( D ) p - S6 / loading control signal from western blots experiments in which a panel of six TNBC PTEN - null lines HCC70 that acquired resistance to AZD8186 or MK2206 were treated with vehicle , vorapaxar ( 10 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) alone or in indicated combinations . values were normalised to vehicle treatment for lines . Mean of 2 independent experiments ± SD . P values calculated by two - paired student t - * P = 0 . 0235 * * P = 0 . 002 . lines used in experiments were : MDA - MB - 468 , HCC70 , HCC1937 , HCC38 , HCC1395 , BT - 549 , HCC70 AZD8186 - resistant HCC70 MK2206 - resistant .

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(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.


( E ) Long - term proliferation of MDA - MB - 468 treated with vorapaxar ( 5 or 2 . 5 μM ) , GDC0941 ( 1 μM ) or lapatinib ( 1 μM ) , alone or in indicated combinations . were treated for two stained by crystal violet . One representative experiment of three is shown .

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(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.


b , Carboxy - terminal V5immunoblot of full - ( 68 kDa ) ( 36 kDa ) ATG16L1β . Silver stain ( bottom gels ) depicts total . Data represent 3 independent experiments . IB , immunoblot .

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b, Carboxy-terminal V5immunoblot of full-length (68 kDa) and cleaved (36 kDa) ATG16L1β. Silver stain (bottom gels) depicts total protein. Data represent 3 independent experiments. IB, immunoblot.


c , TNF - α - mediated processing of humanATG16L1 in monocyte - derived macrophages . Scatter plot of immunoblot densitometry depicts a of : full - ATG16L1 ( Extended Data Fig . 2 ) . n = 7 donors .

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c, TNF-α-mediated processing of humanATG16L1 in monocyte-derived macrophages. Scatter plot of immunoblot densitometry depicts a ratio of cleaved:full-length ATG16L1 (Extended Data Fig. 2). n = 7 donors.


d , TNF - α - mediated processing of murineATG16L1 in macrophages . Immunoblot represents 5 independent experiments . Scatterplot represents data pooled from 5 independent experiments . WT , wild type .

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d, TNF-α-mediated processing of murineATG16L1 in macrophages. Immunoblot represents 5 independent experiments. Scatterplot represents data pooled from 5 independent experiments. WT, wild type.


a , HeLa transfected with control siRNA ( siCTL ) or siCaspase 8 were stimulated with 2 . 0 μM staurosporine or 20 ng ml − 1 TNF + 10 μg ml − 1 CHX .

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a, HeLa cells transfected with control siRNA (siCTL) or siCaspase 8 were stimulated with 2.0 μM staurosporine or 20 ng ml−1 TNF + 10 μg ml−1 CHX.


b , HeLa transfected with control siRNA or siCaspase 9 were stimulated as in a .

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b, HeLa cells transfected with control siRNA or siCaspase 9 were stimulated as in a.


c , HeLa transfected with control , caspase - 3 - or caspase - 7 - specific siRNAs were stimulated with 20 ng ml − 1 TNF + 10 & amp ; amp ; mgr ; g ml − 1 CHX .

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c, HeLa cells transfected with control, caspase-3- or caspase-7-specific siRNAs were stimulated with 20 ng ml−1 TNF + 10 &amp;mgr;g ml−1 CHX.


d , Wild - type or caspase - 3 - knockout macrophages were stimulated with 2 . 0 µM staurosporine or 20 ng ml − 1 TNF + 10 µg ml − 1 CHX . Data in a - c represent 2 independent experiments ; data in d represent 4 independent experiments .

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d, Wild-type or caspase-3-knockout macrophages were stimulated with 2.0 µM staurosporine or 20 ng ml−1 TNF + 10 µg ml−1 CHX. Data in a-c represent 2 independent experiments; data in d represent 4 independent experiments.


a , Intracellular for LC3 ATG16L1 following glucose starvation in presence of bafilomycin A1 . mask in LC3 channel illustrates punctate LC3 , representing LC3 - II . Histogram depicts mean of 10 , 000 per sample . b , of mean ATG16L1 in a , n = 4 mice . Glu . , glucose .

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a, Intracellular staining for LC3 and ATG16L1 following glucose starvation in the presence of bafilomycin A1. White mask in LC3 channel illustrates punctate LC3 staining, representing LC3-II. Histogram depicts mean fluorescence intensity of 10,000 cells per sample. b, Quantification of mean ATG16L1 fluorescence intensity in a, n = 4 mice. Glu., glucose.


c , Immunoblot of phosphorylated ( p - ) AMPK & amp ; amp ; agr ; ( Thr 172 ) lipidated LC3 ( LC3 - II ) .

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c, Immunoblot of phosphorylated (p-) AMPK&amp;agr; (Thr 172) and lipidated LC3 (LC3-II).


d , of mean LC3 - II in murine macrophages , n = 4 mice .

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d, Quantification of mean LC3-II area in murine macrophages, n = 4 mice.


e , Caspase 3 activity in murine macrophages following glucose starvation , n = 3 mice . RLU , relative units .

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e, Caspase 3 activity in murine macrophages following glucose starvation, n = 3 mice. RLU, relative light units.


f , of mean LC3 - II in wild - type ( Casp3 + / + ) or Casp3 - knockout ( Casp3 − / − ) macrophages , n = 3 mice . Data in a - f represent 2 independent experiments .

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f, Quantification of mean LC3-II area in wild-type (Casp3+/+) or Casp3-knockout (Casp3−/−) macrophages, n = 3 mice. Data in a-f represent 2 independent experiments.


a , of ATG16L1 mean by image - flow cytometry of 10 , 000 , n = 4 ( wild - type , WT ) , 5 ( T300A ) . Data represent 3 independent experiments . b , of caspase 3 activity upon glucose starvation of 2 × 104 , n = 5 . Data represent 2 independent experiments . c , of mean LC3 - II of samples in a . Data represent 3 independent experiments .

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a, Quantification of ATG16L1 mean fluorescence intensity by image-based flow cytometry of 10,000 cells, n = 4 (wild-type, WT), 5 (T300A). Data represent 3 independent experiments. b, Quantification of caspase 3 activity upon glucose starvation of 2 × 104 cells, n = 5. Data represent 2 independent experiments. c, Quantification of mean LC3-II area of samples in a. Data represent 3 independent experiments.


d , Visualization of early autophagosomes by transmission electron ( ) . Red arrows , early autophagosomes , characterized by vesicular structures with a double - . Blue arrows , multivesicular lysosomal structures , identified by degraded intravesicular cargo . representative of 10 - 12 per donor , n = 4 ( WT ) , n = 3 ( T300A ) . A total of 29 ( WT ) 26 ( T300A ) containing autophagosomes was analysed . Histogram quantifies autophagic vesicles as a of autophagosome : .

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d, Visualization of early autophagosomes by transmission electron micrographs (TEM). Red arrows, early autophagosomes, characterized by vesicular structures with a double-membrane. Blue arrows, multivesicular lysosomal structures, identified by degraded intravesicular cargo. Micrographs representative of 10-12 images per donor, n = 4 (WT), n = 3 (T300A). A total of 29 (WT) and 26 (T300A) images containing autophagosomes was analysed. Histogram quantifies autophagic vesicles as a ratio of autophagosome:cytosolic area.


a - c , Y . enterocolitica colony forming units ( c . f . u . ) in human murine macrophages infected for 6 h ( 20 m . o . i . ) . Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype , total n = 16 ( non - risk , WT ) , 15 ( T300A , risk ) . Data in b represent 3 independent experiments , n = 4 mice . Data in c represent 3 independent experiments , n = 6 .

