LPS - primed neutrophils of indicated genotypes were left untreated ( Mock ) or stimulated with Flatox ( 3 hr ) ( G , H ) . IL - 1β secretion levels were determined in culture media G ) ; and combined cell lysates and culture media ( Lys + Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) H ) . Cytokine values represent mean ± SEM of n = 3 biological repeats and immunoblots are representative of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of indicated genotypes were left untreated (Mock) or stimulated with Flatox (3 hr) (G, H). IL-1β secretion levels were determined in culture media G); and combined cell lysates and culture media (Lys+Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) H). Cytokine values represent mean ± SEM of n=3 biological repeats and immunoblots are representative of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with LeTx ( A ) , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with LeTx (A), PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( B ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (B) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with ATP ( C ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with ATP (C) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( D ) . PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Error bars represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) . All data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (D). PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Error bars represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni). All data are representative of n=3 biological repeats.


( E ) LPS - primed B6 BMNs were left untreated ( LPS ) or subsequently stimulated with FlaTox ( LPS + FlaTox ) for 3 hr . Data are representative of n = 3 biological repeats . Culture media ( Sup ) and whole cell lysates ( Lys ) were immunoblotted for release of cytosolic GAPDH into culture media . Data are representative of n = 3 biological repeats .

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

(E) LPS-primed B6 BMNs were left untreated (LPS) or subsequently stimulated with FlaTox (LPS+FlaTox) for 3 hr. Data are representative of n=3 biological repeats. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.


( F ) B6 BMNs were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . Culture media ( Sup ) and whole cell lysates ( Lys ) were immunoblotted for release of cytosolic GAPDH into culture media . Data are representative of n = 3 biological repeats .

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

(F) B6 BMNs were left untreated (Mock) or infected with S. Typhimurium at MOI 25. Culture media (Sup) and whole cell lysates (Lys) were immunoblotted for release of cytosolic GAPDH into culture media. Data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) ( A Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of the antibody . All data are representative of n = 3 biological repeats .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) (A Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) ( B , Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of the antibody . All data are representative of n = 3 biological repeats .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) (B, Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) ( C , Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted separately for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) . * with Western blots indicates non - specific cross - reactivity of the antibody . All data are representative of n = 3 biological repeats .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) (C, Whole cell lysates (Lys) and culture media (Sup) were immunoblotted separately for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30). * with Western blots indicates non-specific cross-reactivity of the antibody. All data are representative of n=3 biological repeats.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) D , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) D, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) E , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) E, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) F , PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) F, PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with TcdA ( 3 hr ) G ) , IL - 1β secretion levels were determined by Luminex assay . Values represent mean ± SEM of n = 3 ( G , biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

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 B-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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with TcdA (3 hr) G), IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 (G, biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with FlaTox ( 3 hr ) , H ) IL - 1β secretion levels were determined by Luminex assay . Values represent mean ± SEM of n = 4 ( H ) biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with FlaTox (3 hr) , H) IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=4 (H) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) or stimulated with nigericin ( 100 mins ) I ) . IL - 1β secretion levels were determined by Luminex assay . Values represent mean ± SEM of n = 3 I ) biological repeats . Statistical significance was analysed by two - way ANOVA : * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 .

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 B-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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock) or stimulated with nigericin (100 mins) I). IL-1β secretion levels were determined by Luminex assay. Values represent mean ± SEM of n=3 I) biological repeats. Statistical significance was analysed by two-way ANOVA: *, p ≤0.05; **, p ≤ 0.01.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for cleavage of pro - IL - 1β ( ~ 39 kDa ) into mature IL - 1β ( p17 ) and cleaved GSDMD ( p30 ) ( A ) * with Western blots indicates non - specific bands .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for cleavage of pro-IL-1β (~39 kDa) into mature IL-1β (p17) and cleaved GSDMD (p30) (A) * with Western blots indicates non-specific bands.


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) ( B , PMP was assessed by measurement of SYTOX Green incorporation over time . Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * * , p ≤ 0 . 001 ; ns = non - significant . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) (B, PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with PMA ( 3 hr ) Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for extracellular release of β - actin and

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with PMA (3 hr) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with nigericin ( 100 mins ) Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for extracellular release of β - actin and

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with nigericin ( 100 mins ) G ) PMP was assessed by measurement of SYTOX Green incorporation over time . Values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * * , p ≤ 0 . 001 ; ns = non - significant . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with nigericin (100 mins) G) PMP was assessed by measurement of SYTOX Green incorporation over time. Values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: ***, p ≤ 0.001; ns= non-significant. P-values were corrected for multiple comparisons (Bonferroni).


LPS - primed neutrophils of the indicated genotypes were left untreated ( Mock ) , stimulated with TcDA ( 3 hr ) ( H ) . Whole cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for extracellular release of β - actin and GAPDH H ) .

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

LPS-primed neutrophils of the indicated genotypes were left untreated (Mock), stimulated with TcDA (3 hr) (H). Whole cell lysates (Lys) and culture media (Sup) were immunoblotted for extracellular release of β-actin and GAPDH H).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( A ) Representative confocal micrographs taken at different time points were stained with DAPI ( dark blue nuclei ) and SYTOX Green ( light blue ) . Scale bar : 20 μM .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (A) Representative confocal micrographs taken at different time points were stained with DAPI (dark blue nuclei) and SYTOX Green (light blue). Scale bar: 20 μM.


( B ) Representative confocal micrographs of untreated ( Mock ) , S . Typhimurium - infected or PMA - stimulated wildtype BMNs stained for neutrophil elastase ( Red ) and SYTOX Green ( Green ) ( Scale bar : 10 μM ) .

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

(B) Representative confocal micrographs of untreated (Mock), S. Typhimurium-infected or PMA-stimulated wildtype BMNs stained for neutrophil elastase (Red) and SYTOX Green (Green) (Scale bar: 10 μM).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( C , Cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for the indicated proteins at 1h and 3 hr post - infection . Data information : S . Typh . : S . Typhimurium .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (C, Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . D ) Cell lysates ( Lys ) and culture media ( Sup ) were immunoblotted for the indicated proteins at 1h and 3 hr post - infection . Data information : S . Typh . : S . Typhimurium .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. D) Cell lysates (Lys) and culture media (Sup) were immunoblotted for the indicated proteins at 1h and 3 hr post-infection. Data information: S. Typh.: S. Typhimurium.


