. Data represent mean ± SD. 8-15 female mice per group at an average age of 10.7 ± 1.5 months (Grn-/- n = 8, Trem2-/- n = 10, Double-/- n = 10, WT n = 15) were used. Data in F two-tailed student`s t-test was performed.


( A ) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h , detected by fluorescent microplate reader and confocal microscopy , respectively . Scale bar 20 µm .

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

(A) Summary and representative analysis of NET formation of pPMNs stimulated by 50 nM PMA for 3 h, detected by fluorescent microplate reader and confocal microscopy, respectively. Scale bar 20 µm.


( B ) Representative confocal microscopy images of pPMNs that were treated without ( control ) or with 50 nM PMA for 3 h , and stained for lamin B and DNA as described in panel B . The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope . Scale bar 10 µm .

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 B-MEASURED_VAR 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

(B) Representative confocal microscopy images of pPMNs that were treated without (control) or with 50 nM PMA for 3 h, and stained for lamin B and DNA as described in panel B. The light blue arrows indicate the release of decondensed DNA associated with lamin B molecules from ruptured nuclear envelope. Scale bar 10 µm.


( D ) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice . Vinculin served as loading control .

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(D) Representative and summary of immunoblots of lamin B of bone marrow neutrophils from WT and Lmnb1TG mice. Vinculin served as loading control.


( E ) Summary analysis of PAF - induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h , then fixed by 2 % PFA and stained by Sytox Green , following by detection with fluorescent microplate reader .

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 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

(E) Summary analysis of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, then fixed by 2% PFA and stained by Sytox Green, following by detection with fluorescent microplate reader.


( F ) The endpoint analysis of NET - DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without ( control ) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at 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 .

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 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 B-MEASURED_VAR 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

(F) The endpoint analysis of NET-DNA release index was detected by coincubation of primary mouse peritoneal mPMNs from WT and Lmnb1TG mice without (control) or with 10 µM PAF in medium containing 1 μM Sytox Green dye with recording by a microplate reader at 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.


( G , H ) Representative images ( G ) and summary analysis ( H ) of PAF - induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h , and stained with both cell permeable SYTO Red and cell impermeable Sytox Green , 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 . ( G ) Scale bars , 40 μm .

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 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

(G,H) Representative images (G) and summary analysis (H) of PAF-induced NET formation in mPMNs from WT vs Lmnb1TG mice that were stimulated without or with 10 µM PAF for 3 h, and stained with both cell permeable SYTO Red and cell impermeable Sytox Green, 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. (G) Scale bars, 40 μm.


( A , B ) Representative and summary of immunoblots of full - length lanes of lamin B in human dPMNs that were treated with PMA for 0 , 0 . 5 , 1 , 2 , 3 h during NET formation , or apoptotic dPMNs that were induced by PMA for longer term ( 12 h ) treatment ( A ) . β - actin served as loading

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

(A,B) Representative and summary of immunoblots of full-length lanes of lamin B in human dPMNs that were treated with PMA for 0, 0.5, 1, 2, 3 h during NET formation, or apoptotic dPMNs that were induced by PMA for longer term (12 h) treatment (A). β-actin served as loading control


( C , D ) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0 , 0 . 5 , 1 , 2 , 3 h during NET formation . β - actin served as loading

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

(C,D) Representative and summary immunoblots of lamin B in human dPMNs that were treated with PAF for 0, 0.5, 1, 2, 3 h during NET formation. β-actin served as loading control


( E ) Representative confocal microscopy image of a group of pPMNs with NET formation ( enclosed by a light blue trapezoid ) and their extracellular NETs ( indicated by a purple rectangle square ) released by these cells , as well as the immunoblot analysis of un - cleaved lamin B and cleaved histone H3 with the whole cell lysates , as well as immunoblot analysis of un - cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment . The anti - human lamin B was used in all immunoblots confocal image ( E ) , and the latter was further detected by FITC - labeled 2nd Ab , scale bar , 20 μm .

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

(E) Representative confocal microscopy image of a group of pPMNs with NET formation (enclosed by a light blue trapezoid) and their extracellular NETs (indicated by a purple rectangle square) released by these cells, as well as the immunoblot analysis of un-cleaved lamin B and cleaved histone H3 with the whole cell lysates, as well as immunoblot analysis of un-cleaved lamin B with lysate of the NETs isolated from conditioned medium of pPMNs with NET formation that were induced by 3 h PMA treatment. The anti-human lamin B was used in all immunoblots confocal image (E), and the latter was further detected by FITC-labeled 2nd Ab, scale bar, 20 μm.


