A ) Immunohistochemistry of Csf - 1rflx / wt ; Cx3cr1 - Cre - ( top ) and Csf - 1rflx / wt ; Cx3cr1 - Cre + ( bottom ) mice unilaterally injected with Aβ1 - 42 in the hippocampus and stained for IB4 ( green ) , F4 / 80 ( white ) and Aβ ( red ) . Scale bars indicate 50 μm . B ) Quantification of F4 / 80 and IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . Quantification of immunopositivity in both injected and uninjected hippocampi of Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mice . ( Two - way ANOVA with Sidak ' s test for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of the uninjected hippocampus , obliques ( # ) indicate comparison between Csf - 1rflx / wt ; Cx3cr1 - Cre - and Csf - 1rflx / wt ; Cx3cr1 - Cre + mice , # # or * * p < 0 . 003 , # # # # or * * * * p < 0 . 0001 , n = 5 mice per group , error bars indicate SEM ) .

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A) Immunohistochemistry of Csf-1rflx/wt;Cx3cr1-Cre- (top) and Csf-1rflx/wt;Cx3cr1-Cre+ (bottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). Scale bars indicate 50 μm. B) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice. (Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, obliques (#) indicate comparison between Csf-1rflx/wt;Cx3cr1-Cre- and Csf-1rflx/wt;Cx3cr1-Cre+ mice, ## or ** p < 0.003, #### or **** p < 0.0001, n = 5 mice per group,error bars indicate SEM).


C ) Immunohistochemistry of Csf - 1rflx / wt ; Tie2 - Cre - ( top ) and Csf - 1rflx / wt ; Tie2 - Cre + mice ( ottom ) mice unilaterally injected with Aβ1 - 42 in the hippocampus and stained for IB4 ( green ) , F4 / 80 ( white ) and Aβ ( red ) . D ) Quantification of F4 / 80 and IB4 immunopositivity following intrahippocampal injection of Aβ1 - 42 . Quantification of immunopositivity in both injected and uninjected hippocampi of Csf - 1rflx / wt ; Tie2 - Cre - and Csf - 1rflx / wt ; Tie2 - Cre + mice . ( n = 5 mice per group . Two - way ANOVA with Sidak ' s test for multiple comparisons . Asterisks ( * ) indicate comparison to immunopositivity values of the uninjected hippocampus , * p < 0 . 01 , * * * p < 0 . 0005 , error bars indicate SEM ) ) .

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C) Immunohistochemistry of Csf-1rflx/wt;Tie2-Cre- (top) and Csf-1rflx/wt;Tie2-Cre+ mice (ottom) mice unilaterally injected with Aβ1-42 in the hippocampus and stained for IB4 (green), F4/80 (white) and Aβ (red). D) Quantification of F4/80 and IB4 immunopositivity following intrahippocampal injection of Aβ1-42. Quantification of immunopositivity in both injected and uninjected hippocampi of Csf-1rflx/wt;Tie2-Cre- and Csf-1rflx/wt;Tie2-Cre+ mice. (n = 5 mice per group. Two-way ANOVA with Sidak's test for multiple comparisons. Asterisks (*) indicate comparison to immunopositivity values of the uninjected hippocampus, *p < 0.01, *** p < 0.0005, error bars indicate SEM)).


qPCR of wild type ( blue ) or Csf - 1r + / - ( red ) endothelial cells treated with control , Csf - 1r + / + MCM or Csf - 1r + / - MCM . Statistical analyses of inter - genotype changes . ( * * * * p < 0 . 0001 , * * p < 0 . 005 , Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings , Two - way ANOVA with multiple comparisons and Sidak post - test , error bars indicate SEM ) ) .

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qPCR of wild type (blue) or Csf-1r+/- (red) endothelial cells treated with control, Csf-1r+/+ MCM or Csf-1r+/- MCM. Statistical analyses of inter-genotype changes. (**** p < 0.0001, ** p < 0.005, Scatter plots represent technical replicates of n = 2 independent primary cell isolations and microglia conditionings, Two-way ANOVA with multiple comparisons and Sidak post-test, error bars indicate SEM)).


IHC of ALSP patient post mortem tissue for amyloid - β ( green ) and CLD5 ( red ) , ( B ) human IgG ( green ) and PDGFRβ ( red ) , ( C ) GFAP ( green ) and CLD5 ( red ) . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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IHC of ALSP patient post mortem tissue for amyloid-β (green) and CLD5 (red), (B) human IgG (green) and PDGFRβ (red), (C) GFAP (green) and CLD5 (red). ΔA781_N783 and P824R indicate the CSF-1R variant present.


( D ) Iba1 ( green ) and GFAP ( red ) , ( E ) amyloid - β ( green ) and CD68 ( red ) , ( f ) CD163 ( green ) and CLD5 ( red ) , imaging of an ALSP patient with the ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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(D) Iba1 (green) and GFAP (red), (E) amyloid-β (green) and CD68 (red), (f) CD163 (green) and CLD5 (red), imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


( G ) Fibrinogen ( green ) and CLD5 ( red ) and ( H ) FLAIR imaging of an ALSP patient with the ΔA781 _ N783 CSF - 1R variant . ΔA781 _ N783 and P824R indicate the CSF - 1R variant present .

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(G) Fibrinogen (green) and CLD5 (red) and (H) FLAIR imaging of an ALSP patient with the ΔA781_N783 CSF-1R variant. ΔA781_N783 and P824R indicate the CSF-1R variant present.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( A ) PS1 , PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N ( PS1 ) , BI - HF5C ( PS2 ) and N1660 ( NCT ) , respectively . ( B ) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687 . ( C ) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation / immunoblotting using antibodies 3552 / 2D8 .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(A) PS1, PS2 and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI-HF5C (PS2) and N1660 (NCT), respectively.(B) Full length APP and APP CTFs were analyzed by immunoblotting using antibody 6687. (C) Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( D ) Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 . Pooled clones of HEK293 / sw cells stably transfected with PS1 L166P were used as reference . Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (D) Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8. Pooled clones of HEK293/sw cells stably transfected with PS1 L166P were used as reference. Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( E ) PS1 expression levels were analyzed in cell lysates of HEK293 / sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12 , respectively .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (E) PS1 expression levels were analyzed in cell lysates of HEK293/sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12, respectively.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( F ) Conditioned media were analyzed by ELISA specific for Aβ40 , Aβ42 and Aβ43 . Data represent mean ± s . e . m . ( n = 6 ) . Absolute levels and Aβ ratios are shown .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing. (F) Conditioned media were analyzed by ELISA specific for Aβ40, Aβ42 and Aβ43. Data represent mean ± s.e.m. (n=6). Absolute levels and Aβ ratios are shown.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( G ) Secreted Aβ was analyzed as in ( D ) . To verify individual Aβ species , Aβ standards were co - migrated . Conditioned media were analyzed for individual Aβ species on Tris - Bicine urea SDS - PAGE by combined immunoprecipitation / immunoblotting of Aβ using antibodies 3552 / 2D8 .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(G) Secreted Aβ was analyzed as in (D). To verify individual Aβ species, Aβ standards were co-migrated. Conditioned media were analyzed for individual Aβ species on Tris-Bicine urea SDS-PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8.


Pooled clones ( A - D ) and single cell clones ( E - H ) of HEK293 / sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ - secretase expression and APP processing . ( H ) Total Aβ in conditioned media was analyzed by MALDI - TOF MS following immunoprecipitation with antibody 4G8 . Observed ( Aβ42 , 4513 . 6 ; Aβ43 , 4615 . 3 ) and predicted molecular masses ( Aβ42 , 4514 . 1 ; Aβ43 , 4615 . 2 ) were in good agreement .