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( E ) Cell death was measured by LDH assay 3 h post - infection . Data information : In E , values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (E) Cell death was measured by LDH assay 3 h post-infection. Data information: In E, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( F ) IL - 1β secretion levels were determined by Luminex assay 1 hr post - infection . Data information : In F , values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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 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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (F) IL-1β secretion levels were determined by Luminex assay 1 hr post-infection. Data information: In F, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


Neutrophils of the indicated genotypes were left untreated ( Mock ) or infected with S . Typhimurium at MOI 25 . ( G ) Representative confocal micrographs of neutrophils stained with DAPI ( dark blue nuclei ) and SYTOX Green ( light blue ) taken at different time points post - infection ( scale bar : 20 μM ) . ( H ) PMP was assessed by measurement of SYTOX Green incorporation over time . Data information : In H , values represent mean ± SEM of n = 3 biological repeats . Statistical significance was analysed by two - way ANOVA : * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 . P - values were corrected for multiple comparisons ( Bonferroni ) .

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

Neutrophils of the indicated genotypes were left untreated (Mock) or infected with S. Typhimurium at MOI 25. (G) Representative confocal micrographs of neutrophils stained with DAPI (dark blue nuclei) and SYTOX Green (light blue) taken at different time points post-infection (scale bar: 20 μM). (H) PMP was assessed by measurement of SYTOX Green incorporation over time. Data information: In H, values represent mean ± SEM of n=3 biological repeats. Statistical significance was analysed by two-way ANOVA: **, p ≤ 0.01; ***, p ≤ 0.001. P-values were corrected for multiple comparisons (Bonferroni).


( A Representative images of HUVECs ( A ; n = 6 ) sprouting in a fibrin gel with VEGF ( 25ng / ml ) in the presence or absence of COCO . ( D Quantification of tube surface area of micrographs shown in ( A , ( D : * P = 0 . 0119 ( - vs COCO 30ng / ml ) ; * P = 0 . 0108 ( - vs COCO 60ng / ml )

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

(A Representative images of HUVECs (A; n=6) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. (D Quantification of tube surface area of micrographs shown in (A, (D: *P=0.0119 (- vs COCO 30ng/ml); *P=0.0108 (- vs COCO 60ng/ml)


B ) Representative images of HRMECs ( B ; n = 4 ) sprouting in a fibrin gel with VEGF ( 25ng / ml ) in the presence or absence of COCO . E ) Quantification of tube surface area of micrographs shown in B ) . ( E : * * P = 0 . 0013 ( - vs COCO 30ng / ml ) ; * * * P = 0 . 00026 ( - vs COCO 60ng / ml ) ) .

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

B) Representative images of HRMECs (B; n=4) sprouting in a fibrin gel with VEGF (25ng/ml) in the presence or absence of COCO. E) Quantification of tube surface area of micrographs shown in B). (E: **P=0.0013 (- vs COCO 30ng/ml); ***P=0.00026 (- vs COCO 60ng/ml)).


( C ) Representative images of choroidal explants cultured for 5 days in the presence or absence of COCO . Scale bar , 500 μm . ( F ) Quantification of sprouting surface area of micrographs shown in ( C ; n = 6 ) .

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(C) Representative images of choroidal explants cultured for 5 days in the presence or absence of COCO. Scale bar, 500 μm. (F) Quantification of sprouting surface area of micrographs shown in (C; n=6).


( G ) COCO decreases scratch wound migration after 18h . Scale bar , 150 μm . ( H ) Quantification of wound closure .

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(G) COCO decreases scratch wound migration after 18h. Scale bar, 150 μm. (H) Quantification of wound closure.


( I ) COCO decreases EdU incorporation in cultured HUVECs . ( J ) Quantification of EdU incorporation .

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(I) COCO decreases EdU incorporation in cultured HUVECs. (J) Quantification of EdU incorporation.


( K ) COCO does not induced apoptosis in HUVECs . ( L ) Quantification of apoptotic ( Annexin V - positive ) and viable ( Annexin V - negative ) cells ( n = 4 ) .

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(K) COCO does not induced apoptosis in HUVECs. (L) Quantification of apoptotic (Annexin V-positive) and viable (Annexin V-negative) cells (n= 4).


( B ) Retinal flat mounts of P5 mice injected with PBS , COCO or Flt1Fc are stained with IB4 ( negative images of the fluorescent signal ) . Scale bar , 100 μm . ( C ) Quantification of vascular length and number of branchpoints ( p - values are versus PBS treatment ) . Vessel length : * * P = 0 . 0059 ( PBS vs COCO ) ; * P = 0 . 0169 ( PBS vs Flt1Fc ) . Vessel junctions : * * P = 0 . 0063 ( PBS vs COCO ) ; * P = 0 . 0159 ( PBS vs Flt1Fc ) ; ( n = 4 mice / group ) .

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

(B) Retinal flat mounts of P5 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0059 (PBS vs COCO); *P=0.0169 (PBS vs Flt1Fc). Vessel junctions: **P=0.0063 (PBS vs COCO); *P=0.0159 (PBS vs Flt1Fc); (n=4 mice/group).


( D ) COCO does not increase empty collagen IV sleeves . Scale bar , 50 μm . ( E ) Quantification of % of IsoB4 + vessels to Coll IV + vessels ; ( n = 9 ) .

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(D) COCO does not increase empty collagen IV sleeves. Scale bar, 50 μm. (E) Quantification of % of IsoB4+ vessels to Coll IV+ vessels; (n=9).


( F ) Visualization of pericyte coverage ( NG2 : green ; IsoB4 : red ) in PBS - or COCO - injected retinas . Scale bar , 50 μm . ( G ) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining ( % overlay ) ( n = 8 ) .

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(F) Visualization of pericyte coverage (NG2:green; IsoB4:red) in PBS- or COCO-injected retinas. Scale bar, 50 μm. (G) Quantification of percentage of IsoB4 vascular staining covered by NG2 staining (% overlay) (n=8).


( H ) COCO injections ( P1 ) do not result in apoptosis in P5 retinas . Scale bar , 40 μm . ( I ) Quantification of apoptotic cells in control and COCO - injected mice ( n = 3 ) .

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(H) COCO injections (P1) do not result in apoptosis in P5 retinas. Scale bar, 40 μm. (I) Quantification of apoptotic cells in control and COCO-injected mice (n=3).


( B ) Retinal flat mounts of P28 mice injected with PBS , COCO or Flt1Fc are stained with IB4 ( negative images of the fluorescent signal ) . Scale bar , 100 μm . ( C ) Quantification of vascular length and number of branchpoints ( p - values are versus PBS treatment ) . Vessel length : * * P = 0 . 0019 ( PBS vs COCO ) ; * P = 0 . 0128 ( PBS vs Flt1Fc ) . Vessel junctions : * * P = 0 . 0073 ( PBS vs COCO ) ; * P = 0 . 0302 ( PBS vs Flt1Fc ) ; ( n = 4 ) .