( F ) Representative immunoblots with full - length lanes for lamin B analysis , and pro - caspase - 3 and its activated form caspase - 3 in human dPMNs with NET formation that were treated with PMA for 0 , 0 . 5 , 1 , 2 , 3 h . Anti - human caspase - 3 was used ( F , G ) , and β - actin served as loading

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

(F) Representative immunoblots with full-length lanes for lamin B analysis, and pro-caspase-3 and its activated form caspase-3 in human dPMNs with NET formation that were treated with PMA for 0, 0.5, 1, 2, 3 h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control


( G ) Representative immunoblots display full - length lamin B ( 69 kDa ) and their cleaved fragments ( 45 and 25 kDa ) , and pro - caspase - 3 and its activated form caspase - 3 in the apoptotic human dPMNs that were treated with PMA for 12 , 24h . Anti - human caspase - 3 was used ( F , G ) , and β - actin served as loading

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunoblots display full-length lamin B (69 kDa) and their cleaved fragments (45 and 25 kDa), and pro-caspase-3 and its activated form caspase-3 in the apoptotic human dPMNs that were treated with PMA for 12, 24h. Anti-human caspase-3 was used (F,G), and β-actin served as loading control


( A ) Representative images for the time course of PKCα nuclear translocation , subsequent nuclear envelope rupture , and DNA release in human dPMNs exposed to 50 nM PMA for 5 , 15 , 30 , 60 , and 180 min , then stained concomitantly for DNA ( DAPI ) , nuclear lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , and PKCα ( primary anti - human PKCα , and PE - labeled secondary antibody ) , following by confocal fluorescent microscopy analysis . White - empty arrows indicate cytoplasmic distribution of PKCα at 5 , 15 , and 30 min , yellow arrows indicate site of discontinuity / rupture of nuclear envelope at 60 min , light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min ( A ) . Scale bars , 20 μm . The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining .

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-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 O O O O O O O O O O O O O O O O O O

(A) Representative images for the time course of PKCα nuclear translocation, subsequent nuclear envelope rupture, and DNA release in human dPMNs exposed to 50 nM PMA for 5, 15, 30, 60, and 180 min, then stained concomitantly for DNA (DAPI), nuclear lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and PKCα (primary anti-human PKCα, and PE-labeled secondary antibody), following by confocal fluorescent microscopy analysis. White-empty arrows indicate cytoplasmic distribution of PKCα at 5, 15, and 30 min, yellow arrows indicate site of discontinuity/rupture of nuclear envelope at 60 min, light blue arrows display the sites of nuclear envelope rupture and chromatin release at 180 min (A). Scale bars, 20 μm. The time course started 5 min after PMA stimulation in order to allow the adherence of neutrophils on the bottom of dish for immunocytostaining.


( B ) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti - lamin B , and FITC - labeled secondary antibody .

O O O O O O O O O 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) Summary analysis of the nuclear envelope continuity was analyzed based on staining of the nuclear envelope with primary anti-lamin B, and FITC-labeled secondary antibody.


( C Representative immunoblots of total PKCα and p - PKCα in primary human pPMNs that were treated by PMA ( C ) for 0 , 0 . 5 , 1 , 2 , 3 h .

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

(C Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PMA (C) for 0, 0.5, 1, 2, 3 h.


D ) Representative immunoblots of total PKCα and p - PKCα in primary human pPMNs that were treated by PAF ( D ) for 0 , 0 . 5 , 1 , 2 , 3 h .

D) Representative immunoblots of total PKCα and p-PKCα in primary human pPMNs that were treated by PAF (D) for 0, 0.5, 1, 2, 3 h.


( A ) Representative immunoblots of p - PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h .

O O O O O 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

(A) Representative immunoblots of p-PKCα and total PKCα of isolated nuclei and the corresponding status of lamin B in human dPMNs that were treated with PMA for 0 or 2 h.


( B ) Representative sectional images of nuclei of two neutrophils treated by PMA , stained for lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , p - PKCα ( primary anti - human p - PKCαS657 , and PE - labeled secondary antibody ) , and nuclear DNA ( DAPI ) . The accumulation of p - PKCα ( red arrow pointed ) at the site of ruptured / interrupted ( green arrow ) nuclear lamin B , and swollen / de - condensed DNA ( blue arrow ) were indicated ( b1 ) . A neutrophil ( white empty arrow ) with NET formation that contained the mixture of DNA with p - PKCα and lamin B ( white arrow ) was also demonstrated ( b2 ) . Scale bars , 10 µm .

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

(B) Representative sectional images of nuclei of two neutrophils treated by PMA, stained for lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), p-PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody), and nuclear DNA (DAPI). The accumulation of p-PKCα (red arrow pointed) at the site of ruptured/interrupted (green arrow) nuclear lamin B, and swollen/de-condensed DNA (blue arrow) were indicated (b1). A neutrophil (white empty arrow) with NET formation that contained the mixture of DNA with p-PKCα and lamin B (white arrow) was also demonstrated (b2). Scale bars, 10 µm.


( C ) Immunoblot images of lamin B , then stripped and probed for p - PKCα , of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA .

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(C) Immunoblot images of lamin B, then stripped and probed for p-PKCα, of the homogenates of NETs isolated from conditioned medium of primary human pPMNs with NET formation that were induced by PMA.