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Pooled clones (A-D) and single cell clones (E-H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ-secretase expression and APP processing.(H) Total Aβ in conditioned media was analyzed by MALDI-TOF MS following immunoprecipitation with antibody 4G8. Observed (Aβ42, 4513.6; Aβ43, 4615.3) and predicted molecular masses (Aβ42, 4514.1; Aβ43, 4615.2) were in good agreement.


( A ) PS1 / 2 - / - MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A . Antibody 5E12 was used for the detection of the PS1 CTF .

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(A) PS1/2-/- MEF cells stably transduced with PS1 WT or PS1 L435F were analyzed for PS1 expression and APP processing by immunoblotting as in Fig 1A. Antibody 5E12 was used for the detection of the PS1 CTF.


( B ) Stably transduced PS1 / 2 - / - MEF cells of A were transiently transfected with APPsw - 6myc and conditioned media were analyzed for Aβ species by Tris - Bicine urea SDS - PAGE . Individual Aβ species were verified by co - migration with Aβ standards ( left panel ) and quantified ( right panel ) . Data represent mean ± s . e . m . of n = 3 independently performed transfections . Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis .

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(B) Stably transduced PS1/2-/- MEF cells of A were transiently transfected with APPsw-6myc and conditioned media were analyzed for Aβ species by Tris-Bicine urea SDS-PAGE. Individual Aβ species were verified by co-migration with Aβ standards (left panel) and quantified (right panel). Data represent mean ± s.e.m. of n=3 independently performed transfections. Note that more sample was loaded for the PS1 L435F mutant to facilitate immunoblot analysis.


( C ) Secreted Aβ from cells in B were quantified by ELISA . Data represent mean ± s . e . m . of n = 5 ( PS1 WT ) or 6 ( PS1 L435F ) independently performed transfections .

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(C) Secreted Aβ from cells in B were quantified by ELISA. Data represent mean ± s.e.m. of n=5 (PS1 WT) or 6 (PS1 L435F) independently performed transfections.


Immunohistochemical detection of Aβ40 ( left column ) , Aβ42 ( medium column ) and Aβ43 ( right column ) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation ( cases # 1 and # 2 , 1st and 2nd rows ) , another FAD case with different PS1 mutation ( case # 3 , 3rd row ) and one SAD case ( case # 4 , forth row ) . In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40 . This is in contrast to Aβ plaques of the control FAD and SAD cases , in which solely Aβ42 predominates and Aβ43 is sparse ; even levels of Aβ40 seem to be lower than in the PS1 L435F cases . Note that in both PS1 L435F cases several plaques are larger than those seen in cases # 3 and # 4 , representing cotton wool plaques . Scale bar = 500 µm . Magnification is identical in all pictures .

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Immunohistochemical detection of Aβ40 (left column), Aβ42 (medium column) and Aβ43 (right column) in consecutive frontal cortex paraffin sections of two FAD cases with PS1 L435F mutation (cases #1 and #2, 1st and 2nd rows), another FAD case with different PS1 mutation (case #3, 3rd row) and one SAD case (case #4, forth row). In both PS1 L435F mutation cases the numerous Aβ plaques mainly contain Aβ42 and Aβ43 but less Aβ40. This is in contrast to Aβ plaques of the control FAD and SAD cases, in which solely Aβ42 predominates and Aβ43 is sparse; even levels of Aβ40 seem to be lower than in the PS1 L435F cases. Note that in both PS1 L435F cases several plaques are larger than those seen in cases #3 and #4, representing cotton wool plaques. Scale bar = 500 µm. Magnification is identical in all pictures.


a ) Kaplan - Meier to first fever ( ≥ 38 ° C ) episode within 24 hours of receiving vaccines administered at 4 months of age ; control n = 88 and 4CMenB n = 89 .

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a) Kaplan-Meier to first fever (≥38°C) episode within 24 hours of receiving vaccines administered at 4 months of age; control n=88 and 4CMenB n=89.


b ) Principal component ( PC ) analysis of RNA - sequencing data ( 14837 genes , n = 253 ) from all study time points . The lines connect participant ' s pre - and post - vaccination samples . The ellipses are the two - dimensional ( PC1 and PC2 ) 95 % confidence intervals for each study time point . A contribution plot ( top right ) displays the genes contributing most to PC1 / PC2 , i . e . 0 . 15 implies 0 . 15 % contribution of that variable to the principal components displayed .

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b) Principal component (PC) analysis of RNA-sequencing data (14837 genes, n= 253) from all study time points. The lines connect participant's pre- and post-vaccination samples. The ellipses are the two-dimensional (PC1 and PC2) 95% confidence intervals for each study time point. A contribution plot (top right) displays the genes contributing most to PC1/PC2, i.e. 0.15 implies 0.15% contribution of that variable to the principal components displayed.


c ) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA - sequencing data , with median and interquartile range . P - values were determined from a two sample Wilcoxon rank sum test . The number of individuals in each group is display in the x - axis .

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c) Plotted are the CIBERSORTx neutrophil fractions from whole blood RNA-sequencing data, with median and interquartile range. P-values were determined from a two sample Wilcoxon rank sum test. The number of individuals in each group is display in the x-axis.


d ) Spearman ' s rank correlation between neutrophil counts measure by full blood counts ( n = 142 ) and those estimated by CIBERSORTx , using the LM22 signature matrix .

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d) Spearman's rank correlation between neutrophil counts measure by full blood counts (n= 142) and those estimated by CIBERSORTx, using the LM22 signature matrix.


a ) Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination ( 4 months of age ) 4 hours post - vaccination ( 719 DEGs , n = 28 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . The top 10 genes , ranked by FDR , are labelled .

a) Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination (4 months of age) 4 hours post-vaccination (719 DEGs, n=28). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). The top 10 genes, ranked by FDR, are labelled.


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination b ) Same as ( a ) but 24 hours post - vaccination ( 5553 DEGs , n = 31 )

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Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination b) Same as (a) but 24 hours post-vaccination (5553 DEGs, n=31)


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination c ) Same as ( a ) but 3 days post - vaccination ( 159 DEGs , n = 30 )

Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination c) Same as (a) but 3 days post-vaccination (159 DEGs, n=30)


Volcano plot highlighting differentially expressed genes ( DEGs , false - discovery rate [ FDR ] < 0 . 01 ; red upregulated and blue downregulated ) at each study time point versus pre - vaccination d ) Same as ( a ) but 7 days post - vaccination ( 6 DEGs , n = 36 ) .

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Volcano plot highlighting differentially expressed genes (DEGs, false-discovery rate [FDR] <0.01; red upregulated and blue downregulated) at each study time point versus pre-vaccination d) Same as (a) but 7 days post-vaccination (6 DEGs, n=36).


a ) Top genes differentially regulated between vaccine groups 4 hours post - vaccination . Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 . The expression E value , are the gene expression value derived from the voom - limma workflow ( Law et al . , 2014 ) .

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a) Top genes differentially regulated between vaccine groups 4 hours post-vaccination. Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36. The expression E value, are the gene expression value derived from the voom-limma workflow (Law et al., 2014).


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . b ) Same as ( a ) but top genes differentially regulated between vaccine groups 24 hours post - vaccination .

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Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). b) Same as (a) but top genes differentially regulated between vaccine groups 24 hours post-vaccination.


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . c ) Same as ( a ) but top genes differentially regulated between vaccine groups 3 days post - vaccination .