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

(B) Retinal flat mounts of P28 mice injected with PBS, COCO or Flt1Fc are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints (p-values are versus PBS treatment). Vessel length: **P=0.0019 (PBS vs COCO); *P=0.0128 (PBS vs Flt1Fc). Vessel junctions: **P=0.0073 (PBS vs COCO); *P=0.0302 (PBS vs Flt1Fc); (n=4).


( D ) COCO injections ( P1 , P7 and P14 ) do not result in ONL thinning in P28 retinas . Scale bar , 40 μm . ( E ) Quantification of apoptotic cells ( cleaved caspase - 3 ; CC3 ) in control and COCO - injected mice ( p - values are versus PBS treatment ) . ( n = 4 ) .

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

(D) COCO injections (P1, P7 and P14) do not result in ONL thinning in P28 retinas. Scale bar, 40 μm. (E) Quantification of apoptotic cells (cleaved caspase-3; CC3) in control and COCO-injected mice (p-values are versus PBS treatment). (n=4).


( B ) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 ( negative images of the fluorescent signal ) . Scale bar , 100 μm . ( C ) Quantification of vascular length and number of branchpoints ; ( n = 4 ) .

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

(B) Retinal flat mounts of adult mice injected with PBS or COCO for 5 days are stained with IB4 (negative images of the fluorescent signal). Scale bar, 100 μm. (C) Quantification of vascular length and number of branchpoints; (n=4).


( E ) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4 . Scale bar , 90 μm . ( F ) Quantification of vascular length and number of branchpoints ; ( n = 4 ) .

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(E) Retinal flat mounts of adult mice injected with PBS or COCO for 28 days are stained with IB4. Scale bar, 90 μm. (F) Quantification of vascular length and number of branchpoints; (n=4).


( B ) Retinal flat mounts of P17 OIR mice injected with PBS , COCO or Flt1Fc are stained with IB4 . Red areas highlight vascular tufts .

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(B) Retinal flat mounts of P17 OIR mice injected with PBS, COCO or Flt1Fc are stained with IB4. Red areas highlight vascular tufts.


( C ) Quantification of neovascular tuft area in P17 OIR . * * P = 0 . 0021 ( PBS vs COCO ) ; * P = 0 . 0122 ( PBS vs Flt1Fc ) ; ( n = 6 ) . ( D ) Quantification of central avascular area in P17 OIR ; ( n = 6 ) .

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 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 neovascular tuft area in P17 OIR. **P=0.0021 (PBS vs COCO); *P=0.0122 (PBS vs Flt1Fc); (n=6). (D) Quantification of central avascular area in P17 OIR; (n=6).


( F ) Choroidal flat mounts of adult mice subjected two weeks prior to Laser - CNV injected with PBS , COCO or Flt1Fc stained with IB4 ( green ) and phalloidin ( red ) . Scale bar , 100 μm . ( G ) Quantification of CNV surface area . * P = 0 . 0210 ( PBS vs COCO ) ; * P = 0 . 0113 ( PBS vs Flt1Fc ) ; ( n = 12 - 14 burns from 5 animals / group ) .

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

(F) Choroidal flat mounts of adult mice subjected two weeks prior to Laser-CNV injected with PBS, COCO or Flt1Fc stained with IB4 (green) and phalloidin (red). Scale bar, 100 μm. (G) Quantification of CNV surface area. *P=0.0210 (PBS vs COCO); *P=0.0113(PBS vs Flt1Fc); (n=12-14 burns from 5 animals/group).


( A ) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min . Immunoblots are representative of 4 independent experiments . ( B ) Densitometric analysis of western blot experiments . * P = 0 . 0457 ; ( n = 4 ) .

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(A) Western blot analysis of HUVECs treated 24 hours prior with COCO and stimulated with VEGF for up to 60 min. Immunoblots are representative of 4 independent experiments. (B) Densitometric analysis of western blot experiments. * P=0.0457; (n=4).


( C ) COCO treatment does not alter VEGF receptor expression in HUVECs . Immunoblots are representative of 3 independent experiments . ( D ) Densitometric analysis of western blot experiments ; ( n = 3 ) .

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(C) COCO treatment does not alter VEGF receptor expression in HUVECs. Immunoblots are representative of 3 independent experiments. (D) Densitometric analysis of western blot experiments; (n=3).


( A ) Western blot of whole adult human retina extracts incubated with an anti - human COCO antibody and revealing a unique band at ~ 36 kDa ( arrow ) . Recombinant human COCO was used as positive control .

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

(A) Western blot of whole adult human retina extracts incubated with an anti-human COCO antibody and revealing a unique band at ~36 kDa (arrow). Recombinant human COCO was used as positive control.


( B ) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti - mouse COCO antibody and revealing a unique band at ~ 36 - 38 kDa ( arrow ) . Western blots are representative of 3 independent experiments .

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(B) Western blot of photoreceptors produced from human embryonic stem cells incubated with an anti-mouse COCO antibody and revealing a unique band at ~36-38 kDa (arrow). Western blots are representative of 3 independent experiments.


( C ) Immunofluoresence analysis of adult human retina sections with an anti - mouse COCO antibody . Specific immuno - reactivity was observed in multiple areas ( arrows ) when compared with sections only exposed to the secondary antibody .

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(C) Immunofluoresence analysis of adult human retina sections with an anti-mouse COCO antibody. Specific immuno-reactivity was observed in multiple areas (arrows) when compared with sections only exposed to the secondary antibody.


( D ) RNAscope in situ hybridization ( Dand5 , top ; Negative probe ( dapB ) ; down ) and hematoxylin staining of P5 mouse retinas .

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(D) RNAscope in situ hybridization (Dand5, top; Negative probe (dapB); down) and hematoxylin staining of P5 mouse retinas.


( E ) Dual RNAscope in situ hybridization and visual - arrestin immunohistochemistry of P5 mouse retinas . Images are representative of 4 animals .

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(E) Dual RNAscope in situ hybridization and visual-arrestin immunohistochemistry of P5 mouse retinas. Images are representative of 4 animals.


( F ) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation . Exogenously added COCO was detected using an anti - human COCO antibody , showing co - localization with human mitochondria ( MTCO1 antibody ) .