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a-c, Y. enterocolitica colony forming units (c.f.u.) in human and murine macrophages infected for 6 h (20 m.o.i.). Data in a are pooled from 4 independent cohorts of 3 or 4 donors per genotype, total n = 16 (non-risk, WT), 15 (T300A, risk). Data in b represent 3 independent experiments, n = 4 mice. Data in c represent 3 independent experiments, n = 6.


d , TNF - α , IL - 1β IL - 6 in culture media of WT T316A macrophages infected with 20 m . o . i . Y . enterocolitica . Data represent 2 independent experiments , n = 4 mice .

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d, TNF-α, IL-1β and IL-6 levels in culture media of WT and T316A macrophages infected with 20 m.o.i. Y. enterocolitica. Data represent 2 independent experiments, n = 4 mice.


e , Ileum colon histology ( haematoxylin eosin ) of WT mice . are representative of 3 ( PBS ) 8 ( Y . enterocolitica ) treated mice .

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e, Ileum and colon histology (haematoxylin and eosin staining) of WT mice. Images are representative of 3 (PBS) and 8 (Y. enterocolitica) treated male mice.


f , Measurement of cytokine mRNA transcripts in mesenteric lymph nodes . Data are pooled from 2 independent experiments of 3 mice ( PBS ) 7 mice ( Y . enterocolitica ) per experiment . Transcripts are normalized to Gapdh ( 2 − Dgr ; Ct ) .

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f, Measurement of cytokine mRNA transcripts in mesenteric lymph nodes. Data are pooled from 2 independent experiments of 3 mice (PBS) and 7 mice (Y. enterocolitica) per experiment. Transcripts are normalized to Gapdh (2−Dgr;Ct).


g , Y . enterocolitica c . f . u . in mesenteric lymph nodes of WT T316A knock - in mice 48 h following oral gavage . Data represent 3 independent experiments , n = 9 mice .

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g, Y. enterocolitica c.f.u. in mesenteric lymph nodes of WT and T316A knock-in mice 48 h following oral gavage. Data represent 3 independent experiments, n = 9 male mice.


D . Immunoblot of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) , gasdermin D ( GSDMD ) GAPDH ( loading control ) in WT mutant BMDMs left untreated or assessed 20 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Data are from one three independent experiments (

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D. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11), gasdermin D (GSDMD) and GAPDH (loading control) in WT and mutant BMDMs left untreated or assessed 20 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Data are from one three independent experiments (D


E . Release of IL - 1β , IL - 18 , TNF LDH from BMDMs after treatment as in D . Data information : Each symbol represents an independent biological replicate ( E ) . * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons ( E ) . Data are from three independent experiments E ; mean s . e . m . in

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E. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in D. Data information: Each symbol represents an independent biological replicate (E). **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (E). Data are from three independent experiments E; mean and s.e.m. in E


G . IncuCyte live - imaging of WT Casp11 - / - BMDM viability after infection with M . catarrhalis as in D , or transfection with LPS . Data information : Data are pooled from two ( G ) independent experiments mean s . e . m . in G ) .

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G. IncuCyte live-imaging analysis of WT and Casp11-/- BMDM viability after infection with M. catarrhalis as in D, or transfection with LPS. Data information: Data are pooled from two (G) independent experiments mean and s.e.m. in G).


A . Immunoblot of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) gasdermin D ( GSDMD ) in WT , Nlrp3 - / - , Casp11 - / - Aim2 - / - BMDMs left untreated ( Med . ) or 5 h after transfection with 5 µg of LOS from M . catarrhalis ( O35E ) or 5 µg of LPS from E . coli . Data information : Data are from three independent experiments ( A ,

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A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Nlrp3-/-, Casp11-/- and Aim2-/- BMDMs left untreated (Med.) or 5 h after transfection with 5 µg of LOS from M. catarrhalis (O35E) or 5 µg of LPS from E. coli. Data information: Data are from three independent experiments (A,


B . Release of IL - 1β , IL - 18 , TNF LDH from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B , NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons ( B , Data are pooled from three independent experiments ( B , mean s . e . m . in B ,

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B. Release of IL-1β, IL-18, TNF and LDH from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B, NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B, Data are pooled from three independent experiments (B, mean and s.e.m. in B,


F . Transcript abundance for genes encoding GTPases in WT Ifnar1 - / - BMDMs infected with M . catarrhalis as in C . Transcript abundance is provided in per million ( CPM ) . Data information : Data are pooled from three independent

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F. Transcript abundance for genes encoding GTPases in WT and Ifnar1-/- BMDMs infected with M. catarrhalis as in C. Transcript abundance is provided in counts per million (CPM). Data information: Data are pooled from three independent experiments


G . qRT - PCR of genes encoding family of guanylate binding ( GBP1 - 11 ) in WT Ifnar1 - / - BMDMs left untreated ( Med . ) or following treated as in C . Data information : Each symbol represents an independent biological replicate G ) . NS , no statistical significance ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons G ) ) . Data are from one experiment representative of four ( G ) independent experiments ( mean s . e . m . in G ) .

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G. qRT-PCR analysis of genes encoding the family of guanylate binding proteins (GBP1-11) in WT and Ifnar1-/- BMDMs left untreated (Med.) or following treated as in C. Data information: Each symbol represents an independent biological replicate G). NS, no statistical significance; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test G)). Data are from one experiment representative of four (G) independent experiments (mean and s.e.m. in G).


A . Confocal microscopy of intracellular M . catarrhalis ( green ) GBP1 , GBP2 , GBP3 , GBP5 or GBP7 ( red ) in WT BMDMs left untreated ( Med . ) or assessed 12 h after infection with M . catarrhalis ( Ne11 , MOI 20 ) . B . Quantitation of GBP1 - , GBP2 - , GBP3 - , GBP5 - GBP7 - postive M . catarrhalis in WT BMDMs as treated in A . Data information : Arrowheads indicate colocalised with GBPs . To quantify proportion of GBP - positive in a field of view , we used single red channel ( anti - GBP ) compared this to green channel ( anti - M . catarrhalis ) . number of GBP - positive M . catarrhalis was divided by total number of M . catarrhalis counted to obtain proportion of GBP - positive ( B ) . A total of 100 BMDMs were analysed to quantify proportion of GBP - positive per using confocal microscopy ( B ) Data are from three independent experiments ( A , or are pooled from two ( B ) independent experiments Scale bars , 5 µm ( A ,

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A. Confocal microscopy analysis of intracellular M. catarrhalis (green) and GBP1, GBP2, GBP3, GBP5 or GBP7 (red) in WT BMDMs left untreated (Med.) or assessed 12 h after infection with M. catarrhalis (Ne11, MOI 20). B. Quantitation of GBP1-, GBP2-, GBP3-, GBP5- and GBP7-postive M. catarrhalis in WT BMDMs as treated in A. Data information: Arrowheads indicate bacteria colocalised with GBPs. To quantify the proportion of GBP-positive bacteria in a field of view, we used the single red channel (anti-GBP staining) and compared this to the green channel (anti-M. catarrhalis staining). The number of GBP-positive M. catarrhalis was divided by the total number of M. catarrhalis counted to obtain the proportion of GBP-positive bacteria (B). A total of 100 BMDMs were analysed to quantify the proportion of GBP-positive bacteria per cell using confocal microscopy (B) Data are from three independent experiments (A, or are pooled from two (B) independent experiments Scale bars, 5 µm (A,


F . Recovery of M . catarrhalis ( as colony - forming units ( CFU ) from WT , Gbpchr3 - KO Ifnar1 - / - lung fibroblasts 4 , 8 12 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) . Data information : Each symbol represents an independent biological replicate F ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons Data are from three independent experiments F ; mean s . e . m .

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F. Recovery of M. catarrhalis (as colony-forming units (CFU) from primary WT, Gbpchr3-KO and Ifnar1-/- lung fibroblasts 4, 8 and 12 h after infection with M. catarrhalis (Ne11, MOI 100). Data information: Each symbol represents an independent biological replicate F). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test Data are from three independent experiments F; mean and s.e.m.