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

Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). c) Same as (a) but top genes differentially regulated between vaccine groups 3 days post-vaccination.


Note , only genes differentially expressed in either study group ( concomitant 4CMenB or control vaccines alone ) were included in intergroup analysis . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The FDR was derived by comparing fold - changes in gene expression in the control group with the test group , from baseline to the time point designated , and is reported if statistically significant ( FDR < 0 . 05 ) . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . d ) Same as ( a ) but top genes differentially regulated between vaccine groups 7 days post - vaccination .

Note, only genes differentially expressed in either study group (concomitant 4CMenB or control vaccines alone) were included in intergroup analysis. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The FDR was derived by comparing fold-changes in gene expression in the control group with the test group, from baseline to the time point designated, and is reported if statistically significant (FDR <0.05). P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). d) Same as (a) but top genes differentially regulated between vaccine groups 7 days post-vaccination.


False - discovery rate ( FDR ) are displayed above and were derived from a two sample Wilcoxon rank sum test ( Benjamini - Hochberg correction ) . Vertical lines represent the interquartile range around the median . The red dashed horizontal line is upper limit of the normal CRP reference interval ( 0 - 5mg / l ) . The number of individuals in each group is display in the x - axis .

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

False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). Vertical lines represent the interquartile range around the median. The red dashed horizontal line is upper limit of the normal CRP reference interval (0-5mg/l). The number of individuals in each group is display in the x-axis.


a , b ) Agreement plot . Red = differentially expressed ( DE ) in febrile infants , blue = DE in afebrile infants , purple = DE in febrile and afebrile infants .

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a, b) Agreement plot. Red = differentially expressed (DE) in febrile infants, blue = DE in afebrile infants, purple =DE in febrile and afebrile infants.


c ) Exemplar genes differentially expressed at 4 hours and 24 hours , respectively , between infants who experienced a fever within 24 hours of vaccination and afebrile infants . The plotted lines are the LOESS ( locally estimated scatterplot smoothing ) regression curves with the 95 % confidence intervals in grey . The false - discovery rate ( FDR ) was derived by comparing fold - changes in gene expression between infants who experienced post - vaccination fever and those who remained afebrile . P - values were obtained from the moderated t - statistic , after adjustment for multiple testing ( Benjamini and Hochberg ' s method ) . Pre - vaccination samples n = 125 , 4 hour samples n = 28 , 24 hour samples n = 31 , 3 day sample n = 30 , 7 day samples n = 36 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Exemplar genes differentially expressed at 4 hours and 24 hours, respectively, between infants who experienced a fever within 24 hours of vaccination and afebrile infants. The plotted lines are the LOESS (locally estimated scatterplot smoothing) regression curves with the 95% confidence intervals in grey. The false-discovery rate (FDR) was derived by comparing fold-changes in gene expression between infants who experienced post-vaccination fever and those who remained afebrile. P-values were obtained from the moderated t-statistic, after adjustment for multiple testing (Benjamini and Hochberg's method). Pre-vaccination samples n= 125, 4 hour samples n= 28, 24 hour samples n = 31, 3 day sample n= 30, 7 day samples n= 36.


Boxplots represent the median with interquartile ranges . False - discovery rate ( FDR ) are displayed above and were derived from a two sample Wilcoxon rank sum test ( Benjamini - Hochberg correction ) . N = 6 mice per vaccine group .

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Boxplots represent the median with interquartile ranges. False-discovery rate (FDR) are displayed above and were derived from a two sample Wilcoxon rank sum test (Benjamini-Hochberg correction). N = 6 mice per vaccine group.


Boxplots depicting the change in temperature from baseline to 24 hours after the second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide ( LPS ) and peptidoglycan ( PGN ) preparations , with and without alum , and combinations compared with 1 / 5 of the human dose of 4CMenB . Boxplots represent the median with interquartile ranges . Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed - rank test . * < 0 . 05 , * * < 0 . 01 , * * * * < 0 . 0001 . N = 6 mice per vaccine group .

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

Boxplots depicting the change in temperature from baseline to 24 hours after the second 1μg dose of each of a variety of Escherichia coli lipopolysaccharide (LPS) and peptidoglycan (PGN) preparations, with and without alum, and combinations compared with 1/5 of the human dose of 4CMenB. Boxplots represent the median with interquartile ranges. Vaccine group medians at each timepoint were compared with baseline for that vaccine using a Wilcoxon signed-rank test. * < 0.05, ** < 0.01, **** < 0.0001. N= 6 mice per vaccine group.


( A , B ) CMH2DCFDA staining ( A ) and data quantification ( B ) showing H2O2 level in different aged fly brains with and without treatment with the indicated RET inhibitors ( n = 5 per group ) .

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(A, B) CMH2DCFDA staining (A) and data quantification (B) showing H2O2 level in different aged fly brains with and without treatment with the indicated RET inhibitors (n=5 per group).


( I ) Dose - dependent effect of CPT on RET - ROS in mitochondria isolated from 45 days old flies and respiring under RET condition ( n = 4 ) .

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(I) Dose-dependent effect of CPT on RET-ROS in mitochondria isolated from 45 days old flies and respiring under RET condition (n=4).


( K ) CMH2DCFDA staining showing H2O2 level in young flies treated with DES or DES + CPT ( n = 5 per group ) .

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(K) CMH2DCFDA staining showing H2O2 level in young flies treated with DES or DES + CPT (n=5 per group).


( E - H ) Survival curves comparing control flies and flies fed with food containing 5 µM CPT ( E ) , 10 µM CPT ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( F ) , or 20 µM CPT ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( G ) from 35 days onwards . Bar graphs ( H ) show quantification of mean and maximal lifespan ( n = 3 groups , 20 - 25 flies per group ) , using single factor ANOVA with Scheffe ' s analysis as a post hoc test . : Data are representative of at least three repeats . H ) Data are shown as mean ± Survival curves were analyzed with logrank test along with Wang Allison test for maximum lifespan . Asterisks indicate statistical significance ( * * p < 0 . 01 , * p < 0 . 05 ) . Dashed lines mark time points of 50 % survival .

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 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 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 O O O O O O

(E-H) Survival curves comparing control flies and flies fed with food containing 5 µM CPT (E), 10 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (F), or 20 µM CPT (**p<0.01 logrank, **p<0.01 Wang Allison) (G) from 35 days onwards. Bar graphs (H) show quantification of mean and maximal lifespan (n=3 groups, 20-25 flies per group), using single factor ANOVA with Scheffe's analysis as a post hoc test. : Data are representative of at least three repeats. H) Data are shown as mean ± Survival curves were analyzed with logrank test along with Wang Allison test for maximum lifespan. Asterisks indicate statistical significance (**p< 0.01, *p< 0.05). Dashed lines mark time points of 50% survival.


( C ) Images and data quantification showing accumulation of protein aggregates in the brains of aged flies with or without CPT treatment ( n = 5 ) .

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(C) Images and data quantification showing accumulation of protein aggregates in the brains of aged flies with or without CPT treatment (n=5).


( D ) Images and data quantification showing DA neuron mitochondrial morphology in the brains of aged flies with or without CPT treatment ( n = 5 ) .

(D) Images and data quantification showing DA neuron mitochondrial morphology in the brains of aged flies with or without CPT treatment (n=5).


Images ( E ) showing 4 - HNE levels within or near TH - positive DA neurons in young flies , old flies , and old flies treated with CPT ( n = 4 sets , 5 samples per set ) . Arrows point to HNE signals co - labelled with TH - positive DA neurons .