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(F) Immunofluoresence analysis of HUVECs exposed to COCO for 5 hrs prior to fixation. Exogenously added COCO was detected using an anti-human COCO antibody, showing co-localization with human mitochondria (MTCO1 antibody).


( B ) Differential gene expression heatmap generated by DESeq2 of top - altered genes of HUVECs treated with COCO compared to PBS - treated control cells ( n = 4 independent samples / group ) .

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(B) Differential gene expression heatmap generated by DESeq2 of top-altered genes of HUVECs treated with COCO compared to PBS-treated control cells (n = 4 independent samples/group).


( A ) Seahorse analysis of mitochondrial respiration ( OCR ) and glycolysis ( ECAR ) in HUVECs stimulated with COCO for 1 , 2 and 24 hours . * * P = 0 . 0032 for ECAR , 1hour ; * * P = 0 . 0079 for ECAR , 2hours ; * P = 0 . 0255 for OCR , 2hours ; ( n = 5 ) .

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(A) Seahorse analysis of mitochondrial respiration (OCR) and glycolysis (ECAR) in HUVECs stimulated with COCO for 1, 2 and 24 hours. **P=0.0032 for ECAR, 1hour; **P=0.0079 for ECAR, 2hours; *P=0.0255 for OCR, 2hours; (n=5).


( B ) Media glucose levels ( BioNova analysis ) in HUVECs cultured with COCO for 1 or 6 hours , expressed as % of initial levels * P = 0 . 0459 ; ( n = 4 ) .

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(B) Media glucose levels (BioNova analysis) in HUVECs cultured with COCO for 1 or 6 hours, expressed as % of initial levels *P=0.0459; (n=4).


( C ) Evaluation of ATP content in HUVECs stimulated with COCO for 1 , 6 or 24 hours . * P = 0 . 0120 ( - vs 1hour ) ; * P = 0 . 0109 ( - vs 6hours ) ; * P = 0 . 0219 ( - vs 2 - DG ) ; ( n = 4 ) .

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

(C) Evaluation of ATP content in HUVECs stimulated with COCO for 1, 6 or 24 hours. *P=0.0120 (- vs 1hour); *P=0.0109 (- vs 6hours); *P=0.0219 (- vs 2-DG); (n=4).


( D ) Determination of NAD + / NADH ratio in NADH for 6 or 24 hours with COCO . * * P = 0 . 0028 ( 6hours ) ; * P = 0 . 0207 ( 24hours ) ; ( n = 3 ) .

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

(D) Determination of NAD+/NADH ratio in NADH for 6 or 24 hours with COCO. **P=0.0028 (6hours); *P=0.0207 (24hours); (n=3).


E ) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1 , 3 , 6 , 12 or 24 hours . * P = 0 . 0244 ; ( n = 4 ) .

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E) Evaluation of pyruvate content in HUVECs stimulated with COCO for 1, 3, 6, 12 or 24 hours. *P=0.0244; (n=4).


( F ) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and / or N - acetylcystein . * P = 0 . 0343 ( - vs 48hours - NAC ) ; * P = 0 . 0114 ( - vs H2O2 - NAC ) ; * P = 0 . 0252 ( 48hours - NAC vs 48hours + NAC ) ; * * P = 0 . 0067 ( H2O2 - NAC vs H2O2 + NAC ) ; ( n = 4 ) .

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

(F) Determination of cellular ROS using DCFDA fluorescence in HUVECs treated with COCO and/or N-acetylcystein. *P=0.0343 (- vs 48hours-NAC); *P=0.0114 (- vs H2O2-NAC); *P=0.0252 (48hours-NAC vs 48hours+NAC); **P=0.0067 (H2O2-NAC vs H2O2+NAC); (n=4).


( G ) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO . * P = 0 . 0372 ( - vs 24hours ) ; * P = 0 . 0137 ( - vs 3 - NP ) ; ( n = 3 ) .

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(G) Determination of mitochondrial superoxide using MitoSox fluorescence in HUVECs treated with COCO. *P=0.0372 (- vs 24hours); *P=0.0137 (- vs 3-NP); (n=3).


( H ) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and / or N - acetylcystein . Scale bar , 500 μm . ( I ) Quantification of sprouting surface area of micrographs shown in ( H ) . * P = 0 . 0126 ( - vs COCO - NAC ) ; * P = 0 . 0200 ( COCO - NAC vs COCO + NAC ) ; ( n = 6 ) .

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

(H) Representative images of choroidal explants cultured for 4 days in the presence or absence of COCO and/or N-acetylcystein. Scale bar, 500 μm. (I) Quantification of sprouting surface area of micrographs shown in (H). *P=0.0126 (- vs COCO-NAC); *P=0.0200 (COCO-NAC vs COCO+NAC); (n=6).


A . A549 cells were transfected with 2 μg of poly ( I : C ) for 6 hrs and then infected with ZIKV ( MOI = 3 ) for 18 hrs ( Pre - treatment ) or infected with ZIKV first for 6 hrs and then transfected with poly ( I : C ) for 12 hrs ( Post - treatment ) . Levels of ZIKVgenomic RNA were determined by qRT - PCR . Values are expressed as mean ± standard error . * * * P < 0 . 001 ( Student ' s t - test ) , N =

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

A. A549 cells were transfected with 2 μg of poly(I:C) for 6 hrs and then infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment) or infected with ZIKV first for 6 hrs and then transfected with poly(I:C) for 12 hrs (Post-treatment). Levels of ZIKVgenomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. ***P<0.001 (Student's t-test), N=3


B . A549 cells infected with ZIKV ( MOI = 3 ) for 6 hrs were transfected with 2 μg poly ( I : C ) for 12 hrs after which levels of ifn -

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

B. A549 cells infected with ZIKV (MOI=3) for 6 hrs were transfected with 2 μg poly(I:C) for 12 hrs after which levels of ifn-


C . A549 cells were infected with ZIKV ( MOI = 5 ) for 16 hrs and then transfected with an IFIT1 promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later cell lysates were harvested and subjected to luciferase assay . Values are expressed as mean of two independent experiments , N =

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

C. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2


D . HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins . Sixteen hours later they were transfected with an IFIT1 promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later cell lysates were harvested and subjected to luciferase assay . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) , N =

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

D. HEK293T cells were transfected with plasmids encoding each of the 10 ZIKV proteins. Sixteen hours later they were transfected with an IFIT1 promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test), N=4