( D , E ) Representative and summary immunoblot ( IB ) detection of phospho - lamin B and total lamin B with the lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from human dPMNs that were treated either by PMA ( D , E ) for 0 or 3 h . Data represent mean ± SD ( n = 3 - 5 biological replicates ) for E . P * < 0 . 05 between groups as indicated . Comparison between two groups was analyzed by student t test .

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

(D,E) Representative and summary immunoblot (IB) detection of phospho-lamin B and total lamin B with the lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were treated either by PMA (D,E) for 0 or 3 h. Data represent mean ± SD (n=3-5 biological replicates) for E. P*<0.05 between groups as indicated. Comparison between two groups was analyzed by student t test.


( A ) Summary and representative immunoblots of total PKCα and p - PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h , and then treated without or with PMA ( A ) for 3 h . Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit ( A )

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 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

(A) Summary and representative immunoblots of total PKCα and p-PKCα in dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated without or with PMA (A) for 3 h. Panels A is summary analyses of immunoblots that were calculated based on the arbitrary unit (A)


( B ) Immunoblot ( IB ) detection of the phospho - lamin B and total lamin B with lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h , and then treated by PMA ( B ) for 3 h .

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

(B) Immunoblot (IB) detection of the phospho-lamin B and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from human dPMNs that were pretreated without or with PKC inhibitor Go6976 for 1 h, and then treated by PMA (B) for 3 h.


( C ) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h , and then treated without or with PMA for 3 h , followed by staining of lamin B ( primary anti - lamin B , and FITC - labeled secondary antibody ) , and phosphorylated PKCα ( primary anti - human p - PKCαS657 , and PE - labeled secondary antibody ) . The green / white arrows indicate the site of nuclear envelope rupture . Scale bars , 10 μm ( C ) .

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(C) Confocal microscopy images of human dPMNs that were pretreated without or with PKCα Go6976 for 1 h, and then treated without or with PMA for 3 h, followed by staining of lamin B (primary anti-lamin B, and FITC-labeled secondary antibody), and phosphorylated PKCα (primary anti-human p-PKCαS657, and PE-labeled secondary antibody). The green/white arrows indicate the site of nuclear envelope rupture. Scale bars, 10 μm (C).


( E ) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B ( WT control ) , or mutants with single or multiple defined point mutations at PKCα - consensus - phosphorylation sites ( S395A , S405A , S408A ) of lamin B , and treated for 3 h by 10 μM PAF in medium containing cell - permeable dye SYTO Red ( 500 nM ) for the total cell count , while the cells with NET formation were stained by cell impermeable dye SYTOX Blue ( 5 μM ) . Then the images were taken with Olympus confocal microscopy , followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation . percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ ( E )

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(E) The endpoint analysis of % cells with NET formation was detected with dPMNs that were transfected with plasmids of either wildtype lamin B (WT control), or mutants with single or multiple defined point mutations at PKCα-consensus-phosphorylation sites (S395A, S405A, S408A) of lamin B, and treated for 3 h by 10 μM PAF in medium containing cell-permeable dye SYTO Red (500 nM) for the total cell count, while the cells with NET formation were stained by cell impermeable dye SYTOX Blue (5 μM). Then the images were taken with Olympus confocal microscopy, followed by automated quantification of NETs using ImageJ for quantification of % cells with NET formation. percentage of cells with NET formation by immunofluorescent imaging quantification using ImageJ (E)


( A ) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα - / - , PKCα + / - , and their littermate WT ( PKCα + / + ) mice . Vinculin served as loading control .

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(A) Summary and representative immunoblots of PKCα expressions in mouse bone marrow neutrophils from PKCα-/-, PKCα+/-, and their littermate WT (PKCα+/+) mice. Vinculin served as loading control.


( B ) Representative immunoblot ( IB ) detection of the phospho - lamin B ( Ser ) and total lamin B with lamin B protein purified by immunoprecipitation ( IP ) with anti - lamin B from mouse peritoneal mPMNs from WT vs PKCα - / - mice , stimulated without or with PAF for 3 h .

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(B) Representative immunoblot (IB) detection of the phospho-lamin B (Ser) and total lamin B with lamin B protein purified by immunoprecipitation (IP) with anti-lamin B from mouse peritoneal mPMNs from WT vs PKCα-/- mice, stimulated without or with PAF for 3 h.


( C ) Summary analyses of NET formation in peritoneal mPMNs , from WT , PKCα + / - , or PKCα - / - mice , which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2 % PFA fixation and fluorometric microplate analysis .

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(C) Summary analyses of NET formation in peritoneal mPMNs, from WT, PKCα+/-, or PKCα-/- mice, which were treated without or with either 5 µM or 10 µM PAF for 3 h following by 2% PFA fixation and fluorometric microplate analysis.


( 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 .

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

(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 .

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).