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Images (E) showing 4-HNE levels within or near TH-positive DA neurons in young flies, old flies, and old flies treated with CPT (n=4 sets, 5 samples per set). Arrows point to HNE signals co-labelled with TH- positive DA neurons.


( D ) CMH2DCFDA staining of H2O2 and quantification of signal intensity in the muscle tissues of 50 - day old control flies and flies with NDUFS2 or NDUFS3 knockdown ( n = 5 per group ) .

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(D) CMH2DCFDA staining of H2O2 and quantification of signal intensity in the muscle tissues of 50-day old control flies and flies with NDUFS2 or NDUFS3 knockdown (n=5 per group).


( F ) Survival curves of wild type flies with or without NMN treatment from 35 days onwards ( * * p < 0 . 01 logrank , * * P < 0 . 01 Wang Allison ) ( n = 3 groups , 20 - 25 flies per group ) .

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(F) Survival curves of wild type flies with or without NMN treatment from 35 days onwards (**p<0.01 logrank, **P<0.01 Wang Allison) (n=3 groups, 20-25 flies per group).


( H ) Images and data quantification showing accumulation of protein aggregates in the muscle of young flies , old flies , and old flies treated with CPT ( n = 5 per group ) .

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(H) Images and data quantification showing accumulation of protein aggregates in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).


( A , B ) Survival curves showing effect of neuronal dSirt2 knockdown on fly lifespan under normal and CPT - treatment conditions ( n = 3 groups , 20 - 25 flies per group ) . ( C , D ) Survival curves showing effect of muscle dFoxo knockdown on fly lifespan under normal and CPT - treatment conditions ( n = 3 groups , 20 - 25 flies per group ) . ( E , F ) Survival curves showing effect of neuronal ATG1 knockdown on fly lifespan under normal and CPT - treatment conditions ( n = 3 groups , 20 - 25 flies per group ) .

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(A, B) Survival curves showing effect of neuronal dSirt2 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (C, D) Survival curves showing effect of muscle dFoxo knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group). (E, F) Survival curves showing effect of neuronal ATG1 knockdown on fly lifespan under normal and CPT-treatment conditions (n=3 groups, 20-25 flies per group).


( H ) Images and data quantification in single factor ANOVA with Scheffe ' s analysis as a post hoc test showing lysotracker staining in the muscle of young flies , old flies , and old flies treated with CPT ( n = 5 per group ) .

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(H) Images and data quantification in single factor ANOVA with Scheffe's analysis as a post hoc test showing lysotracker staining in the muscle of young flies, old flies, and old flies treated with CPT (n=5 per group).


( A , B ) Survival curves showing effect of CPT treatment on the lifespan of Mhc > APP . C99 flies ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( A ) and elav > APP . BACE flies ( * * p < 0 . 01 logrank , * * p < 0 . 01 Wang Allison ) ( B ) ( n = 3 groups , 20 - 25 flies per group ) .

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(A, B) Survival curves showing effect of CPT treatment on the lifespan of Mhc>APP.C99 flies (**p<0.01 logrank, **p<0.01 Wang Allison) (A) and elav>APP.BACE flies (**p<0.01 logrank, **p<0.01 Wang Allison) (B) (n=3 groups, 20-25 flies per group).


( E ) Immunofluorescence images and data quantification showing effect of CPT on the formation of ubiquitin - and 6E10 - positive amyloid aggregates in the brain of elav > APP . BACE flies ( n = 5 ) .

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(E) Immunofluorescence images and data quantification showing effect of CPT on the formation of ubiquitin- and 6E10-positive amyloid aggregates in the brain of elav>APP.BACE flies (n=5).


( G ) Quantification of the effect of CPT on DA neuron number in the PPL1 cluster of elav > APP / BACE fly brain ( n = 4 sets , 5 brains per set ) .

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(G) Quantification of the effect of CPT on DA neuron number in the PPL1 cluster of elav>APP/BACE fly brain (n=4 sets, 5 brains per set).


( A , B ) Quantification of effect of CPT on ROS level as detected by staining with the H2O2 dye CMH2DCFDA ( A ) , and on NAD + / NADH ratio ( B ) in control , FAD , and DS iPSC - derived neurons .

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(A, B) Quantification of effect of CPT on ROS level as detected by staining with the H2O2 dye CMH2DCFDA (A), and on NAD+/NADH ratio (B) in control, FAD, and DS iPSC-derived neurons.


( D ) Survival curves showing effect of CPT on lifespan of PS19 tauopathy mice ( * p < 0 . 05 logrank ) ( n = 9 in vehicle group , 8 in CPT group ) .

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(D) Survival curves showing effect of CPT on lifespan of PS19 tauopathy mice (*p<0.05 logrank) (n=9 in vehicle group, 8 in CPT group).


( f ) Percent survival of 15 d old plants with and without SA187 under NHS , HS and LAT conditions . Data information : percent survival measuremts after recovery at 22 ° C for 4 days . For percent survival , plants with green leaves were scored as live plant out of 12 plants for each biological repeat in different temperature treatments . All treatments are compared with direct 44 ° C heat stressed plants . Plots represent the mean of 3 biological replicates bars representSE Asterisks indicate a statistical difference based on the Mann - Whitney test ( * P < 0 . 05 )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percent survival of 15 d old plants with and without SA187 under NHS, HS and LAT conditions. Data information: percent survival measuremts after recovery at 22°C for 4 days. For percent survival, plants with green leaves were scored as live plant out of 12 plants for each biological repeat in different temperature treatments. All treatments are compared with direct 44°C heat stressed plants. Plots represent the mean of 3 biological replicates bars representSE Asterisks indicate a statistical difference based on the Mann-Whitney test (* P < 0.05 )


( a , b ) Dynamics of HSFA2 and HSP101 transcript levels in control ( NHS , NHS + 187 ) , 44 ° C heat - stressed non - colonized and SA187 - colonized ( HS , HS + 187 ) , thermoprimed ( LAT ) as well as thermoprimed and SA187 - colonized plants ( LAT + 187 ) at 1 h , 24 h , 48 h , 72 h and 96 h of recovery at 22 ° C . SA187 - colonized ( HS + 187 ) and thermoprimed ( LAT ) plants showed higher transcript levels in comparison to plants exposed at 44 ° C HS for 30 min ( HS ) after 1 h of recovery at 22 ° C . Data information : All plots represent the means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference based on Student ' s t - test ( * P ≤ 0 .

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(a,b) Dynamics of HSFA2 and HSP101 transcript levels in control (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed and SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C. SA187-colonized (HS+187) and thermoprimed (LAT) plants showed higher transcript levels in comparison to plants exposed at 44°C HS for 30 min (HS) after 1 h of recovery at 22°C. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05


( c , d ) . Transcript levels of heat stress memory genes HSP18 . 1 and APX2 in control plants ( NHS , NHS + 187 ) , 44 ° C heat - stressed non - colonized and SA187 - colonized ( HS , HS + 187 ) , thermoprimed ( LAT ) as well as thermoprimed SA187 - colonized plants ( LAT + 187 ) at 1 h , 24 h , 48 h , 72 h and 96 h of recovery at 22 ° C . Transcript levels were normalized to tubulin as reference gene and the respective 22 ° C NHS plants were harvested at the same time points . All treatments are compared with direct 44 ° C HS treatment for statistical significance . Data information : All plots represent the means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference based on Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,d). Transcript levels of heat stress memory genes HSP18.1 and APX2 in control plants (NHS, NHS+187), 44°C heat-stressed non-colonized and SA187-colonized (HS, HS+187), thermoprimed (LAT) as well as thermoprimed SA187-colonized plants (LAT+187) at 1 h, 24 h, 48 h, 72 h and 96 h of recovery at 22°C . Transcript levels were normalized to tubulin as reference gene and the respective 22°C NHS plants were harvested at the same time points. All treatments are compared with direct 44°C HS treatment for statistical significance. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001


( g ) Relative enrichment of H3K4me3 at APX2 and HSP18 . 2 in control non - primed ( NP ) , SA187 - colonized non - primed plants ( NP + 187 ) , 37 ° C - primed ( P ) and 37 ° C - primed SA187 - colonized plants ( P + 187 ) at 24 and 72 h after priming as determined by chromatin immunoprecipitation - qPCR for the indicated regions of APX2 and HSP18 . 2 . Amplification values were normalized to input and H3 and region 1 of non - primed ( NP ) plants . Data information : All plots represent the means of 3 biological replicates . Error bars represent SE . Asterisks indicate a statistical difference based on Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P and P + 187 treatments ) .