E . A549 cells were infected with ZIKV ( MOI = 5 ) for 16 hrs and then transfected with the indicated promoter - driven firefly luciferase plasmids and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 1 μg of poly ( I : C ) . Eight hours later cell lysates were harvested and subjected to luciferase assay . Values are expressed as mean of two independent experiments , N =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells were infected with ZIKV (MOI=5) for 16 hrs and then transfected with the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 1 μg of poly(I:C). Eight hours later cell lysates were harvested and subjected to luciferase assay. Values are expressed as mean of two independent experiments, N=2


F . HEK293T cells were transfected with plasmids encoding individual ZIKV proteins , the indicated promoter - driven firefly luciferase plasmids and a constitutively active renilla luciferase construct ( pRL - TK ) , as well as 0 . 4 μg of poly ( I : C ) . Twenty - four hours later cell lysates were harvested and subjected to luciferase assay . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. HEK293T cells were transfected with plasmids encoding individual ZIKV proteins, the indicated promoter-driven firefly luciferase plasmids and a constitutively active renilla luciferase construct (pRL-TK), as well as 0.4 μg of poly(I:C). Twenty-four hours later cell lysates were harvested and subjected to luciferase assay. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


A . A549 cells were treated with IFN - α ( 100 U / mL ) , IFN - λ ( 200 ng / mL ) or IFN - γ ( 10 U / mL ) for 6 hrs after which they were infected with ZIKV ( MOI = 3 ) for 18 hrs ( Pre - treatment ) . Alternatively , cells were first infected with ZIKV for 6 hrs followed by treatment with IFN for 12 hrs ( Post - treatment ) . Total RNA was isolated and levels of ZIKV genomic RNA were determined by qRT - PCR . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells were treated with IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN-γ (10 U/mL) for 6 hrs after which they were infected with ZIKV (MOI=3) for 18 hrs (Pre-treatment). Alternatively, cells were first infected with ZIKV for 6 hrs followed by treatment with IFN for 12 hrs (Post-treatment). Total RNA was isolated and levels of ZIKV genomic RNA were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


B . A549 cells were infected with ZIKV ( MOI = 3 ) for 6 hrs prior to addition of IFN - α ( 100 U / mL ) , IFN - λ ( 200 ng / mL ) or IFN - γ ( 10 U / mL ) for 12 hrs . Total RNA was isolated and levels of ifit1 RNAs were determined by qRT - PCR . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

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

B. A549 cells were infected with ZIKV (MOI=3) for 6 hrs prior to addition of IFN-α (100 U/mL), IFN-λ (200 ng/mL) or IFN-γ (10 U/mL) for 12 hrs. Total RNA was isolated and levels of ifit1 RNAs were determined by qRT-PCR. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


C . A549 cells were infected with ZIKV ( MOI = 5 ) for 6 hrs and then transfected with ISRE - or GAS - promoter - driven firefly luciferase together with a constitutively active renilla luciferase construct . Sixteen hours later , cells were treated with IFN - α ( 100 U / ml ) or IFN - α ( 10 U / ml ) for 2 hrs . Cells lysates were harvested and relative luciferase activity from ISRE - and GAS - promoters were determined . Values are expressed as mean ± standard error . * P < 0 . 05 ( Student ' s t - test ) ; N =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells were infected with ZIKV (MOI=5) for 6 hrs and then transfected with ISRE- or GAS- promoter-driven firefly luciferase together with a constitutively active renilla luciferase construct. Sixteen hours later, cells were treated with IFN-α (100 U/ml) or IFN-α (10 U/ml) for 2 hrs. Cells lysates were harvested and relative luciferase activity from ISRE- and GAS-promoters were determined. Values are expressed as mean ± standard error. *P<0.05 (Student's t-test); N=3


D . HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter - driven firefly luciferase plasmid ( pGL3B / 561 ) and a constitutively active renilla luciferase construct ( pRL - TK ) . After 24 hrs they were treated with IFN - α ( 100 U / mL ) for 10 hrs . Cell lysates were then harvested and subjected to luciferase assays . C = capsid ; E = envelope . Values are expressed as mean ± standard error . * P < 0 . 05 ( Student ' s t - test ) ; N =

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

D. HEK293T cells were transfected with ZIKV protein expression constructs together with an ISRE promoter-driven firefly luciferase plasmid (pGL3B/561) and a constitutively active renilla luciferase construct (pRL-TK). After 24 hrs they were treated with IFN-α (100 U/mL) for 10 hrs. Cell lysates were then harvested and subjected to luciferase assays. C = capsid; E = envelope. Values are expressed as mean ± standard error. *P<0.05 (Student's t-test); N=3


A . A549 cells were infected with ZIKV ( MOI = 5 ) for 24 - 48 hrs after which cell lysates were processed by SDS - PAGE and immunoblotting . Representative panels are shown . The experiment was repeated three times .

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

A. A549 cells were infected with ZIKV (MOI=5) for 24-48 hrs after which cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.


B . A549 cells were infected with ZIKV ( MOI = 5 ) for 24 hrs and then treated with DMSO , MG132 ( 20 µM ) or epoxomicin ( Epox ) ( 400 nM ) for 12 hrs . Cell lysates were processed by SDS - PAGE and immunoblotting . Representative panels are shown . The experiment was repeated three times .

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

B. A549 cells were infected with ZIKV (MOI=5) for 24 hrs and then treated with DMSO, MG132 (20 µM) or epoxomicin (Epox) (400 nM) for 12 hrs. Cell lysates were processed by SDS-PAGE and immunoblotting. Representative panels are shown. The experiment was repeated three times.


C . A549 cells ( MOI = 2 ) or Human primary fetal astrocytes ( HFA ) ( MOI = 5 ) were infected with ZIKV for 24 hrs and 48 hrs respectively , after which IFN - α ( 100 U / mL ) was added for 2 hrs . Cells were then fixed and processed for indirect immunofluorescence . Images were acquired on a spinning disk confocal microscope with a 40X objective . Dashed line white circles indicated ZIKV - infected cells . Representative panels are shown . The experiment was repeated two times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A549 cells (MOI=2) or Human primary fetal astrocytes (HFA) (MOI=5) were infected with ZIKV for 24 hrs and 48 hrs respectively, after which IFN-α (100 U/mL) was added for 2 hrs. Cells were then fixed and processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. Dashed line white circles indicated ZIKV-infected cells. Representative panels are shown. The experiment was repeated two times.


D . Mouse embryonic fibroblasts ( MEF ) were infected with ZIKV ( MOI = 5 ) for 48 hrs after which IFN - α ( 200 U / mL ) was added for 2 hrs . Samples were processed for immunofluorescence assay as described in panel C . Representative panels are shown . The experiment was repeated two times .