G . Quantitation of intracellular by Transmission electron microscopy ( ) in WT , Gbpchr3 - KO Ifnar1 - / - BMDMs infected with M . catarrhalis ( Ne11 , MOI 50 ) for 12 h . Each symbol represents an independent experiment ( E F ) . Data information : A total of 100 BMDMs were analysed to quantify intracellular burden using electron microscopy ( G ) . Data are from one experiment representative of two ( G ) independent experiments mean s . e . m .

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G. Quantitation of intracellular bacteria by Transmission electron microscopy (TEM) in WT, Gbpchr3-KO and Ifnar1-/- BMDMs infected with M. catarrhalis (Ne11, MOI 50) for 12 h. Each symbol represents an independent experiment (E and F). Data information: A total of 100 BMDMs were analysed to quantify the intracellular bacterial burden using electron microscopy (G). Data are from one experiment representative of two (G) independent experiments mean and s.e.m.


A . Immunoblot of caspase - 1 ( Casp - 1 ) , caspase - 11 ( Casp - 11 ) gasdermin D ( GSDMD ) in WT , Gbp1 - / - , Gbp2 - / - , Gbp3 - / - , Gbp5 - / - , Gbp7 - / - , Gbp4 / 8 / 9 - / - , Gbp11 - / - Casp11 - / - BMDMs left untreated ( Med . ) or assessed 10 h after infection with M . catarrhalis ( Ne11 , MOI 100 ) , or 5 h after transfection with 5 µg LPS from E . coli .

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

A. Immunoblot analysis of caspase-1 (Casp-1), caspase-11 (Casp-11) and gasdermin D (GSDMD) in WT, Gbp1-/-, Gbp2-/-, Gbp3-/-, Gbp5-/-, Gbp7-/-, Gbp4/8/9-/-, Gbp11-/- and Casp11-/- BMDMs left untreated (Med.) or assessed 10 h after infection with M. catarrhalis (Ne11, MOI 100), or 5 h after transfection with 5 µg LPS from E. coli.


B . Release of IL - 1β , IL - 18 , LDH , TNF , IL - 6 KC from BMDMs after treatment as in A . Data information : Each symbol represents an independent biological replicate ( B ) . NS , no statistical significance ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons ( B ) ) . Data are are pooled from three independent experiments ( B ; mean s . e . m . in B ) .

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

B. Release of IL-1β, IL-18, LDH, TNF, IL-6 and KC from BMDMs after treatment as in A. Data information: Each symbol represents an independent biological replicate (B). NS, no statistical significance; * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B)). Data are are pooled from three independent experiments (B; mean and s.e.m. in B).


B . of OD600 of M . catarrhalis in BHI media over 6 h in presence of solvent control ( Sol . Ctrl . ) , 80 µg / mL recombinant GBP2 or 50 µg / mL kanamycin . Data information : * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons ( B Data are from one experiment representative of two ( A ) or three ( B , Each independent experiment consists of three ( B , 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Quantification of OD600 of M. catarrhalis in BHI media over 6 h in the presence of solvent control (Sol. Ctrl.), 80 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test (B Data are from one experiment representative of two (A) or three (B, Each independent experiment consists of three (B, technical replicates.


C . of ATP from M . catarrhalis in BHI media over 6 h in presence of Sol . Ctrl . , 30 µg / mL recombinant GBP2 or 50 µg / mL kanamycin . Data information : Data are pooled from two ( C ) independent experiments Each independent experiment consists of two ( C ) technical replicates .

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 B-CONTROLLED_VAR 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

C. Quantification of ATP from M. catarrhalis in BHI media over 6 h in the presence of Sol. Ctrl., 30 µg/mL recombinant GBP2 or 50 µg/mL kanamycin. Data information: Data are pooled from two (C) independent experiments Each independent experiment consists of two (C) technical replicates.


D . Viability of M . catarrhalis ( as a of CFU in relation to Sol . Ctrl . ) assessed 6 h after incubation with recombinant GBP2 at 3 , 30 or 300 µg / mL . Data information : * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 ( one - way ANOVA with Dunnett ' s multiple - comparisons D ) Data are from one experiment representative of three D , independent experiments Each independent experiment consists of three D , technical replicates .

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D. Viability of M. catarrhalis (as a percentage of CFU in relation to Sol. Ctrl.) assessed 6 h after incubation with recombinant GBP2 at 3, 30 or 300 µg/mL. Data information: ** P<0.01; *** P<0.001; **** P<0.0001 (one-way ANOVA with Dunnett's multiple-comparisons test D) Data are from one experiment representative of three D, independent experiments Each independent experiment consists of three D, technical replicates.


A , B . burden of IL - 18 in spleen of WT mice ( n = 10 ) Casp11 - / - mice ( n = 10 ) after M . catarrhalis infection . C , D . burden of IL - 18 in spleen of WT mice ( n = 10 ) Nlrp3 - / - mice ( n = 11 ) after M . catarrhalis infection . E , F . burden of IL - 18 in spleen of WT mice ( n = 10 ) Gbp1 - / - mice ( n = 9 ) after M . catarrhalis infection . G , H . burden of IL - 18 in spleen of WT mice ( n = 10 ) Gbp2 - / - mice ( n = 8 ) after M . catarrhalis infection . I , J . burden of IL - 18 in spleen of WT mice ( n = 10 ) Gbp3 - / - mice ( n = 8 ) after M . catarrhalis infection . K , L . burden of IL - 18 in spleen of WT mice ( n = 5 ) Gbp5 - / - mice ( n = 8 ) after M . catarrhalis infection . Data information : A mix of mice 6 - 8 old were used in each experiment were injected i . p . with 2 × 107 CFU of M . catarrhalis analysed after 6 h . Each symbol represents an mouse ( A - L ) . Each panel represents data from a single experiment ( mean s . e . m . in A - L ) . Each experiment was performed at least two times . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( two - t - ( A - L ) ) .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Casp11-/- mice (n=10) after M. catarrhalis infection. C, D. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Nlrp3-/- mice (n=11) after M. catarrhalis infection. E, F. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp1-/- mice (n=9) after M. catarrhalis infection. G, H. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp2-/- mice (n=8) after M. catarrhalis infection. I, J. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=10) and Gbp3-/- mice (n=8) after M. catarrhalis infection. K, L. Bacterial burden and concentration of IL-18 in the spleen of WT mice (n=5) and Gbp5-/- mice (n=8) after M. catarrhalis infection. Data information: A mix of male and female mice 6-8 weeks old were used in each experiment and were injected i.p. with 2×107 CFU of M. catarrhalis and analysed after 6 h. Each symbol represents an individual mouse (A-L). Each panel represents data from a single experiment (mean and s.e.m. in A-L). Each experiment was performed at least two times. * P<0.05; ** P<0.01; *** P<0.001 (two-tailed t-test (A-L)).


C Frequency of C179 + or V5 - tag + 293T 24h after transfection of AAV - vector plasmids as measured by flow cytometry . Symbols represent single experiments bars mean ± SE ( n = 3 ) .

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C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3).


D Immunoblot of 293T 72 h after transduction with AAV - vectors at a MOI of 106 . Antigen expression was detected with an anti - V5 - tag antibody . Equal loading was controlled with a GAPDH antibody ( n = 3 ) .

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D Immunoblot of 293T cells 72 h after transduction with AAV-vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3).


Total IgG ELISA titers expressed as under curve ( AUC ) against homologous Cal / 7 / 9 of pre - immune pre - challenge sera of of indicated vaccine groups ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 ) . Background reactivity of pooled AAV - GFP pre - challenge serum is shown as dashed . ELISAs were performed in technical duplicates .

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Total IgG ELISA titers expressed as area under the curve (AUC) against homologous Cal/7/9 virus of pre-immune and pre-challenge sera of individual animals of the indicated vaccine groups (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11). Background reactivity of pooled AAV-GFP pre-challenge serum is shown as dashed line. ELISAs were performed in technical duplicates.


IgA ELISA titers in post - challenge lung homogenates against Cal / 7 / 9 of mice of indicated vaccine groups ( AAV - HA , - cHA , - GFP , - NP , WIV n = 11 ) . of Cal / 7 / 9 ( blue symbols ) PR8 low dose ( red symbols ) challenge groups . ELISAs were performed in technical duplicates .

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IgA ELISA titers in post-challenge lung homogenates against Cal/7/9 of individual mice of the indicated vaccine groups (AAV-HA, -cHA, -GFP, -NP, WIV n = 11). of the Cal/7/9 (blue symbols) and PR8 low dose (red symbols) challenge groups. ELISAs were performed in technical duplicates.


A Immunoblot of purified H1N1 A / California / 7 / 2009 ( Cal / 7 / 9 ) , A / Brevig Mission / 1 / 1918 ( BM / 1 / 1918 ) , A / Puerto Rico / 8 / 1934 ( PR8 ) or A / Brisbane / 59 / 2007 ( Bris / 59 / 7 ) separated under reducing denaturing conditions . Uncleaved HA0 cleavage products HA1 HA2 were detected with pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 ) as indicated ( n = 3 ) .

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

A Immunoblot analysis of purified H1N1 viruses A/California/7/2009 (Cal/7/9), A/Brevig Mission/1/1918 (BM/1/1918), A/Puerto Rico/8/1934 (PR8) or A/Brisbane/59/2007 (Bris/59/7) separated under reducing and denaturing conditions. Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre-challenge sera (AAV-HA, -cHA, -GFP , WIV n = 18, AAV-NP n = 11) as indicated (n = 3).


A Immunoblot of purified H1N1 A / California / 7 / 2009 ( Cal / 7 / 9 ) , A / Brevig Mission / 1 / 1918 ( BM / 1 / 1918 ) , A / Puerto Rico / 8 / 1934 ( PR8 ) or A / Brisbane / 59 / 2007 ( Bris / 59 / 7 ) separated under reducing denaturing conditions . Uncleaved HA0 cleavage products HA1 HA2 were detected with pooled pre - challenge sera ( AAV - HA , - cHA , -

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

A Immunoblot analysis of purified H1N1 viruses A/California/7/2009 (Cal/7/9), A/Brevig Mission/1/1918 (BM/1/1918), A/Puerto Rico/8/1934 (PR8) or A/Brisbane/59/2007 (Bris/59/7) separated under reducing and denaturing conditions. Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre-challenge sera (AAV-HA, -cHA, -GFP


C to F Binding of C179 ( C ) , or antibodies present in AAV - HA ( D ) , AAV - cHA ( E ) or WIV ( F ) pooled pre - challenge sera ( n = 18 mice per group ) to Cal / 7 / 9 or PR8 after incubation of virions at pH = 7 . 2 , 5 . 8 , 5 . 4 , 5 . 0 , 4 . 4 or 4 . 4 + DTT to induce conformational changes in HA or remove HA1 subdomain . Mean ± SD ( n = 3 , in technical triplicates ) . Statistical significance between pH = 7 . 2 other conditions was determined using Kruskal - Wallis with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

C to F Binding of C179 (C), or antibodies present in AAV-HA (D), AAV-cHA (E) or WIV (F) pooled pre-challenge sera (n = 18 mice per group) to Cal/7/9 or PR8 virus after incubation of the virions at pH = 7.2, 5.8, 5.4, 5.0, 4.4 or 4.4+DTT to induce conformational changes in HA or remove the HA1 subdomain. Mean ± SD (n = 3, in technical triplicates). Statistical significance between pH = 7.2 and other conditions was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05, **p<0.01).


A B Influenza specific activation of murine FcγRI , FcγRII , FcγRIII FcγRIV by antibodies in pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 mice per group ) . MDCKII were infected with Cal / 7 / 9 ( A ) or PR8 ( B ) before mouse serum FcγR - expressing reporter were added . Influenza specific Fc - FcγR interaction induces production of IL - 2 in FcγR - expressing reporter , which was measured by anti - IL2 ELISA ( n = 3 , technical duplicates ) . Mean ± SE .

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A and B Influenza virus specific activation of the murine FcγRI, FcγRII, FcγRIII and FcγRIV by antibodies in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11 mice per group). MDCKII cells were infected with Cal/7/9 (A) or PR8 (B) before mouse serum and FcγR-expressing reporter cells were added. Influenza specific Fc-FcγR interaction induces the production of IL-2 in the FcγR-expressing reporter cell, which was measured by anti-IL2 ELISA (n = 3, technical duplicates). Mean ± SE.


C HA - specific activation of FcγR by AAV - HA , AAV - cHA or WIV pooled pre - challenge sera ( n = 18 mice per group ) . MDCKII were transfected with wildtype HA ( pAAV - HA ) or a stalk - only HA ( pAAV - mHL1 ) before mouse serum FcγR - I - expressing reporter were added ( n = 2 , technical duplicates ) . Mean ± SE .

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

C HA-specific activation of FcγR by AAV-HA, AAV-cHA or WIV pooled pre-challenge sera (n = 18 mice per group). MDCKII cells were transfected with wildtype HA (pAAV-HA) or a stalk-only HA (pAAV-mHL1) before mouse serum and FcγR-I-expressing reporter cells were added (n = 2, technical duplicates). Mean ± SE.


A to F Mice were vaccinated three times with AAV - vectors or two times with WIV then challenged with a lethal dose of homologous influenza ( Cal / 7 / 9 ) ( A , B ; n = 5 ) or two different lethal doses of heterologous PR8 ( C , D ; n = 6 E , F ; n = 7 ) . Survival was monitored over a 14 - day period is depicted as Kaplan - Meier plot ( A , C , E ) . loss was determined during 14 - day period , which is shown in relation to at day 0 ( B , D , F ) . Mean ± SE . Statistical significance between negative control group ( AAV - GFP ) or WIV group ( E ) each vaccine group was determined using Log - Rank ( Mantel - Cox ) ( * p < 0 . 05 , * * p < 0 . 01 ) .

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A to F Mice were vaccinated three times with AAV-vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus (Cal/7/9) (A, B; n = 5) or two different lethal doses of the heterologous PR8 strain (C, D; n = 6 and E, F; n = 7). Survival was monitored over a 14-day period and is depicted as Kaplan-Meier plot (A, C, E). Weight loss was determined during the 14-day period, which is shown in relation to the weight at day 0 (B, D, F). Mean ± SE. Statistical significance between negative control group (AAV-GFP) or WIV group (E) and each vaccine group was determined using Log-Rank (Mantel-Cox) test (*p<0.05, **p<0.01).


B Cal / 7 / 9 - specific serum antibody titers in sera at indicated time points . Black lines : , red lines : mean titer . Statistical significance between pre - immune immune sera was determined using Friedman with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) . lower table indicates Cal / 7 / 9 - specific MN50 HAI mean titers . Assays were performed in technical duplicates .

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B Cal/7/9-specific serum antibody titers in individual sera at indicated time points. Black lines: individual animals, red lines: mean titer. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). The lower table indicates Cal/7/9-specific MN50 and HAI mean titers. Assays were performed in technical duplicates.


C to E Clinical score ( C ) , ( D ) , relative ( E ) during challenge period . Statistical significance between pre - immune immune sera was determined using Friedman with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ± SD

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C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD


C to E Clinical score ( C ) , ( D ) , relative ( E ) during challenge period . Statistical significance between pre - immune immune sera was determined using Friedman with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ±

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F titers in nasal turbinates ( NT ) , trachea or lung homogenates . Statistical significance between AAV - GFP vaccine groups was determined using Kruskal - Wallis with Dunn ' s multiple comparison testing ( * p < 0 . 05 ) . Lines indicate GMT . Assays were performed in technical duplicates .

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F Virus titers in nasal turbinates (NT), trachea or lung homogenates. Statistical significance between AAV-GFP and vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05). Lines indicate the GMT. Assays were performed in technical duplicates.


G H of submucosal ( SMG ) ( G ) or bronchi ( H ) with an anti - H1N1 polyclonal antibody ( bar : 100 µm ; red : influenza antigen ( neufuchsin ) ; blue : nuclei ( haematoxylin ) ) .

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G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)).


I J H & E stained histological sections of bronchi ( I ) lung parenchyma with alveolar epithelial ( AEC , J ) . Arrowheads : areas of suppurative bronchitis / bronchiolitis ; arrows : interstitial pneumonia , necrosis of AEC ( bar : ( I ) 100 µm ; ( J ) 50 µm ) .

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I and J H&E stained histological sections of bronchi (I) and lung parenchyma with alveolar epithelial cells (AEC, J). Arrowheads: areas of suppurative bronchitis/bronchiolitis; arrows: interstitial pneumonia, necrosis of AEC (bar: (I) 100 µm; (J) 50 µm).


( B ) - effect relationships of PARP1 inhibition by thioparib , talazoparib , olaparib , as assayed by histone - ELISA . Data are obtained from three biological replicates depicted as mean ± . ( C ) - dependent increase in PARP1 - binding by three PARP inhibitors using DSB FA assays . FA was measured 60 min after adding NAD + , ΔFA values represent changes in PARP1 - binding . Data from four biological replicates are presented as mean ± . (

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(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. (C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM. (


( F ) ( G ) Effects of thioparib on BRCA1 - deficient MDA - MB - 436 ( F ) BRCA2 - deficient Capan - 1 ( G ) xenografts . Mice bearing MDA - MB - 436 tumors were dosed orally with 10 mg / kg thioparib , 100 mg / kg olaparib , or vehicle ( Veh ) once daily for 3 ; mice bearing Capan - 1 tumors were dosed orally with 5 or 25 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( Veh ) once daily for 3 ( n = 6 ) . Data are shown as mean ± . Statistical was performed by two - way ANOVA . * * * P < 0 . 0001 . Representative of xenograft tumors are shown , mice tails represent complete regression of tumors .

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(F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.


( C ) IC50 values of thioparib in six acquired PARPi - resistant lines . Numbers 1 - 6 represent PARPi - resistant Capan - 1 / OP , Capan - 1 / TP , MDA - MB - 436 / OP , MDA - MB - 436 / TP , U251 / OP , U251 / TP , respectively . were treated with indicated PARP inhibitors for 7 days then subjected to SRB . IC50 values are expressed as mean ± SD from three separate experiments .

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(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.


( E ) Efficacy of thioparib in PDX model BR - 05 - 0028 ( BRCA1 - deficient ) . Mice bearing BR - 05 - 0028 tumors derived from a with breast cancer ( n = 6 ) were treated with 10 or 30 mg / kg thioparib or 100 mg / kg olaparib for 6 . Data are depicted as mean ± . * * * P < 0 . 0001 .

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(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.


( B ) - dependent effects of thioparib on viability of malignant hematologic . were treated with thioparib or other PARP inhibitors for 72 h then subjected to CCK8 . Data from three independent experiments were presented as mean ± .

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(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.


( D ) Kaplan - Meier survival curve of M - NSG mice transplanted with JeKo - 1 treated with 10 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( n = 10 ) . Statistical was performed by log - rank ( Mantel - Cox ) . * * * P < 0 . 0001 , ns : P = 0 . 3526 .

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(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.


( B ) Western blotting of γH2AX phosphor - RPA32 in exposed to thioparib , talazoparib , or olaparib for 12 h .

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(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.


( E ) ( F ) . Thioparib caused changes in apoptosis - related in JeKo - 1 or THP - 1 , as determined by western blotting . were exposed to thioparib , talazoparib , olaparib , or Cpd 391 for 24 h then subjected to western blotting .

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(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.


( G ) ( H ) . Depletion of PARP1 in HT - 29 Capan - 1 / TP resulted in a decrease in damage thioparib resistance . upper panel shows changes in γH2AX phospho - RPA32 in HT - 29 PARP1 knockout ( KO ) clones ( G ) or Capan - 1 / TP PARP1 KO clones ( H ) their parental after exposure to thioparib for 24 h . lower panel shows IC50 values of thioparib in these PARP1 KO clones parental . were treated with thioparib for 5 or 7 d IC50 values were determined by SRB from three separate experiments . Data are presented as mean ± SD . ThP : Thioparib , OP : Olaparib , TP : Talazoparib , 391 : Cpd 391 .

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(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.


( A ) HR repair assays in U2OS - DR - GFP treated with increasing concentrations of thioparib . were incubated with indicated drugs at time of I - SceI introduction for 48 h . GFP - positive were analyzed after I - SceI transfection for 72 h by flow cytometry . ATR inhibitor VE - 821 was used as a positive control . Three independent experiments were performed data are expressed as mean ± SD . Statistical was performed by one - way ANOVA , groups were compared with control . * * * P < 0 . 0001 , * P = 0 . 0205 , 0 . 0101 ( from left to right ) .

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(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).


( C ) Representative of RAD51 foci in U2OS - DR - GFP with or without radiation . Graphs ( right panel ) show of with ≥ 5 RAD51 foci in vehicle - drug - treated . Scale bar : 5 μm . Data are obtained from three biological replicates presented as mean ± SD . Statistical was performed by one - way ANOVA . Left to right : P < 0 . 0001 , = 0 . 0033 , < 0 . 0001 , = 0 . 0006 , = 0 . 0031 , = 0 . 0015 .

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(C) Representative images of RAD51 foci in U2OS-DR-GFP cells with or without radiation. Graphs (right panel) show quantification of cells with ≥ 5 RAD51 foci in vehicle- and drug-treated cells. Scale bar: 5 μm. Data are obtained from three biological replicates and presented as mean ± SD. Statistical analysis was performed by one-way ANOVA. Left to right: P < 0.0001, = 0.0033, < 0.0001, = 0.0006, = 0.0031, = 0.0015.


( H ) HR repair . U2OS - DR - GFP ( WT ) PARP1 KO ( KO1 KO2 ) were treated with indicated drugs for 48 h collected after 72 h of I - SceI transfection as in ( A ) . VE - 821 served as a positive control . PARP1 knockout efficiency was evaluated by western blotting . Data from three biological replicates are presented as mean ± SD . Statistical was performed by one - way ANOVA , each group was compared with control . * * * P < 0 . 0001 , * P = 0 . 0143 , 0 . 0483 , ns : P = 0 . 3061 , 0 . 8269 , 0 . 9997 , 0 . 3906 ( from left to right ) .

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(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).


( C ) Effects of thioparib on CXCL9 , CXCL10 , IL15 mRNA in JeKo - 1 . Data from three independent experiments are shown as mean ± SD . Statistical was performed by two - way ANOVA . Left panel : * P = 0 . 0367 , * * P = 0 . 0011 ; median panel : * * * P = 0 . 0002 , < 0 . 0001 , < 0 . 0001 ; right panel : * * * P = 0 . 0005 , 0 . 0009 , 0 . 0001 ( from left to right ) .

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(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).


( D ) of p - STAT1 , p - TBK1 , γH2AX , phospho - RPA32 in HT - 29 parent , PARP1 − / − ( # KO1 ) , PARP7 − / − lines after 24 or 48 h thioparib treatment .

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(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.


( F ) Western blot of p - STAT1 , p - TBK1 , γH2AX in HT - 29 parent , STING − / − , TBK1 − / − after 24 or 48 h thioparib treatment .