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(g) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2. Amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. Data information: All plots represent the means of 3 biological replicates. Error bars represent SE. Asterisks indicate a statistical difference based on Student's t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments).


( b , c ) Fresh weight and percent survival of Col - 0 , hsfa2 , hsfa1q and ein3 - 1 plants in HS , HS + 187 , LAT and LAT + 187 treatments . Due to the dwarf size of hsfa1q mutants , LAT treatment was performed on d 18 and HS at d 20 . All treatments are compared with plants upon 44 ° C HS . Data information : All plots represent the means of 3 biological replicates ( n = 36 , 12 plants per biological repeat ) . Error bars represent SD of three biological repeats . Asterisks indicate a statistical difference based on the Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,c) Fresh weight and percent survival of Col-0, hsfa2, hsfa1q and ein3-1 plants in HS, HS+187, LAT and LAT+187 treatments. Due to the dwarf size of hsfa1q mutants, LAT treatment was performed on d 18 and HS at d 20. All treatments are compared with plants upon 44°C HS. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


( e ) Percent survival of Col - 0 plants with and without ACC under HS , HS + ACC , LAT and LAT + ACC conditions . Data information : All plots represent the means of 3 biological replicates ( n = 36 , 12 plants per biological repeat ) . Error bars represent SD of three biological repeats . Asterisks indicate a statistical difference based on the Student ' s t - test ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ) .

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(e) Percent survival of Col-0 plants with and without ACC under HS, HS+ACC, LAT and LAT+ACC conditions. Data information: All plots represent the means of 3 biological replicates (n=36, 12 plants per biological repeat). Error bars represent SD of three biological repeats. Asterisks indicate a statistical difference based on the Student's t-test (* P≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001).


( c ) Relative enrichment of H3K4me3 at APX2 and HSP18 . 2 in control non - primed ( NP ) , SA187 - colonized non - primed plants ( NP + 187 ) , 37 ° C - primed ( P ) and 37 ° C - primed SA187 - colonized plants ( P + 187 ) at 24 and 72 h after priming as determined by chromatin immunoprecipitation - qPCR for the indicated regions of APX2 and HSP18 . 2 ( Fig 3F ) . Data information : For H3K4me3 enrichment analysis , amplification values were normalized to input and H3 and region 1 of non - primed ( NP ) plants . All treatments are compared with direct 44 ° C HS treatment for statistical significance . All plots represent the means of 3 biological replicates . Error bars indicate SE . Asterisks indicate a statistical difference based on the Student ' s t - test * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P and P + 187 treatments .

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(c) Relative enrichment of H3K4me3 at APX2 and HSP18.2 in control non-primed (NP), SA187-colonized non-primed plants (NP+187), 37°C-primed (P) and 37°C-primed SA187-colonized plants (P+187) at 24 and 72 h after priming as determined by chromatin immunoprecipitation-qPCR for the indicated regions of APX2 and HSP18.2 (Fig 3F). Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.


( f ) Relative enrichment of H3K4me3 levels at APX2 and HSP18 . 2 memory genes in hsfa2 mutant . Data information : For H3K4me3 enrichment analysis , amplification values were normalized to input and H3 and region 1 of non - primed ( NP ) plants . All treatments are compared with direct 44 ° C HS treatment for statistical significance . All plots represent the means of 3 biological replicates . Error bars indicate SE . Asterisks indicate a statistical difference based on the Student ' s t - test * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * P ≤ 0 . 001 for differences between NP in comparison to NP + 187 , P and P + 187 treatments .

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(f) Relative enrichment of H3K4me3 levels at APX2 and HSP18.2 memory genes in hsfa2 mutant. Data information: For H3K4me3 enrichment analysis, amplification values were normalized to input and H3 and region 1 of non-primed (NP) plants. All treatments are compared with direct 44°C HS treatment for statistical significance. All plots represent the means of 3 biological replicates. Error bars indicate SE. Asterisks indicate a statistical difference based on the Student's t-test *P ≤ 0.05; **P ≤ 0.01; **P ≤ 0.001 for differences between NP in comparison to NP+187, P and P+187 treatments.


( b ) Relative percent abundance of bacterial phyla in the soil and the wheat root endosphere without ( R - mock ) and with SA187 treatment ( R - 187 ) . Data information : Microbiome analysis was performed in triplicates .

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(b) Relative percent abundance of bacterial phyla in the soil and the wheat root endosphere without (R-mock) and with SA187 treatment (R-187). Data information: Microbiome analysis was performed in triplicates.


B . Western blot analysis of the dose - dependent expression of ZBP1 in HT29 / TO - ZBP1WT and HT29 / TO - ZBP1Zα1α2mut cells treated with indicated concentrations of Dox for 24 h . Blots are representative of three biological repeats .

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B. Western blot analysis of the dose-dependent expression of ZBP1 in HT29 / TO-ZBP1WT and HT29 / TO-ZBP1Zα1α2mut cells treated with indicated concentrations of Dox for 24 h. Blots are representative of three biological repeats.


I . Western blot analysis of cells from the same wells as used in ( H ) for ZBP1 expression levels , representative of four biological replicates .

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I. Western blot analysis of cells from the same wells as used in (H) for ZBP1 expression levels, representative of four biological replicates.


L . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with DMSO or 500 ng / mL Dox for 24 hours , or HT29 chemotaxis buffer ( control buffer ) containing equal volume and amount of DMSO or Dox as the conditioned media . Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO or Dox ( n = 10 biological replicates ) , or conditioned media from DMSO - treated cells ( n = 11 biological replicates ) or Dox - treated cells ( n = 12 biological replicates ) , where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils from four donors . An unpaired t - test was used to test for statistical differences between indicated conditions . * * * * p < 0 . 0001 .

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L. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with DMSO or 500 ng/mL Dox for 24 hours, or HT29 chemotaxis buffer (control buffer) containing equal volume and amount of DMSO or Dox as the conditioned media. Data is presented as individual values with grand mean of the migrated percentage induced by control buffer containing DMSO or Dox (n = 10 biological replicates), or conditioned media from DMSO-treated cells (n = 11 biological replicates) or Dox-treated cells (n = 12 biological replicates), where the number of biological replicates is defined by the total number of independent cell cultures tested on primary neutrophils from four donors. An unpaired t-test was used to test for statistical differences between indicated conditions. **** p < 0.0001.


M . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h . Data is presented as individual values with mean and S . E . M . ( n = 3 biological replicates of conditioned media ) . One - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p = 0 . 4129 ) ; * * * p = 0 . 0004 .