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

D. Mouse embryonic fibroblasts (MEF) were infected with ZIKV (MOI=5) for 48 hrs after which IFN-α (200 U/mL) was added for 2 hrs. Samples were processed for immunofluorescence assay as described in panel C. Representative panels are shown. The experiment was repeated two times.


E . STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV ( MOI = 5 ) for another 48 hrs . The ZIKV replication was measured by qRT - PCR .

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

E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The ZIKV replication was measured by qRT-PCR.


E . STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV ( MOI = 5 ) for another 48 hrs . The silencing efficiency was determined by immunoblotting . Representative panels are shown . The experiment was repeated three times .

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

E. STAT2 levels in MEFs were decreased by transfection with siRNAs for 48 hrs after which the cells were infected with ZIKV (MOI=5) for another 48 hrs. The silencing efficiency was determined by immunoblotting. Representative panels are shown. The experiment was repeated three times.


A . A549 cells were transfected with control plasmid pcDNA 3 . 1 or plasmids encoding FLAG - tagged WT or mutant NS5 proteins . At 48 hrs post - transfection cells were treated with IFN - α ( 100 U / mL ) for 2 hrs and then processed for indirect immunofluorescence . Images were acquired on a spinning disk confocal microscope with a 40X objective . NS5 positive cells are indicated by dashed white circles . Representative panels are shown . The experiment was repeated three times . B . Quantification of nuclearSTAT2 was performed using Volocity image analyses software . A minimum of 20 cells were counted for each sample . N . D . = not detected . Values are expressed as mean ± standard error . * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) ; N =

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

A. A549 cells were transfected with control plasmid pcDNA 3.1 or plasmids encoding FLAG-tagged WT or mutant NS5 proteins. At 48 hrs post-transfection cells were treated with IFN-α (100 U/mL) for 2 hrs and then processed for indirect immunofluorescence. Images were acquired on a spinning disk confocal microscope with a 40X objective. NS5 positive cells are indicated by dashed white circles. Representative panels are shown. The experiment was repeated three times.B. Quantification of nuclearSTAT2 was performed using Volocity image analyses software. A minimum of 20 cells were counted for each sample. N.D.= not detected. Values are expressed as mean ± standard error. *P<0.05, **P<0.01 (Student's t-test); N=3


C . A549 cells were transfected with plasmids encoding FLAG - tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin ( 400 nM ) for 24 hrs . Cells were harvested and process for immunoprecipitation ( IP ) using a mouse anti - FLAG or anti - myc antibody followed by SDS - PAGE and Immunoblotting . Representative panels are shown . The experiment was repeated three times .

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

C. A549 cells were transfected with plasmids encoding FLAG-tagged ZIKVNS5 or FLAG alone for 24 hrs and then treated with epoxomicin (400 nM) for 24 hrs. Cells were harvested and process for immunoprecipitation (IP) using a mouse anti-FLAG or anti-myc antibody followed by SDS-PAGE and Immunoblotting. Representative panels are shown. The experiment was repeated three times.


D . A549 cells were transfected with plasmids encoding FLAG - tagged WT , mutant NS5 proteins or FLAG alone for 48 hrs . Cells were harvested and process for immunoprecipitation ( IP ) using rabbit anti - STAT2 followed by SDS - PAGE and immunoblotting . Arrows indicate co - immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2 . Representative panels are shown . The experiment was repeated two times .

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

D. A549 cells were transfected with plasmids encoding FLAG-tagged WT, mutant NS5 proteins or FLAG alone for 48 hrs. Cells were harvested and process for immunoprecipitation (IP) using rabbit anti-STAT2 followed by SDS-PAGE and immunoblotting. Arrows indicate co-immunoprecipitated WT NS5 and NS5 mutants that are in a complex with STAT2. Representative panels are shown. The experiment was repeated two times.


( A ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 12 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 16 ) mice ( age 9 - 12 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . Mice were monitored for up to 200 days post - treatment with tamoxifen . Data are presented as % survival post - treatment with tamoxifen and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test ; * * * * p < 0 . 0001 .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Bcl-xfl/fl;RosaCreERT2+/Ki (n=12) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=16) mice (age 9-12 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. Mice were monitored for up to 200 days post-treatment with tamoxifen. Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.


( B ) Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 24 ) or , as controls , Bcl - xfl / fl ( n = 9 ) and RosaCreERT2 + / Ki ( n = 6 ) mice ( males and females , aged 8 - 14 weeks , numbers also indicated in the figure legend ) were lethally γ - irradiated ( 2 x 5 . 5 Gy , 3 h apart ) and reconstituted with bone marrow from UBC - GFP mice ( referred to as GFP - Chimeras ) . After 8 weeks , reconstituted mice were treated with tamoxifen ( 200 mg / kg body weight administered in 3 daily doses oral gavage ) and monitored for up to 200 days ( termination of the experiment ) . Data are presented as % survival post - treatment with tamoxifen and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test ; * * * * p < 0 . 0001 .

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

(B) Bcl-xfl/fl;RosaCreERT2+/Ki (n=24) or, as controls, Bcl-xfl/fl (n=9) and RosaCreERT2+/Ki (n=6) mice (males and females, aged 8-14 weeks, numbers also indicated in the figure legend) were lethally γ-irradiated (2 x 5.5 Gy, 3 h apart) and reconstituted with bone marrow from UBC-GFP mice (referred to as GFP-Chimeras). After 8 weeks, reconstituted mice were treated with tamoxifen (200 mg/kg body weight administered in 3 daily doses oral gavage) and monitored for up to 200 days (termination of the experiment). Data are presented as % survival post-treatment with tamoxifen and statistical significance was assessed using the Mantel-Cox (Log-rank) test; ****p<0.0001.


( C - F ) Total counts of ( C ) platelets , ( D ) red blood cells ( RBC ) , ( E ) hemoglobin ( HGB ) content and ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 . ( G ) Spleen weights were measured in sick mice ( at sacrifice ) or , for the healthy control mice , at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents an individual mouse and numbers are indicated . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 .

O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001. (G) Spleen weights were measured in sick mice (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse and numbers are indicated. Statistical significance was assessed using the Student's t-test; ****p<0.0001.


( C - F ) Total counts of ( C ) platelets , ( D ) red blood cells ( RBC ) , ( E ) hemoglobin ( HGB ) content and ( F ) hematocrit ( HCT ) of tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimera ( n = 21 ) or , as controls , Bcl - xfl / fl ; GFP - Chimera ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimera ( n = 6 ) mice were determined by ADVIA . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * * * * p < 0 . 0001 .