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(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.


( H ) Immunohistochemistry of CD4 , CD8 , CD45 expression in MC38 tissue sections . of positive of CD4 , CD8 , CD45 were shown . Scale bar : 25 μm . Data from ten technical replicates from each of two mice are presented as mean ± SD . Statistical was performed by unpaired t - . * * * P < 0 . 0001 , ns : P = 0 . 1958 .

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(H) Immunohistochemistry staining of CD4, CD8, and CD45 expression in MC38 tumor tissue sections. Quantification of cells positive of CD4, CD8, CD45 were shown. Scale bar: 25 μm. Data from ten technical replicates from each of the two mice are presented as mean ± SD. Statistical analysis was performed by unpaired t-test. ***P < 0.0001, ns: P = 0.1958.


Western blot of soluble insoluble fractions ( Red - yellow rectangles , highlights CTF - 35 CTF - 25 respectively in insoluble fraction ) .

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Western blot analysis of soluble and insoluble fractions (Red- and yellow rectangles, highlights CTF-35 and CTF-25 respectively in the insoluble fraction).


Immunofluorescent showing of phosphorylated TDP - 43 ( pTDP - 43 ) . Scale bar : 20µm . Presented immunofluorescent of control ( C2 ) mutant TDP - 43 ( P2 ) . I represents its corresponding profile plot of selected ( square ) region . Scale bar : 10µm , J pooled control ( C0 = 10 , C1 = 10 C2 = 10 ) n = 30 pooled mutant TDP - 43 ( P1 = 10 , P2 = 10 P3 = 10 ) n = 30 from each differentiation . Unpaired Mann - Whitney - .

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Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP-43 (pTDP-43). Scale bar: 20µm. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). I represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10µm, J quantification pooled control (C0=10, C1=10 and C2=10) n=30 and pooled mutant TDP-43 (P1=10, P2=10 and P3=10) n=30 cells from each differentiation. Unpaired Mann-Whitney-test.


Western blots showing nucleo - fractionation of WT - mCherry MUT - mCherry iPSC - derived motor neurons with F ( left panel ) of total mCherry total untagged TDP - 43 on α - tubulin ( right panel ) relative mCherry untagged TDP - 43 on total mCherry total untagged TDP - 43

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Western blots showing nucleo-cytoplasmic fractionation of WT-mCherry and MUT-mCherry iPSC-derived motor neurons with F (left panel) quantification of total mCherry and total untagged TDP-43 on α-tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively


Representative kymographs from control mutant TDP - 43 iPSC - derived motor neurons . x - axis represent in µm while on y - axis time in seconds is revealed . Stationary mitochondria are visible as straight vertical lines , while moving mitochondria are depicted as skewed lines . B of of moving mitochondria .

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Representative kymographs from control and mutant TDP-43 iPSC-derived motor neurons. The x-axis represent distance in µm while on the y-axis time in seconds is revealed. Stationary mitochondria are visible as straight vertical lines, while moving mitochondria are depicted as skewed lines. B Quantification of percentage of moving mitochondria.


Immunofluorescent showing of pTDP - 43 in motor neurons . Scale bar : 10µm its perspective plot in F . G of E , each dot represents one analysed , unpaired Mann - Whitney - .

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Immunofluorescent analysis showing fluorescence intensity of pTDP-43 in motor neurons. Scale bar: 10µm and its perspective intensity plot in F. G Quantification of E, each dot represents one analysed cell, unpaired Mann-Whitney-test.


axonal transport . of of moving mitochondria ( J ) , number of moving mitochondria ( K ) number of stationary mitochondria ( L ) normalized to neurite 100µm .

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axonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K) and number of stationary mitochondria (L) normalized to neurite length 100µm.


Total lysates were fractionated into soluble insoluble fractions analysed by Western blot , representative Western blots of soluble insoluble fraction without C with D HDAC6 inhibitor treatment .

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Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot, representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment.


Immunofluorescent of TDP - 43 in control , mutant TDP - 43 mutant TDP - 43 + HDAC6 inhibitor after 12 h treatment . Presented immunofluorescent of control ( C2 ) mutant TDP - 43 ( P2 ) . Scale bar : 25µm . B Profile plot of control , mutant TDP - 43 mutant TDP - 43 + HDAC6 inhibitor . C of nucleo - ( N / C ) of TDP - 43 in motor neurons , each dot represents one analyzed , unpaired Mann - Whitney - .

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Immunofluorescent analysis of TDP-43 in control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). Scale bar: 25µm. B Profile intensity plot of control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor. C Quantification of nucleo-cytoplasmic ratio (N/C) fluorescent intensity of TDP-43 in motor neurons, each dot represents one analyzed cell, unpaired Mann-Whitney-test.


of relative mCherry TDP - 43 on total fraction , paired - t - .

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Quantification of relative nuclear mCherry TDP-43 on total fraction, ratio paired-t-test.


Quantification of percentage of moving mitochondria,


P . falciparum symptomatic ( n = 30 ) asymptomatic ( n = 40 ) infected individuals , as well as - microscopy PCR - negative healthy immune controls ( n = 31 ) were recruited for study . A - F . Clinical parameters determined in study include ( A ) , gender ( B ) , parasitemia ( C ) , hemoglobin ( g / dL blood ) ( D ) , hematocrit ( E ) , platelet ( F ) . Boxes represent 25th to 75th percentiles , whiskers show range ( minimum to maximum ) , lines represent median . Significance was determined by Kruskal - Wallis with Dunn ' s multiple comparisons ( A , D - F ) , Chi - square ( B ) , Mann - Whitney ( C ) using 30 ( symptomatic ) , 40 ( asymptomatic ) 31 ( healthy immune controls ) biological replicates . * * * * p < 0 . 0001 .

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P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. A-F. Clinical parameters determined in the study include age (A), gender (B), parasitemia (C), hemoglobin (g/dL blood) (D), hematocrit (E), and platelet count (F). Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median. Significance was determined by the Kruskal-Wallis test with Dunn's multiple comparisons (A, D-F), the Chi-square test (B), and the Mann-Whitney test (C) using 30 (symptomatic), 40 (asymptomatic) and 31 (healthy immune controls) biological replicates. ****p<0.0001.


P . falciparum symptomatic ( n = 30 ) asymptomatic ( n = 40 ) infected individuals , as well as - microscopy PCR - negative healthy immune controls ( n = 31 ) were recruited for study . G . Antibody titers specific for P . falciparum lysate . Bars represent mean ± SD of using 29 ( symptomatic ) , 35 ( asymptomatic ) 26 ( healthy immune controls ) biological replicates . dotted depicts average antibody background of malaria - naive healthy Melbourne controls . H . Mean antibody titers as determined by ELISA to P . falciparum recombinant EBA - 175 , EBA - 140 , PfRh2 , PfRh4 , PfRh5 , PfRipr .

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P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. G. Antibody titers specific for P. falciparum parasite lysate. Bars represent the mean ± SD of using 29 (symptomatic), 35 (asymptomatic) and 26 (healthy immune controls) biological replicates. The dotted line depicts the average antibody background levels of malaria-naive healthy Melbourne controls. H. Mean antibody titers as determined by ELISA to the P. falciparum recombinant proteins EBA-175, EBA-140, PfRh2, PfRh4, PfRh5, and PfRipr.


PBMCs from P . falciparum symptomatic ( n = 16 ) asymptomatic ( n = 24 ) infected individuals as well as healthy immune controls ( n = 24 ) were stained with a panel of metal - labelled antibodies analysed by CyTOF . tSNE was performed FlowSOM clustering was used to identify sub - populations within gated : A . TH1 - like memory CD4 + T ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 + ) B . TH2 - like memory CD4 + T ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 - ) C . Circulating memory TFH ( CD19 - CD3 + CD4 + CD45RA - CXCR5 + ) D . Classical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 + ) E . Atypical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 - CD21 - ) F . Activated MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 - ) tSNE plots in top panel display density represent pooled data for each group , while lower panel shows a projection of FlowSOM clusters on a tSNE plot . Heatmaps show median marker expression for each FlowSOM cluster .

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PBMCs from P. falciparum symptomatic (n=16) and asymptomatic (n=24) infected individuals as well as healthy immune controls (n=24) were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated: A. TH1-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3+) B. TH2-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3-) C. Circulating memory TFH cells (CD19-CD3+CD4+CD45RA-CXCR5+) D. Classical MBCs (CD3-CD19+CD20+CD10-CD27+CD21+) E. Atypical MBCs (CD3-CD19+CD20+CD10-CD27-CD21-) F. Activated MBCs (CD3-CD19+CD20+CD10-CD27+CD21-) The tSNE plots in the top panel display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster.


Unsupervised identification of differentially abundant high - dimensional populations between clinical groups was performed using CITRUS ( FDR < 5 % ) . between P . falciparum symptomatic infected individuals asymptomatic participants or healthy immune controls . A - E . tSNE plots of clinical groups viSNE plots depicting expression of selected surface markers , relative abundance phenotype of differentially abundant clusters identified among CXCR3 + CCR6 - memory CD4 + T ( A ) , CXCR3 - CCR6 - memory CD4 + T ( B ) , circulating memory TFH ( C ) , classical MBCs ( D ) , atypical MBCs ( E ) . tSNE plots in top or each panel display density represent pooled data for each group as calculated in clustering while viSNE plots on each top panel depict relevant marker expression on tSNE overlays . lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay , whiskers showing range ( minimum to maximum ) , with lines representing median of 16 ( symptomatic ) , 24 ( asymptomatic ) 24 ( healthy immune controls ) biological replicates , while pink histograms illustrate marker expression in identified differentially abundant populations , relative to background expression , shown in lilac .

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Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac.


PBMCs from P . falciparum symptomatic ( n = 6 ) asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for - seq . A . Estimated proportions of PBMC sub - populations determined from - type deconvolution from study participant transcriptional profiles .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. A. Estimated proportions of PBMC sub-populations determined from cell-type deconvolution from study participant transcriptional profiles.


PBMCs from P . falciparum symptomatic ( n = 6 ) asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for - seq . D . Blood transcriptional module ( BTM ) showing significant BTMs differentially enriched for pairwise comparisons ( FDR < 15 % ) .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. D. Blood transcriptional module (BTM) analysis showing significant BTMs differentially enriched for pairwise comparisons (FDR<15%).


Gene expression profiles of PBMCs from P . falciparum symptomatic healthy immune controls were compared . A . Unsupervised hierarchical clustering heat map of 922 differentially expressed genes ( DEGs ) between symptomatic P . falciparum malaria healthy immune controls using complete method Euclidian measure of . B . Mean - difference plot displaying genes differentially expressed between symptomatic P . falciparum malaria healthy immune controls . Each gene is plotted as a single point determined by log - fold - change average transcript abundance . Red genes are overrepresented , blue genes are underrepresented in symptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. A. Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes (DEGs) between symptomatic P. falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance. B. Mean-difference plot displaying genes differentially expressed between symptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in symptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic healthy immune controls were compared . F . Upstream regulator of 922 DEGs between symptomatic P . falciparum malaria healthy immune controls . red lines represent a significant activation z - score of ± 2 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. F. Upstream regulator analysis of the 922 DEGs between symptomatic P. falciparum malaria and healthy immune controls. The red lines represent a significant activation z-score of ±2.


Gene expression profiles of PBMCs from P . falciparum symptomatic healthy immune controls were compared . Hierarchical clustering ( G ) displaying DEGs in symptomatic P . falciparum malaria healthy immune controls predicted to be controlled by IFN - γ IL - 13 upstream regulators .

Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. Hierarchical clustering heatmap (G) displaying DEGs in symptomatic P. falciparum malaria and healthy immune controls predicted to be controlled by IFN-γ and IL-13 upstream regulators.


Gene expression profiles of PBMCs from P . falciparum symptomatic asymptomatic infected participants were compared . C . Heat map of 418 DEGs in P . falciparum symptomatic asymptomatic infected participants as well as in healthy immune controls . E . Volcano plot displaying selected genes clusters 1 2 from in C scaled by Log2 - fold - change - Log10 ( P - value ) differentially expressed between P . falciparum symptomatic asymptomatic infected individuals . Genes in red are overrepresented in symptomatic malaria genes in blue are overrepresented in asymptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. C. Heat map of the 418 DEGs in P. falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls. E. Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2-fold-change and -Log10(P-value) differentially expressed between P. falciparum symptomatic and asymptomatic infected individuals. Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic asymptomatic infected participants were compared . F . Mean RPKMs + of selected genes in P . falciparum malaria symptomatic ( n = 6 ) asymptomatic ( n = 5 ) individuals . Dotted red lines depict transcriptional of healthy immune controls , Mann - Whitney of biological replicates , * p < 0 . 05 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. F. Mean RPKMs + SEM of selected genes in P. falciparum malaria symptomatic (n=6) and asymptomatic (n=5) individuals. Dotted red lines depict transcriptional levels of healthy immune controls, Mann-Whitney test of biological replicates,*p<0.05.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals healthy immune controls were compared . A . Hierarchical clustering heat map of 171 differentially expressed genes ( DEGs ) in P . falciparum asymptomatic healthy immune control participants . C . Mean - difference plot displaying DEGs between asyptomatic P . falciparum malaria healthy immune controls . Each gene is plotted as a single point determined by log - fold - change average transcript abundance . Red genes are overrepresented , blue genes are underrepresented in asymptomatic malaria .

Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. A. Hierarchical clustering heat map of the 171 differentially expressed genes (DEGs) in P. falciparum asymptomatic and healthy immune control participants. C. Mean-difference plot displaying DEGs between asyptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in asymptomatic malaria.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals healthy immune controls were compared . E . Mean RPKMs + of selected genes in P . falciparum malaria asymptomatic individuals ( n = 5 ) healthy immune controls ( n = 6 ) . Dotted red lines depict transcriptional of P . falciparum - infected symptomatic individuals , Mann - Whitney of biological replicates , * p < 0 . 05 .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. E. Mean RPKMs + SEM of selected genes in P. falciparum malaria asymptomatic individuals (n=5) and healthy immune controls (n=6). Dotted red lines depict transcriptional levels of P. falciparum-infected symptomatic individuals, Mann-Whitney test of biological replicates, *p<0.05.


A . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM + activated MBCs . B . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T - bet + CD4 + T .

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A. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM+ activated MBCs. B. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T-bet+ CD4+ T cells.


B - G . PBMCs from P . falciparum symptomatic ( n = 6 ) asymptomatic ( n = 6 ) infected individuals as well as healthy immune controls ( n = 6 ) were stained with a panel of antibodies analyzed by flow cytometry . tSNE plots ( B ) display density of pooled data for each group , ( C ) shows a projection of FlowSOM clusters on a tSNE plot , ( D ) is a depicting celluar phenotype of clusters of CTLA - 4 + CD3 + CD19 - . Bar plots representing mean relative abundance of biological replicates + of relevant clusters among clinical groups ( E ) , Mann - Whitney , * p < 0 . 05 . panel on left shows average CTLA - 4 mean ( MFI ) or 6 biological replicates in cluster 4 + , Mann - Whitney , * * p < 0 . 01 , while representative histograms of CTLA - 4 expression in cluster 4 are shown on right ( F ) . Marker within clusters 1 6 ( G ) .

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B-G. PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=6) infected individuals as well as healthy immune controls (n=6) were stained with a panel of antibodies and analyzed by flow cytometry. tSNE plots (B) display cell density of the pooled data for each group, (C) shows a projection of the FlowSOM clusters on a tSNE plot, and (D) is a heatmap depicting the celluar phenotype of clusters of CTLA-4+CD3+CD19- cells. Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups (E), Mann-Whitney test, *p<0.05. The panel on the left shows the average CTLA-4 mean fluorescent intensity (MFI) or 6 biological replicates in cluster 4 + SEM, Mann-Whitney test, **p<0.01, while representative histograms of CTLA-4 expression in cluster 4 are shown on the right (F). Marker intensity within clusters 1 and 6 (G).


Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA , B Aβ40 Aβ42 concentrations in CSF of APP23 mice ( heterozygous ; 3 ( n = 14 ) , 6 ( n = 12 ) , 8 ( n = 10 ) , 12 ( n = 11 ) , 19 ( n = 9 ) , 25 ( n = 6 ) months of ) . CSF Aβ40 ( F ( 1 , 56 ) = 22 . 351 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 56 ) = 38 . 597 , P < 0 . 001 ) followed a significant quadratic trend .

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Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA, B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice (heterozygous; 3 (n = 14), 6 (n = 12), 8 (n = 10), 12 (n = 11), 19 (n = 9), and 25 (n = 6) months of age). CSF Aβ40 (F(1, 56) = 22.351, P < 0.001) as well as CSFAβ42 (F(1, 56) = 38.597, P < 0.001) followed a significant quadratic trend.


C Aβ42 / 40 in CSF of APP23 mice showed a delayed decrease with ( F ( 1 , 56 ) = 53 . 894 , P < 0 . 001 ) .

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C Aβ42/40 ratio in CSF of APP23 mice showed a delayed decrease with age (F(1, 56) = 53.894, P < 0.001).


D , E Aβ40 Aβ42 concentrations in CSF of APP24 mice ( homozygous ; 2 ( n = 13 ) , 3 - 4 ( n = 16 ) , 7 - 8 ( n = 15 ) , 18 - 19 ( n = 14 ) , 24 ( n = 16 ) , 30 ( n = 18 ) months of ) . CSF Aβ40 followed a significant quadratic trend ( F ( 1 , 86 ) = 6 . 678 , P = 0 . 011 ) CSF Aβ42 best fitted a cubic trend ( F ( 1 , 86 ) = 30 . 599 , P < 0 . 001 ) .

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D, E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice (homozygous; 2 (n = 13), 3-4 (n = 16), 7-8 (n = 15), 18-19 (n = 14), 24 (n = 16), and 30 (n = 18) months of age). CSF Aβ40 followed a significant quadratic trend (F(1, 86) = 6.678, P = 0.011) and CSF Aβ42 best fitted a cubic trend (F(1, 86) = 30.599, P < 0.001).


F Aβ42 / 40 in CSF of APP24 mice showed a delayed decrease with ( F ( 1 , 86 ) = 64 . 936 , P < 0 . 001 ) .

F Aβ42/40 ratio in CSF of APP24 mice showed a delayed decrease with age (F(1, 86) = 64.936, P < 0.001).


G , H Aβ40 Aβ42 in CSF of APP51 mice ( heterozygous ; 3 ( n = 6 ) , 15 ( n = 8 ) , 24 ( n = 8 ) months of ; 22 mice in total ) . CSF Aβ40 ( F ( 1 , 19 ) = 37 . 349 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 19 ) = 107 . 670 , P < 0 . 001 ) followed a significant quadratic trend .

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G, H Aβ40 and Aβ42 in the CSF of female APP51 mice (heterozygous; 3 (n = 6), 15 (n = 8), and 24 (n = 8) months of age; 22 mice in total). CSF Aβ40 (F(1, 19) = 37.349, P < 0.001) as well as CSFAβ42 (F(1, 19) = 107.670, P < 0.001) followed a significant quadratic trend.


I Aβ42 / 40 in CSF of APP51 mice showed a delayed decrease with ( F ( 1 , 19 ) = 26 . 367 , P < 0 . 001 ) .

I Aβ42/40 ratio in CSF of APP51 mice showed a delayed decrease with age (F(1, 19) = 26.367, P < 0.001).


HumanAβ in CSF brain of APP transgenic miceA APP23CSFAβ40 Aβ42 in same as shown in Fig1 . CSFAβ42 Aβ40 are expressed as percentages of measured in youngest group .

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HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1. CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group.


B , C Aβ40 Aβ42 ( pmol / g wet brain ) in FA - soluble brain extract from same APP23mice showed a robust increase with ; ANOVA revealed a significant cubic trend ( F ( 1 , 56 ) = 221 . 114 , P < 0 . 001 F ( 1 , 56 ) = 370 . 947 , P < 0 . 001 , respectively ) .

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B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23mice showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 56) = 221.114, P < 0.001 and F(1, 56) = 370.947, P < 0.001, respectively).


D APP24 CSF Aβ40 Aβ42 in same shown in Fig1 as of youngest group .

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D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group.


E , F Aβ40 Aβ42 ( pmol / g wet brain ) in brain from same APP24mice also showed a robust increase with ; ANOVA revealed a significant cubic trend ( F ( 1 , 86 ) = 202 . 173 , P < 0 . 001 F ( 1 , 86 ) = 139 . 941 , P < 0 . 001 , respectively ) .

E, F Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP24mice also showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 86) = 202.173, P < 0.001 and F(1, 86) = 139.941, P < 0.001, respectively).


G APP51 CSF Aβ40 Aβ42 in same shown in Fig1 as percentages of in youngest group .

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G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group.


H , I Aβ40 Aβ42 ( pmol / g wet brain ) in brain from same APP51 mice showed a robust increase with ; ANOVA revealed a significant quadratic trend ( F ( 1 , 19 ) = 12 . 960 , P = 0 . 002 F ( 1 , 19 ) = 19 . 366 , P < 0 . 001 , respectively ) .

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H, I Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP51 mice showed a robust increase with age; ANOVA revealed a significant quadratic trend (F(1, 19) = 12.960, P = 0.002 and F(1, 19) = 19.366, P < 0.001, respectively).


Aβplaque pathology in brains of APP23miceAβimmunostaining ( CN3 antibody , blue ) in 25 - μm sagittal brain sections shows only sparse Aβ deposits primarily in frontal cortex of 6 - to 8 - month - old APP23mice . At 12 months thereafter , there is a progressive increase in plaque number a progressive involvement of different brain regions . Insets highlight plaque characteristics at different ages . Scale bar , 100 μm . mean number of Aβ plaques per section per hemibrain increased with in 3 - to 12 - month - old mice . Only four mice were analyzed in 3 - month - old group , as APP23mice do not develop plaques at this ( Sturchler - Pierrat et al , 1997 ) . 6 - , 8 - , 12 - month - old groups included 12 , 10 , 11 mice , respectively ( these are same mice that were used for CSF brainAβ measurements ) . Note that Aβ plaques became too numerous often could no longer be individually distinguished in groups > 12 months of . Data are represented as group means ± .

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Aβplaque pathology in the brains of APP23miceAβimmunostaining (CN3 antibody, dark blue) in 25-μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6- to 8-month-old APP23mice. At 12 months and thereafter, there is a progressive increase in plaque number and size and a progressive involvement of different brain regions. Insets highlight the plaque characteristics at the different ages. Scale bar, 100 μm.The mean number of Aβ plaques per section per hemibrain increased with age in 3- to 12-month-old mice. Only four mice were analyzed in the 3-month-old age group, as APP23mice do not develop plaques at this age (Sturchler-Pierrat et al, 1997). The 6-, 8-, and 12-month-old age groups included 12, 10, and 11 mice, respectively (these are the same mice that were used for CSF and brainAβ measurements). Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age. Data are represented as group means ± SEM.