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M. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with DMSO or 20 µM zVAD + 10 μM Nec1s for 24 h. Data is presented as individual values with mean and S.E.M. (n = 3 biological replicates of conditioned media). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.4129); *** p = 0.0004.


C . Transwell migration of primary neutrophils towards conditioned media from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox in combination with indicated inhibitors for 24 h . Data is presented as individual values with grand mean ( n = 3 biological replicates of conditioned media from GSK ' 872 and NSA - treated wells , n = 6 biological replicates of conditioned media from other conditions ) . Two - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . * * * * p < 0 . 0001 ; ns = not significant ( p > 0 . 96 ) .

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C. Transwell migration of primary neutrophils towards conditioned media from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox in combination with indicated inhibitors for 24 h. Data is presented as individual values with grand mean (n = 3 biological replicates of conditioned media from GSK'872 and NSA-treated wells, n = 6 biological replicates of conditioned media from other conditions). Two-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. **** p < 0.0001; ns = not significant (p > 0.96).


D . CXCL8 concentration in the culture media of HT29 / TO - ZBP1WT cells transfected with siRNA targeting mismatch sequence ( MM ) or RIPK3 and treated with Dox at indicated concentrations for 24 h . Data is presented as mean with S . E . M . ( n = 4 biological replicates ) . Unpaired t - tests were used to test for statistical differences between indicated conditions . * * p = 0 . 0079 . Cell lysates from one biological replicate were analyzed by Western blotting .

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D. CXCL8 concentration in the culture media of HT29 / TO-ZBP1WT cells transfected with siRNA targeting mismatch sequence (MM) or RIPK3 and treated with Dox at indicated concentrations for 24 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Unpaired t-tests were used to test for statistical differences between indicated conditions. ** p = 0.0079. Cell lysates from one biological replicate were analyzed by Western blotting.


E . Cytokine concentration in the culture media of HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT cells treated with 0 , 50 or 500 ng / mL Dox . data is plotted as mean with S . E . M . ( n = 6 biological replicates ) . Brown - Forsythe and Welch ANOVA tests and Dunnet ' s T3 multiple comparisons test were used to test for statistical differences between indicated conditions . Cell lysates were analyzed by Western blotting for ZBP1 levels . Blots are representative of six biological replicates . ns = not significant ( p > 0 . 2 ) ; * * * p = 0 . 0003 for CXCL8 , p = 0 . 0008 for CXCL1 ; * * * * p < 0 . 0001 .

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E. Cytokine concentration in the culture media of HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0, 50 or 500 ng/mL Dox. data is plotted as mean with S.E.M. (n = 6 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. Cell lysates were analyzed by Western blotting for ZBP1 levels. Blots are representative of six biological replicates. ns = not significant (p > 0.2); *** p = 0.0003 for CXCL8, p = 0.0008 for CXCL1; **** p < 0.0001.


G , NF - κB activity in HEK293FT cells with and without stable expression of full - length ( FL ) RIPK3 - 2xFV or RIPK3 ∆ C - 2xFV ( ∆ C ) transfected with dual luciferase reporters and ZBP1 or empty vector ( EV ) as indicated . Reporter activities were measured 24 h after transfection . Data is presented as mean with S . E . M ( n = 3 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p > 0 . 05 ) ; * * * * p < 0 . 0001 . ( G ) Cell lysates were analyzed by Western blotting . Blots are representative of three biological replicates . Asterisk indicates background signals of the antibody .

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G, NF-κB activity in HEK293FT cells with and without stable expression of full-length (FL) RIPK3-2xFV or RIPK3∆C-2xFV (∆C) transfected with dual luciferase reporters and ZBP1 or empty vector (EV) as indicated. Reporter activities were measured 24 h after transfection. Data is presented as mean with S.E.M (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p > 0.05); **** p<0.0001. (G) Cell lysates were analyzed by Western blotting. Blots are representative of three biological replicates. Asterisk indicates background signals of the antibody.


A . Western blot analysis of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . Blots are representative of three biological replicates . Immunoprecipitated material was treated or not with USP21 ( 1 μM ) for deubiquitination . Input loaded was 5 % for ZBP1 and 1 % for co - immunoprecipitants . Asterisk indicates antibody heavy chain signal in IP samples .

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A. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Blots are representative of three biological replicates. Immunoprecipitated material was treated or not with USP21 (1 μM) for deubiquitination. Input loaded was 5% for ZBP1 and 1% for co-immunoprecipitants. Asterisk indicates antibody heavy chain signal in IP samples.


B . Enrichment of Ub - conjugates by GST - 1xUBA for analysis of ubiquitination status of RIPK1 , ZBP1 and RIPK3 in HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16h . Blots are representative of at least three biological replicates . After enrichment , samples were treated or not with USP21 ( 1 μM ) for deubiquitination . Asterisk indicates unspecific bands . Arrows indicate RIPK3 signal .

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B. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1, ZBP1 and RIPK3 in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16h. Blots are representative of at least three biological replicates. After enrichment, samples were treated or not with USP21 (1 μM) for deubiquitination. Asterisk indicates unspecific bands. Arrows indicate RIPK3 signal.


C . Enrichment of Ub - conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST - 1xUBA or linkage - specific SUBs in HT29 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h .

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C. Enrichment of Ub-conjugates for analysis of the ubiquitination status of RIPK1 and ZBP1 using GST-1xUBA or linkage-specific SUBs in HT29 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h.


D . Enrichment of Ub - conjugates by GST - 1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29 , HT29 / TO - ZBP1WT and HT29 / RIPK1 - KO aA3 / TO - ZBP1WT cells treated with 500 ng / mL Dox for 16 h . Blots are representative of four biological replicates . Asterisk indicates antibody background signal .

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D. Enrichment of Ub-conjugates by GST-1xUBA for analysis of ubiquitination status of RIPK1 and ZBP1 in HT29, HT29 / TO-ZBP1WT and HT29 / RIPK1-KO aA3 / TO-ZBP1WT cells treated with 500 ng/mL Dox for 16 h. Blots are representative of four biological replicates. Asterisk indicates antibody background signal.


E . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and GFP ( Ctrl ) or GFP - tagged SUBs as indicated . Luciferase reporter activities were measured 24 h after transfection and normalized to GFP + EV - transfected condition . Data is plotted as mean with S . E . M . ( n = 4 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1 / GFP - transfected condition . * * * * p < 0 . 0001 . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , GFP or GFP - tagged SUBs and Actin . Blots are representative of three biological replicates .

E. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and GFP (Ctrl) or GFP-tagged SUBs as indicated. Luciferase reporter activities were measured 24 h after transfection and normalized to GFP+EV-transfected condition. Data is plotted as mean with S.E.M. (n = 4 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between the indicated condition and ZBP1/GFP-transfected condition. **** p < 0.0001. Cell lysates were analyzed by Western blotting to determine expression of ZBP1, GFP or GFP-tagged SUBs and Actin. Blots are representative of three biological replicates.


F , NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and variants of OTULIN . Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1 / EV - transfected cells . Data is plotted as mean with S . E . M . Multiple Welch t - tests were used to test for statistical differences between the indicated condition and the ZBP1 / EV - transfected condition . ( F ) n = 4 biological replicates . * * p = 0 . 0012 , ns = not significant ( p = 0 . 2342 ) . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , or OTULIN , and Actin . Blots are representative of ( F ) four biological replicates .

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F, NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of OTULIN. Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (F) n = 4 biological replicates. ** p = 0.0012, ns = not significant (p = 0.2342). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, or OTULIN, and Actin. Blots are representative of (F) four biological replicates.


G . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with dual luciferase reporters , ZBP1 or EV , and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1 / EV - transfected cells . Data is plotted as mean with S . E . M . Multiple Welch t - tests were used to test for statistical differences between the indicated condition and the ZBP1 / EV - transfected condition . ( G ) n = 3 biological replicates . * p = 0 . 0137 ; ns = not significant ( p = 0 . 8374 ) . Cell lysates were analyzed by Western blotting to determine expression of ZBP1 , CYLD and Actin . Blots are representative of ( G ) three biological replicates .

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G. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with dual luciferase reporters, ZBP1 or EV, and variants of CYLD Luciferase reporter activities were measured 24 h after transfection and normalized to ZBP1/EV-transfected cells. Data is plotted as mean with S.E.M. Multiple Welch t-tests were used to test for statistical differences between the indicated condition and the ZBP1/EV-transfected condition. (G) n = 3 biological replicates. * p = 0.0137; ns = not significant (p = 0.8374). Cell lysates were analyzed by Western blotting to determine expression of ZBP1, CYLD and Actin. Blots are representative of (G) three biological replicates.


A . NF - κB activity in HEK293FT / RIPK3 - 2xFV cells transfected with siRNA targeting HOIP ( siHOIP ) or a mismatch sequence ( siMM ) , and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP ( control ) . Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM + ZBP1 samples . Data is plotted as mean with S . E . M . ( n = 4 biological replicates ) . A Welch ' s t - test was used to test for the statistical difference between indicated conditions . * * * * p < 0 . 0001 . HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting . Blots are representative of three biological replicates .

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A. NF-κB activity in HEK293FT / RIPK3-2xFV cells transfected with siRNA targeting HOIP (siHOIP) or a mismatch sequence (siMM), and 48 h later transfected with dual luciferase reporters and ZBP1 or GFP (control). Luciferase reporter activities were measured 24 h after transfection and normalized to the siMM+ZBP1 samples. Data is plotted as mean with S.E.M. (n = 4 biological replicates). A Welch's t-test was used to test for the statistical difference between indicated conditions. **** p < 0.0001. HOIP knockdown and ZBP1 expression levels were analyzed by Western blotting. Blots are representative of three biological replicates.


D - F . Western blot analysis of anti - FLAG ( ZBP1 ) immunoprecipitation from HT29 or HT29 / TO - ZBP1WT or HT29 / RIPK1 - KO clone aA3 / TO - ZBP1WT cells treated with 0 or 500 ng / mL Dox for 16 h . Asterisk indicates antibody heavy chain signal in IP samples . Blots are representative of at least ( D ) two and ( E - F ) three biological replicates .

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D-F. Western blot analysis of anti-FLAG (ZBP1) immunoprecipitation from HT29 or HT29 / TO-ZBP1WT or HT29 / RIPK1-KO clone aA3 / TO-ZBP1WT cells treated with 0 or 500 ng/mL Dox for 16 h. Asterisk indicates antibody heavy chain signal in IP samples. Blots are representative of at least (D) two and (E-F) three biological replicates.


B . Western blot analysis of HCT116 / RIPK3 - 2xFV cells ( FL and ∆ C ) treated with 100 nM dimerizer . Blots are representative of two biological replicates .

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B. Western blot analysis of HCT116 / RIPK3-2xFV cells (FL and ∆C) treated with 100 nM dimerizer. Blots are representative of two biological replicates.


D . CXCL8 concentration in the culture media of HCT116 / RIPK3 - 2xFV cells ( WT and RHIMmut ) treated with 100 nM dimerizer for 24 h . In RIPK3 - RHIMmut three key residues , VQV , of the RHIM region was changed to AAAA . Data is plotted as mean with S . E . M . ( n = 5 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between indicated time points . * * p = 0 . 0011 ; ns = not significant ( p = 0 . 9301 ) . Inset : Western blot analysis to determine RIPK3 expression levels in one biological replicate . Line indicates that image was cut and spliced to remove non - relevant lanes from the scanned blot .

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D. CXCL8 concentration in the culture media of HCT116 / RIPK3-2xFV cells (WT and RHIMmut) treated with 100 nM dimerizer for 24 h. In RIPK3-RHIMmut three key residues, VQV, of the RHIM region was changed to AAAA. Data is plotted as mean with S.E.M. (n = 5 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated time points. ** p = 0.0011; ns = not significant (p = 0.9301). Inset: Western blot analysis to determine RIPK3 expression levels in one biological replicate. Line indicates that image was cut and spliced to remove non-relevant lanes from the scanned blot.


H . Relative mRNA levels of CXCL8 in HCT116 / RIPK3 - 2xFV cells pretreated with 100 nM CpA for 0 . 5 h before treatment with 0 or 100 nM dimerizer for 3 h . Data is presented as mean with S . E . M . ( n = 3 biological replicates ) . One - way ANOVA and Sidak ' s multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO + dimerizer - treated condition . * * * * p < 0 . 0001 . ( H ) Cell lysates were analyzed by Western blotting for cIAP1 levels . Blots are representative of two biological replicates .

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H. Relative mRNA levels of CXCL8 in HCT116 / RIPK3-2xFV cells pretreated with 100 nM CpA for 0.5 h before treatment with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 3 biological replicates). One-way ANOVA and Sidak's multiple comparisons tests were used to test for statistical differences between indicated conditions and DMSO+dimerizer-treated condition. **** p < 0.0001. (H) Cell lysates were analyzed by Western blotting for cIAP1 levels. Blots are representative of two biological replicates.


J . Relative CXCL8 mRNA levels in WT or HOIP - knockout HCT116 / RIPK3 - 2xFV cells treated with 0 or 100 nM dimerizer for 3 h . Data is plotted as mean with S . E . M . ( n = 3 biological replicates ) . A Welch ' s t - test was used to test for the statistical difference as indicated . * * * p = 0 . 0010 . Cell lysates were loaded for Western blot analysis .

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J. Relative CXCL8 mRNA levels in WT or HOIP-knockout HCT116 / RIPK3-2xFV cells treated with 0 or 100 nM dimerizer for 3 h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). A Welch's t-test was used to test for the statistical difference as indicated. *** p = 0.0010. Cell lysates were loaded for Western blot analysis.


K . Relative CXCL8 mRNA levels in HCT116 / Tet - On - GFP - K63 - SUB / RIPK3 - 2xFV , HCT116 / Tet - On - GFP - M1 - SUB / RIPK3 - 2xFV and HCT116 / Tet - On - GFP / RIPK3 - 2xFV cells treated with 0 or 100 ng / mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h . Data is presented as mean with S . E . M . ( n = 4 biological replicates ) . Brown - Forsythe and Welch ANOVA tests and Dunnet ' s T3 multiple comparisons test were used to test for statistical differences between indicated conditions . ns = not significant ( p = 0 . 9997 ) ; * * p = 0 . 0030 ; * p = 0 . 0366 . Cells were analyzed by Western blotting for the inducible - expression levels of GFP ( control ) and GFP - SUBs . Blots are representative of three biological replicates .

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K. Relative CXCL8 mRNA levels in HCT116 / Tet-On-GFP-K63-SUB / RIPK3-2xFV, HCT116 / Tet-On-GFP-M1-SUB / RIPK3-2xFV and HCT116 / Tet-On-GFP / RIPK3-2xFV cells treated with 0 or 100 ng/mL Dox for 48 h before stimulated with 0 or 100 nM dimerizer for 3 h. Data is presented as mean with S.E.M. (n = 4 biological replicates). Brown-Forsythe and Welch ANOVA tests and Dunnet's T3 multiple comparisons test were used to test for statistical differences between indicated conditions. ns = not significant (p = 0.9997); ** p = 0.0030; * p = 0.0366. Cells were analyzed by Western blotting for the inducible-expression levels of GFP (control) and GFP-SUBs. Blots are representative of three biological replicates.