O O 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 B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Total counts of (C) platelets, (D) red blood cells (RBC), (E) hemoglobin (HGB) content and (F) hematocrit (HCT) of tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) and Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimera (n=21) or, as controls, Bcl-xfl/fl;GFP-Chimera (n=6) and RosaCreERT2+/Ki;GFP-Chimera (n=6) mice were determined by ADVIA. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; ****p<0.0001.


( H ) Representative image of enlarged spleens from two Bcl - xfl / fl ; RosaCreERT2Ki / + mice and age - matched control Bcl - xfl / fl and RosaCreERT2 + / Ki mice ( 34 days after treatment ) .

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

(H) Representative image of enlarged spleens from two Bcl-xfl/fl;RosaCreERT2Ki/+ mice and age-matched control Bcl-xfl/fl and RosaCreERT2+/Ki mice (34 days after treatment).


( I ) Histological analysis of H & E - stained sections of spleens of tamoxifen - treated mice of the indicated genotypes .

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

(I) Histological analysis of H&E-stained sections of spleens of tamoxifen-treated mice of the indicated genotypes.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 8 ) or , as controls , Bcl - xfl / fl ( n = 6 ) and RosaCreERT2 + / Ki ( n = 12 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , or as controls , Bcl - xfl / fl ; GFP - Chimeras ( n = 6 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . ( A ) Total white blood cell counts ( WBC ) were analyzed by ADVIA in sick mice or at the termination of the experiment ( healthy control mice ) . Data are presented as mean ± SEM . Each data point represents an individual mouse and n numbers are indicated above . Statistical significance was assessed using the Student ' s t - test . No statistically significant differences were observed .

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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=8) or, as controls, Bcl-xfl/fl (n=6) and RosaCreERT2+/Ki (n=12) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), or as controls, Bcl-xfl/fl;GFP-Chimeras (n=6) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Total white blood cell counts (WBC) were analyzed by ADVIA in sick mice or at the termination of the experiment (healthy control mice). Data are presented as mean ±SEM. Each data point represents an individual mouse and n numbers are indicated above. Statistical significance was assessed using the Student's t-test. No statistically significant differences were observed.


( B ) The neutrophil / lymphocyte ratio ( NLR ) is presented as mean ± SEM for sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) or healthy control RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents an individual mouse . Statistical significance was assessed using the Student ' s t - test ; * p < 0 . 05 .

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

(B) The neutrophil/lymphocyte ratio (NLR) is presented as mean ±SEM for sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21) or healthy control RosaCreERT2+/Ki;GFP-Chimeras (n=6) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents an individual mouse. Statistical significance was assessed using the Student's t-test; *p<0.05.


( C ) Histological analysis of H & E - stained sections of the sternum of sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras or healthy wild - type and RosaCreERT2 + / Ki ; GFP - Chimera control mice at the indicated time points post - treatment with tamoxifen ( dpt = days post - treatment ) .

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

(C) Histological analysis of H&E-stained sections of the sternum of sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras or healthy wild-type and RosaCreERT2+/Ki;GFP-Chimera control mice at the indicated time points post-treatment with tamoxifen (dpt=days post-treatment).


( D ) Histological analysis of H & E - stained sections of the livers of sick mice or age - matched healthy control wild - type mice or healthy RosaCreERT2 + / Ki ; GFP - Chimeras at the termination of the experiment ( dpt = days post - treatment ) .

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

(D) Histological analysis of H&E-stained sections of the livers of sick mice or age-matched healthy control wild-type mice or healthy RosaCreERT2+/Ki;GFP-Chimeras at the termination of the experiment (dpt=days post-treatment).


( E ) ALT ( left panel ) , AST ( middle panel ) and bilirubin levels ( right panel ) in the serum were determined in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 18 ) or in healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 4 ) at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test ; * * p < 0 . 01 . nd = not detected .

O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ALT (left panel), AST (middle panel) and bilirubin levels (right panel) in the serum were determined in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=18) or in healthy RosaCreERT2+/Ki;GFP-Chimeras (n=4) at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; **p<0.01. nd=not detected.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) and RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles . ( A ) Kidney weights were measured in sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( at sacrifice ) or , for the healthy control mice , at the termination of the experiment . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test ; * p < 0 . 05 , * * * p < 0 . 001 .

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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles. (A) Kidney weights were measured in sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (at sacrifice) or, for the healthy control mice, at the termination of the experiment. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test; *p<0.05, ***p<0.001.


Bcl - xfl / fl ; RosaCreERT2 + / Ki ( n = 7 ) or as controls , Bcl - xfl / fl ( n = 8 ) and RosaCreERT2 + / Ki ( n = 16 ) mice as well as Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 5 ) , Bcl - xfl / fl ; GFP - Chimeras ( n = 8 ) and RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) ( age 8 - 14 weeks , males and females ) were treated with tamoxifen ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) to induce CreERT2 - mediated deletion of the floxed Bcl - x alleles .

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

Bcl-xfl/fl;RosaCreERT2+/Ki (n=7) or as controls, Bcl-xfl/fl (n=8) and RosaCreERT2+/Ki (n=16) mice as well as Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=5), Bcl-xfl/fl;GFP-Chimeras (n=8) and RosaCreERT2+/Ki;GFP-Chimeras (n=6) (age 8-14 weeks, males and females) were treated with tamoxifen (200 mg/kg/body weight administered in 3 daily doses by oral gavage) to induce CreERT2-mediated deletion of the floxed Bcl-x alleles.


( B ) Picture showing both kidneys from age - matched healthy control wild - type mice ( left ) and sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) .

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

(B) Picture showing both kidneys from age-matched healthy control wild-type mice (left) and sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right).


( C ) Histological analysis of H & E - stained sections of the kidneys of age - matched healthy control mice or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras as indicated , showing from left to right , shrunken scarred kidney , segmental chronic tubulo - interstitial disease , accumulation of cellular debris and amorphous material within the collecting ducts of the papilla . The higher magnification shows tubular epithelial degeneration , apoptosis and segmental secondary glomerular sclerosis . Pictures are representative of at least 5 mice for each genotype and treatment .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of age-matched healthy control mice or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras as indicated, showing from left to right, shrunken scarred kidney, segmental chronic tubulo-interstitial disease, accumulation of cellular debris and amorphous material within the collecting ducts of the papilla. The higher magnification shows tubular epithelial degeneration, apoptosis and segmental secondary glomerular sclerosis. Pictures are representative of at least 5 mice for each genotype and treatment.


( D ) Multiphoton analysis of fixed sections of the kidneys of age - matched healthy RosaCreERT2 + / Ki ; GFP - Chimeras ( left ) or sick Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( right ) . Pictures are representative of at least 3 mice for each genotype and treatment . green = second harmony , blue = short wavelength autofluorescence .

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

(D) Multiphoton analysis of fixed sections of the kidneys of age-matched healthy RosaCreERT2+/Ki;GFP-Chimeras (left) or sick Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (right). Pictures are representative of at least 3 mice for each genotype and treatment. green=second harmony, blue=short wavelength autofluorescence.


( A ) TUNEL staining of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at the indicated time points post - tamoxifen treatment ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) cells . ( B ) TUNEL + cells were quantified using a personalized script for the Image J software . Kidney sections were divided into ~ 40 microscopic images and the total numbers of blue and brown ( TUNEL + ) nuclei were determined . The percentages of TUNEL + nuclei were calculated for each picture . The average value for each kidney section was then determined and plotted as a single data point . Error bars represent SEM from at least 3 independent samples for each genotype and treatment . Statistical significance was assessed using one - way ANOVA analysis with Tukey ' s multiple comparisons test ( comparing control RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 2 , 24 h post - γ - irradiation n = 9 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ( 24 h post - tamoxifen n = 6 , 24 h post - γ - irradiation n = 5 ) and control RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 7 , End point n = 8 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( 24 h post - tamoxifen n = 8 , End point n = 6 ) ( * * * p < 0 . 001 ) .

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

(A) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells. (B) TUNEL+ cells were quantified using a personalized script for the Image J software. Kidney sections were divided into ~40 microscopic images and the total numbers of blue and brown (TUNEL+) nuclei were determined. The percentages of TUNEL+ nuclei were calculated for each picture. The average value for each kidney section was then determined and plotted as a single data point. Error bars represent SEM from at least 3 independent samples for each genotype and treatment. Statistical significance was assessed using one-way ANOVA analysis with Tukey's multiple comparisons test (comparing control RosaCreERT2+/Ki (24 h post-tamoxifen n=2, 24 h post-γ-irradiation n=9) with Bcl-xfl/fl;RosaCreERT2+/Ki (24 h post-tamoxifen n=6, 24 h post-γ-irradiation n=5) and control RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=7, End point n=8) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (24 h post-tamoxifen n=8, End point n=6) (***p<0.001).


( A ) TUNEL staining of kidney sections from control RosaCreERT2 + / Ki , Bcl - xfl / fl ; RosaCreERT2 + / Ki , RosaCreERT2 + / Ki ; GFP - Chimeras or Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras at the indicated time points post - tamoxifen treatment ( 200 mg / kg / body weight administered in 3 daily doses by oral gavage ) or γ - irradiation ( 2 x 550 Rad ) , as indicated . Slides were counterstained with hematoxylin . Arrow heads indicate examples of TUNEL + ( apoptotic ) cells .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TUNEL staining of kidney sections from control RosaCreERT2+/Ki, Bcl-xfl/fl;RosaCreERT2+/Ki, RosaCreERT2+/Ki;GFP-Chimeras or Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras at the indicated time points post-tamoxifen treatment (200 mg/kg/body weight administered in 3 daily doses by oral gavage) or γ-irradiation (2 x 550 Rad), as indicated. Slides were counterstained with hematoxylin. Arrow heads indicate examples of TUNEL+ (apoptotic) cells.


( A , B ) Data are presented as % survival and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . The survival curves for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras

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

(A, B) Data are presented as % survival and statistical significance was assessed using the Mantel-Cox (Log-rank) test comparing tamoxifen-treated Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=25) with Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=25), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=18), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=15) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=22). *p<0.05, ****p<0.0001. The survival curves for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras


( A , B ) Data are presented as % survival and statistical significance was assessed using the Mantel - Cox ( Log - rank ) test comparing tamoxifen - treated Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 25 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 25 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 18 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 15 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 22 ) . * p < 0 . 05 , * * * * p < 0 . 0001 . The survival curves for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP -

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


( C ) Histological analysis of H & E - stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points ( dpt = days post - tamoxifen treatment ) . Pictures are representative of at least 3 mice for each genotype and treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological analysis of H&E-stained sections of the kidneys of sick mice of the indicated genotypes at the indicated time points (dpt = days post-tamoxifen treatment). Pictures are representative of at least 3 mice for each genotype and treatment.


( D - F ) ( D ) Total red blood cell ( RBC ) count , ( E ) hematocrit ( HCT ) and ( F ) hemoglobin ( HGB ) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls . Data are presented as mean ± SEM . Each data point represents one individual mouse . Statistical significance was assessed using Student ' s t - test comparing tamoxifen - treated RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 6 ) with Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras ( n = 21 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; GFP - Chimeras ( n = 20 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim - / - ; GFP - Chimeras ( n = 12 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma + / - ; GFP - Chimeras ( n = 15 ) , Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Puma - / - ; GFP - Chimeras ( n = 10 ) and Bcl - xfl / fl ; RosaCreERT2 + / Ki ; Bim + / - ; Puma + / - ; GFP - Chimeras ( n = 17 ) ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Data for the Bcl - xfl / fl ; RosaCreERT2 + / Ki ; GFP - Chimeras and RosaCreERT2 + / Ki ; GFP - Chimeras

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

(D-F) (D) Total red blood cell (RBC) count, (E) hematocrit (HCT) and (F) hemoglobin (HGB) content were determined by ADVIA in the blood of sick mice of the indicated genotypes or at the termination of the experiment for healthy controls. Data are presented as mean ±SEM. Each data point represents one individual mouse. Statistical significance was assessed using Student's t-test comparing tamoxifen-treated RosaCreERT2+/Ki;GFP-Chimeras (n=6) with Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras (n=21), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim+/-;GFP-Chimeras (n=20), Bcl-xfl/fl;RosaCreERT2+/Ki;Bim-/-;GFP-Chimeras (n=12), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma+/-;GFP-Chimeras (n=15), Bcl-xfl/fl;RosaCreERT2+/Ki;Puma-/-;GFP-Chimeras (n=10) and Bcl-xfl/fl;RosaCreERT2+/Ki; Bim+/-;Puma+/-;GFP-Chimeras (n=17) ; ***p<0.001; ****p<0.0001. Data for the Bcl-xfl/fl;RosaCreERT2+/Ki;GFP-Chimeras and RosaCreERT2+/Ki;GFP-Chimeras