M . Relative CXCL8 mRNA levels in HCT116 / RIPK3 - 2xFV cells stably knocked down against mismatch ( shMM ) or two different sites of caspase - 8 ( shCASP8 - A and shCASP8 - B ) , pretreated with DMSO , 10 µM Nec1s , 10 µM GSK ' 872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h . Data is plotted as mean with S . E . M . ( n = 3 biological replicates ) . Two - way ANOVA and Tukey ' s multiple comparison tests were used to test for statistical differences between indicated conditions . * * * p = 0 . 0001 ; * * p = 0 . 0035 . Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase - 8 levels .

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M. Relative CXCL8 mRNA levels in HCT116 / RIPK3-2xFV cells stably knocked down against mismatch (shMM) or two different sites of caspase-8 (shCASP8-A and shCASP8-B), pretreated with DMSO, 10 µM Nec1s, 10 µM GSK'872 or 1 µM NSA for 1h followed by treatment with 0 or 100 nM dimerizer for 3h. Data is plotted as mean with S.E.M. (n = 3 biological replicates). Two-way ANOVA and Tukey's multiple comparison tests were used to test for statistical differences between indicated conditions. *** p = 0.0001; ** p = 0.0035. Cell lysates from one biological replicate were analyzed by Western blotting to determine caspase-8 levels.


A . Western blot analysis of ZBP1 , RIPK1 , RIPK3 , MLKL levels in Calu - 3 cell lines with stable expression of shRNAs targeting ZBP1 or a mismatch sequence ( shMM ) , untreated ( NT ) or treated with 10 ng / mL IFNβ for 48 h . Blots are representative of two biological replicates .

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A. Western blot analysis of ZBP1, RIPK1, RIPK3, MLKL levels in Calu-3 cell lines with stable expression of shRNAs targeting ZBP1 or a mismatch sequence (shMM), untreated (NT) or treated with 10 ng/mL IFNβ for 48 h. Blots are representative of two biological replicates.


A . BAZ2A expression is higher in ESC + 2i than in differentiated cells ( neural progenitors , NPC ) . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ) . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and NPC . Tubulin is shown as a protein loading control .

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A. BAZ2A expression is higher in ESC+2i than in differentiated cells (neural progenitors, NPC). Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Right panel. Western blot showing BAZ2A protein levels in ESC+2i and NPC. Tubulin is shown as a protein loading control.


B . BAZ2A associates with chromatin of ESC and NPC . Chromatin - bound ( Chrom . ) and soluble ( Sol . ) fractions of equivalent cell number of ESC + 2i and NPCs were analyzed by western blot for BAZ2A levels . Total , total protein . Tubulin and histones are shown as loading and fractionation control .

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B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control.


C . siRNA - knockdown efficiency of BAZ2A shown by qRT - PCR and western blot . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to each ESC line . Average values of three independent experiments . Error bars represent s . d . and statistical significance ( P - values ) was calculated using the paired two - tailed t - test ( * * * * < 0 . 0001 ) . Right panel . Western blot showing BAZ2A protein levels . Tubulin is shown as a protein loading control .

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C. siRNA-knockdown efficiency of BAZ2A shown by qRT-PCR and western blot. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to each ESC line. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (**** < 0.0001). Right panel. Western blot showing BAZ2A protein levels. Tubulin is shown as a protein loading control.


D . BAZ2A knockdown affects proliferation of ESC + 2i but not of ESC + serum . Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * < 0 . 01 ; ns , non - significant ) .

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D. BAZ2A knockdown affects proliferation of ESC+2i but not of ESC+serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01; ns, non-significant).


E . BAZ2A is expressed at similar levels in both ESC + 2i and ESC + serum . Left panel . BAZ2A mRNA levels were measured by qRT - PCR and normalized to Rps12 mRNA and to ESC + 2i . Average values of three independent experiments . Error bars represent s . d . Right panel . Western blot showing BAZ2A protein levels in ESC + 2i and ESC + serum . Tubulin and histone H3 are shown as a protein loading controls .

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E. BAZ2A is expressed at similar levels in both ESC+2i and ESC+serum. Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. Right panel. Western blot showing BAZ2A protein levels in ESC+2i and ESC+serum. Tubulin and histone H3 are shown as a protein loading controls.


F . BAZ2A is required for the differentiation of ESC + 2i but not of ESC + serum . Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10 % serum and in the absence of LIF .

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F. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF.


G . Quantification of differentiated cells . Values represent relative number of differentiated cells from three independent experiments relative to control cells . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * * * < 0 . 001 ; ns , non - significant ) .

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G. Quantification of differentiated cells. Values represent relative number of differentiated cells from three independent experiments relative to control cells. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001; ns, non-significant).


H . Western blot showing BAZ2A protein levels in two BAZ2A - KO ESC lines obtained via CRISPr / Cas9 directly in ESC + serum . PARP1 is shown as a protein loading control .

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H. Western blot showing BAZ2A protein levels in two BAZ2A-KO ESC lines obtained via CRISPr/Cas9 directly in ESC+serum. PARP1 is shown as a protein loading control.


I . Cell proliferation is not affected in BAZ2A - KO ESC + serum . Data represent relative cell numbers 2 days culture starting with the same number of cells . Average values of four independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( ns , non - significant ) .

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I. Cell proliferation is not affected in BAZ2A-KO ESC+serum. Data represent relative cell numbers 2 days culture starting with the same number of cells. Average values of four independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns, non-significant).


J . Representative images of wt and Baz2a - KO ESC + serum before and after transition in 2i conditions .

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J. Representative images of wt and Baz2a-KO ESC+serum before and after transition in 2i conditions.


A . Volcano plot showing fold change ( log2 values ) in transcript level of ESC + 2i and ESC + serum with two consecutive treatments with siRNA - control and siRNA - Baz2a , each one lasting for 4 days . Gene expression values of three replicates were averaged and selected for 1 . 5 fold changes and P < 0 . 05 . Statistical significance ( P - values ) was calculated using R package DEseq2 .

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A. Volcano plot showing fold change (log2 values) in transcript level of ESC+2i and ESC+serum with two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Gene expression values of three replicates were averaged and selected for 1.5 fold changes and P < 0.05. Statistical significance (P-values) was calculated using R package DEseq2.


B . Validation by qRT - PCR of genes regulated by BAZ2A in ESC + 2i but not in ESC + serum ( upregulated genes in ESC + 2i upon BAZ2A - KD are labeled in blue , downregulated genes are in red ) . Nanog , Rex1 and Actin B ( ActB ) are shown as genes not regulated by BAZ2A . mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA - Control . Average values of three independent experiments . Error bars represent s . d . Statistical significance ( P - values ) for the experiments was calculated using the paired two - tailed t - test ( * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ) . Data without P values are statistically non significant .

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B. Validation by qRT-PCR of genes regulated by BAZ2A in ESC+2i but not in ESC+serum (upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red). Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001). Data without P values are statistically non significant.


A . Western blot showing equal BAZ2A levels in wt - ESC and F / H - BAZ2A ESC line containing FLAG - HA sequences at the N - terminus of both Baz2a alleles . Whole cell lysates from equivalent amounts of cells were analysed . SNF2H , and Tubulin are shown as protein loading controls .

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A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls.