D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes . Tyro3 , Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting . Arrows indicate the corresponding protein band ( s ) . The secondary antibody against goat IgG generates a ~ 115 kDa non - specific band . The ~ 115 kDa band in the Axl image is the Axl - specific band overlapping the non - specific band . The ~ 115 kDa band in the Mertk image is the non - specific band . Axl and Mertk expression was knocked down by two siRNAs . GAPDH expression is shown as an internal control . E Quantification of siRNA knockdown in cultured astrocytes ( n = 3 independent experiments , one - way ANOVA with repeated measures ) . Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes . Knockdown efficiency of Axl and Mertk was quantified from western blots . Results are normalized to Gapdh or GAPDH , and are shown as ratios to the value of non - transfected astrocytes .

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D Expression of TAM family members and their knockdown by siRNA in cultured astrocytes. Tyro3, Axl and Metk in cultured astrocytes or hippocampal tissue were examined by western blotting. Arrows indicate the corresponding protein band(s). The secondary antibody against goat IgG generates a ~115 kDa non-specific band. The ~115 kDa band in the Axl image is the Axl-specific band overlapping the non-specific band. The ~115 kDa band in the Mertk image is the non-specific band. Axl and Mertk expression was knocked down by two siRNAs. GAPDH expression is shown as an internal control. E Quantification of siRNA knockdown in cultured astrocytes (n = 3 independent experiments, one-way ANOVA with repeated measures). Knockdown of Tyro3 mRNA was examined by qPCR because of the very low expression level even in control astrocytes. Knockdown efficiency of Axl and Mertk was quantified from western blots. Results are normalized to Gapdh or GAPDH, and are shown as ratios to the value of non-transfected astrocytes.


F , G Flow cytometric analysis of microglial debris uptake by cultured astrocytes . After knockdown of TAM family members , astrocytes were co - cultured with fluorescence - labelled apoptotic microglia . Fluorescence of internalized microglial debris in GLAST + astrocytes was quantified by flow cytometry ( n = 4 independent experiments , one - way ANOVA with repeated measures ) . Results are shown as ratios to the value of non - transfected astrocytes .

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F, G Flow cytometric analysis of microglial debris uptake by cultured astrocytes. After knockdown of TAM family members, astrocytes were co-cultured with fluorescence-labelled apoptotic microglia. Fluorescence of internalized microglial debris in GLAST+ astrocytes was quantified by flow cytometry (n = 4 independent experiments, one-way ANOVA with repeated measures). Results are shown as ratios to the value of non-transfected astrocytes.


B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w . Sections were stained with anti - Iba1 or anti - CD11b antibodies ( red ) . Scale bar , 100 µm . C Quantification of microglial number ( n = 5 animals per group , one - way ANOVA ) . Note that Irf8 - / - mice were analyzed by CD11b immunostaining , while other strains were analyzed by Iba1 staining .

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B Representative images of microglial distribution in the cerebral cortex of WT and mutant mice at 3 w. Sections were stained with anti-Iba1 or anti-CD11b antibodies (red). Scale bar, 100 µm. C Quantification of microglial number (n = 5 animals per group, one-way ANOVA). Note that Irf8-/- mice were analyzed by CD11b immunostaining, while other strains were analyzed by Iba1 staining.


D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) and anti - cleaved caspase - 3 ( blue ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Arrows indicate cleaved caspase - 3 + apoptotic cells . Insets are higher magnification images around apoptotic cells . Scale bar , 20 µm and 5 µm ( insets ) . E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice . 3D images reconstructed from confocal images were analyzed ( n = 36 - 50 cells from five animals per group , Kruskal - Wallis test with post hoc Dunn ' s test ) . F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice ( n = 36 - 50 cells from five animals per group , one - way ANOVA with post hoc Tukey ' s test ) .

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D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets). E Percentage of spontaneous apoptotic cells surrounded by microglia in WT and mutant mice. 3D images reconstructed from confocal images were analyzed (n = 36-50 cells from five animals per group, Kruskal-Wallis test with post hoc Dunn's test). F Percentage of spontaneous apoptotic cells surrounded by astrocyte processes in WT and mutant mice (n = 36-50 cells from five animals per group, one-way ANOVA with post hoc Tukey's test).


D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8 - / - mice at 3 w . Sections were stained with anti - GFAP ( green ) , anti - CD11b ( red ) and anti - cleaved caspase - 3 ( blue ) antibodies . 3D images ( lower row ) were reconstructed from confocal images ( upper row ) using Imaris software . Arrows indicate cleaved caspase - 3 + apoptotic cells . Insets are higher magnification images around apoptotic cells . Scale bar , 20 µm and 5 µm ( insets ) .

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D Representative images of a spontaneous apoptotic cell and surrounding glial cells in the cerebral cortex of WT and Irf8-/- mice at 3 w. Sections were stained with anti-GFAP (green), anti-CD11b (red) and anti-cleaved caspase-3 (blue) antibodies. 3D images (lower row) were reconstructed from confocal images (upper row) using Imaris software. Arrows indicate cleaved caspase-3+ apoptotic cells. Insets are higher magnification images around apoptotic cells. Scale bar, 20 µm and 5 µm (insets).


Representative images of HeLa ( B ) or HCT - 8 ( C ) cells infected with Shigella WT pre - treated or not with arsenite , analyzed at the indicated times post - infection Cfu quantification of intracellular bacteria in HeLa and HCT - 8 cells pre - treated or not with arsenite and infected with

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Representative images of HeLa (B) or HCT-8 (C) cells infected with Shigella WT pre-treated or not with arsenite, analyzed at the indicated times post-infection Cfu quantification of intracellular bacteria in HeLa and HCT-8 cells pre-treated or not with arsenite and infected with Shigella


Representative images ( E ) and cfu quantification ( F ) of intracellular bacteria in HeLa cells infected with Shigella WT after pre - treatment with TNF - α , H2O2 , anisomycin , hypoxia and corresponding controls , analyzed at 0 . 5

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Representative images (E) and cfu quantification (F) of intracellular bacteria in HeLa cells infected with Shigella WT after pre-treatment with TNF-α, H2O2, anisomycin, hypoxia and corresponding controls, analyzed at 0.5 hpi


Representative images ( G ) and cfu quantification ( H ) of intracellular Shigella in HeLa cells pre - treated with arsenite , anisomycin , stressors plus NAC , and corresponding

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Representative images (G) and cfu quantification (H) of intracellular Shigella in HeLa cells pre-treated with arsenite, anisomycin, stressors plus NAC, and corresponding controls


Representative images ( A ) and cfu quantification ( B ) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre - treated or not with arsenite . Ruffle formation induced by Shigella WT in panel ( A ) is indicated by white

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Representative images (A) and cfu quantification (B) of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated or not with arsenite. Ruffle formation induced by Shigella WT in panel (A) is indicated by white arrowheads


Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre - treated with anisomycin or DMSO ( control )

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Cfu quantification of Shigella WT or ΔipaB mutant strain bound to HeLa cells pre-treated with anisomycin or DMSO (control)


Representative images ( D ) and cfu quantification ( E ) of Shigella WT or ΔipaB mutant bound to HCT - 8 cells pre - treated or not with

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Representative images (D) and cfu quantification (E) of Shigella WT or ΔipaB mutant bound to HCT-8 cells pre-treated or not with arsenite


Representative image ) , cf and qRT - PCR quantification of intracellular Shigella in HeLa cells pre - treated with arsenite in the presence or not of amitriptyline , and corresponding

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Representative image ), cf and qRT-PCR quantification of intracellular Shigella in HeLa cells pre-treated with arsenite in the presence or not of amitriptyline, and corresponding controls


Representative image cf and qRT - PC quantification of intracellular Shigella in HeLa cells pre - treated wit anisomycin in the presence or not of amitriptyline , and corresponding

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Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells pre-treated wit anisomycin in the presence or not of amitriptyline, and corresponding controls


Representative image cf and qRT - PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre - treated or not with arsenite prior to

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Representative image cf and qRT-PC quantification of intracellular Shigella in HeLa cells transfected with ASM siRNA or control siRNA and pre-treated or not with arsenite prior to infection


3D reconstruction of representative images of ASM ( J , K ) or ceramide ( L , M ) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls . ASM , ceramide and Hoechst staining were surface converted by voxel

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3D reconstruction of representative images of ASM (J, K) or ceramide (L, M) staining in HeLa cells treated with arsenite or anisomycin and corresponding controls. ASM, ceramide and Hoechst staining were surface converted by voxel distance


Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite , TNF - α , H2O2 or anisomycin ; β - actin was used as loading

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Western blot analysis of p38 MAPK phosphorylation in HeLa cells treated with arsenite, TNF-α, H2O2 or anisomycin; β-actin was used as loading control


Representative image of intracellular Shigella in HeLa cells pre - treated with arsenite in the absence or presence of the p38 inhibitor SB203580 , and corresponding

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Representative image of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


cfu quantificatio of intracellular Shigella in HeLa cells pre - treated with arsenite in the absence or presence of the p38 inhibitor SB203580 , and corresponding

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cfu quantificatio of intracellular Shigella in HeLa cells pre-treated with arsenite in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre - treated or not with arsenite prior to

O O O O B-ORGANISM O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O

Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not with arsenite prior to infection


cfu quantificatio of intracellular Shigella in HeLa cells pre - treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580 , and corresponding

cfu quantificatio of intracellular Shigella in HeLa cells pre-treated wit anysomycin in the absence or presence of the p38 inhibitor SB203580, and corresponding controls


Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre - treated or not wit anisomycin prior to

Cfu quantification of intracellular Shigella in HeLa cells transfected with p38 siRNA or control siRNA and pre-treated or not wit anisomycin prior to infection


3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite ) , in the presence or absence of SB203580 , and corresponding controls . Ceramide and Hoechst staining were surface converted by voxel

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3D reconstruction of representative images of ceramide staining in HeLa cells treated with arsenite ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance


3D reconstruction of representative images of ceramide staining in HeLa cells treated wit anisomyci ) , in the presence or absence of SB203580 , and corresponding controls . Ceramide and Hoechst staining were surface converted by voxel

3D reconstruction of representative images of ceramide staining in HeLa cells treated wit anisomyci ), in the presence or absence of SB203580, and corresponding controls. Ceramide and Hoechst staining were surface converted by voxel distance


Representative images ( A ) , cfu ( B and qRT - PCR quantification of intracellular Salmonella in HeLa cells pre - treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0 . 5

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Representative images (A), cfu (B and qRT-PCR quantification of intracellular Salmonella in HeLa cells pre-treated with arsenite or anisomycin and corresponding controls Infection was performed with Salmonella WT and analyzed at 0.5 hpi


Representative images of bacteria bound to HeLa cells pre - treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10

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Representative images of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT or Δ4 mutant strain for 10 min


Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre - treated or not with arsenite followed by incubation with Salmonella WT ( E ) or Δ4 mutant strain ( F ) for 10

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Violin plots showing the distribution of the number of bacteria bound per infected cell in HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella WT (E) or Δ4 mutant strain (F) for 10 min


Representative images ( G ) and cfu quantification ( H ) of bacteria bound to HeLa cells pre - treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC , ΔfliC / pFliC or ΔflhC for 10

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Representative images (G) and cfu quantification (H) of bacteria bound to HeLa cells pre-treated or not with arsenite followed by incubation with Salmonella mutant strains ΔfliC, ΔfliC/pFliC or ΔflhC for 10 min


Representative images ( I ) and cfu quantification ( J ) of Listeria bound to HeLa cells pre - treated or not with arsenite followed by incubation with the bacteria for 20

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Representative images (I) and cfu quantification (J) of Listeria bound to HeLa cells pre-treated or not with arsenite followed by incubation with the bacteria for 20 min


Representative images ( K ) and cfu quantification ( L ) of Yersinia bound / internalized by HeLa cells pre - treated or not with arsenite followed by 70 min incubation with the

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Representative images (K) and cfu quantification (L) of Yersinia bound/internalized by HeLa cells pre-treated or not with arsenite followed by 70 min incubation with the bacteria


Schematic representation of the experimental design for reinfection experiments . Cells were infected with Shigella - mCherry ( primary infection ) or mock - treated . At 3 hpi , cells were re - infected with Shigella expressing GFP ( secondary infection ) . Secondary infection was analyzed at 0 . 5 hpi ( i . e . 1 h after addition of bacteria to cells ) Representative image and cfu quantification of re - infection assays with Shigella W of HeLa cells primarily infected with Shigella WT or ΔipaB / Inv mutant strain , or mock -

O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O O O O O 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-EXP_ASSAY O O O O O O O O O B-ORGANISM O O B-CELL_LINE O O O O B-ORGANISM O O O O O O O O O O O

Schematic representation of the experimental design for reinfection experiments. Cells were infected with Shigella-mCherry (primary infection) or mock-treated. At 3 hpi, cells were re-infected with Shigella expressing GFP (secondary infection). Secondary infection was analyzed at 0.5 hpi (i.e. 1 h after addition of bacteria to cells) Representative image and cfu quantification of re-infection assays with Shigella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated


Representative image and cfu quantificatio of re - infection assays wit Salmonella W of HeLa cells primarily infected with Shigella WT or ΔipaB / Inv mutant strain , or mock -

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Representative image and cfu quantificatio of re-infection assays wit Salmonella W of HeLa cells primarily infected with Shigella WT or ΔipaB/Inv mutant strain, or mock-treated


Representative images ( F ) and cfu quantification ( G ) of the re - infection assays with Salmonella ΔfliC , ΔfliC / pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock -

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Representative images (F) and cfu quantification (G) of the re-infection assays with Salmonella ΔfliC, ΔfliC/pFliC or ΔflhC mutant strains in HeLa cells primarily infected with Shigella WT or mock-treated


Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment . I and J . Representative images ( I ) and cfu quantification ( J ) of the secondary infection with Shigella WT , in cells transfected with ASM siRNA or control

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Schematic representation of the experimental design for reinfections upon knockdown or inhibitor treatment. I and J. Representative images (I) and cfu quantification (J) of the secondary infection with Shigella WT, in cells transfected with ASM siRNA or control siRNA


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Cfu quantification of the secondary infection with Shigella WT in HeLa cells following amitriptyline


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Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin transfectio with MAPK p38 siRN


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Cfu quantification of the secondary infection with Shigella WT in HeLa cells followin SB203580 treatmen


3D reconstruction of representative images of ASM ( A ) or ceramide ( B ) staining in HeLa cells infected with Shigella WTor mock - treated , analyzed at 3 hpi . ASM , ceramide , Hoechst and Shigella staining were surface converted by voxel

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3D reconstruction of representative images of ASM (A) or ceramide (B) staining in HeLa cells infected with Shigella WTor mock-treated, analyzed at 3 hpi. ASM, ceramide, Hoechst and Shigella staining were surface converted by voxel distance


ASM enzymatic activity measurement in HeLa cells infected with Shigella - WT or mock - treated , analyzed at 3 hpi . The ASM enzymatic activity was determined in the membrane fraction corresponding to 3 . 0 × 105 cells per

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ASM enzymatic activity measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi. The ASM enzymatic activity was determined in the membrane fraction corresponding to 3.0×105 cells per condition


ceramide measurement in HeLa cells infected with Shigella - WT or mock - treated , analyzed at 3 hpi Ceramide is shown normalized to mock - treated

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ceramide measurement in HeLa cells infected with Shigella-WT or mock-treated, analyzed at 3 hpi Ceramide is shown normalized to mock-treated cells


Representative spinning - disk confocal images of colon tissue sections of non - infected ( E and F ) and Shigella infected ( G and H ) guinea pigs , collected at 4 hpi . E and G are montages of multiple image fields ; dashed boxes in ( E and G ) are shown enlarged in ( F and H ) ,

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Representative spinning-disk confocal images of colon tissue sections of non-infected (E and F) and Shigella infected (G and H) guinea pigs, collected at 4 hpi. E and G are montages of multiple image fields; dashed boxes in (E and G) are shown enlarged in (F and H), respectively


Mean fluorescence intensity of ceramide signal in surface epithelial cells ( outermost tissue layer ) of non - infected or Shigella - infected guinea pig colon tissue

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Mean fluorescence intensity of ceramide signal in surface epithelial cells (outermost tissue layer) of non-infected or Shigella-infected guinea pig colon tissue sections


A The mRNA value of AKT2 was analyzed in the GEO Profiles from the liver explant of Hepatitis B virus ( HBV ) - associated acute liver failure ( ALF ) patients ( GDS4387 / 225471 _ s _ at ) , and from bronchial epithelial cells with pandemic and seasonal H1N1 influenza virus infections in vitro ( GDS4855 / 203808 _ at ) . normal , n = 10 ; HBV , n = 17 ; control , n = 3 ; influenza A , n = 9 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using unpaired t - test Error bars mean ±

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A The mRNA value of AKT2 was analyzed in the GEO Profiles from the liver explant of Hepatitis B virus (HBV)-associated acute liver failure (ALF) patients (GDS4387/225471_s_at), and from bronchial epithelial cells with pandemic and seasonal H1N1 influenza virus infections in vitro (GDS4855/203808_at). normal, n=10; HBV, n=17; control, n=3; influenza A, n=9. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using unpaired t-test Error bars mean ± SD


C The qRT - PCR analysis of Akt2 mRNA in PEMs stimulated with lipo - poly ( I : C ) ( 1 μg / mL ) , lipo - poly ( A : T ) ( 1 μg / mL ) , lipo - ISD ( 3 μg / mL ) or HSV - 1 ( MOI , 1 ) for 6 hours . n = 3 , respectively . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using one - way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM ) .

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C The qRT-PCR analysis of Akt2 mRNA in PEMs stimulated with lipo-poly(I:C) (1 μg/mL), lipo-poly(A:T) (1 μg/mL), lipo-ISD (3 μg/mL) or HSV-1 (MOI, 1) for 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using one-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).


D The mRNA of Akt2 ( red line ) and Ifnb1 ( black line ) were analyzed by qRT - PC in the mock and VSV ( MOI , 1 ) or LM ( MOI , 1 ) - stimulated PEMs or BMDMs at the indicated time . Mock and VSV , n = 4 ; mock and LM , n ≥ 3 .

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D The mRNA of Akt2 (red line) and Ifnb1 (black line) were analyzed by qRT-PC in the mock and VSV (MOI, 1) or LM (MOI, 1)-stimulated PEMs or BMDMs at the indicated time. Mock and VSV, n=4; mock and LM, n≥3.


G - H PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 ( 10 μM ) ( G ) for 2 hours , or knocked down of Akt2 with siAkt2 - 1 ( 20 nM ) for 48 hours ( H ) , then the mRNA level of Ifnb1 was measured by qRT - PCR after lipo - poly ( I : C ) , lipo - poly ( A : T ) , lipo - ISD , VSV , or HSV - 1 stimulation for another 6 hours . n = 3 , respectively . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM ) .

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G-H PEMs were pretreated with DMSO or AKT2 inhibitor CCT128930 (10 μM) (G) for 2 hours, or knocked down of Akt2 with siAkt2-1 (20 nM) for 48 hours (H), then the mRNA level of Ifnb1 was measured by qRT-PCR after lipo-poly(I:C), lipo-poly(A:T), lipo-ISD, VSV, or HSV-1 stimulation for another 6 hours. n=3, respectively. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent biological replicates Error bars mean ± SEM).


A - B qRT - PCR detection for the mRNA expression of Ifnb1 ( A ) , Ifna4 , Cxcl10 and Ccl5 ( B ) in WT ( n = 3 ) and Akt2 KO ( n = 3 ) PEMs stimulated with lipo - poly ( I : C ) , VSV , lipo - poly ( A : T ) , lipo - ISD or HSV - 1 for 6 hours . C The expression of Ifnb1 mRNA level by qRT - PCR in WT ( n = 3 ) and Akt2 KO ( n = 3 ) PEMs treated with poly ( I : C ) ( 10 μg / mL ) for 6 hours ( left panel ) or LPS ( 1 μg / mL ) for 2 hours ( right panel ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA test Data are from three independent experiments Error bars mean ±

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A-B qRT-PCR detection for the mRNA expression of Ifnb1 (A), Ifna4, Cxcl10 and Ccl5 (B) in WT (n=3) and Akt2 KO (n=3) PEMs stimulated with lipo-poly(I:C), VSV, lipo-poly(A:T), lipo-ISD or HSV-1 for 6 hours. C The expression of Ifnb1 mRNA level by qRT-PCR in WT (n=3) and Akt2 KO (n=3) PEMs treated with poly(I:C) (10 μg/mL) for 6 hours (left panel) or LPS (1 μg/mL) for 2 hours (right panel). Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are from three independent experiments Error bars mean ± SEM


H Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours , then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT - PCR ( left panel ) . Every dot represents three zebrafish embryos . Horizontal square bracket shows the statistical analysis of the comparison with ' PBS mock ' , the rest shows the comparison with ' PBS VSV ' . PBS mock , n = 5 ; PBS VSV , n = 13 ; AKT2 VSV , n = 7 ; AKT2 - T309A / S474A VSV , n = 9 . H & E staining ( middle panel ) and survival rates ( Kaplan - Meier curve ) ( right panel ) were collected from zebrafish larvae after VSV micro - injection for 18 hours or longer . The arrows indicated the VSV - infected eye and skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA test or log - rank ( Mantel - Cox ) test Data are from three independent biological replicates Error bars mean ± SD ) .

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H Zebrafish larvae were overexpressed the indicated protein for 48 hours and challenged with VSV for another 6 hours, then the mRNA levels of ifn1 in zebrafish larvae were measured by qRT-PCR (left panel). Every dot represents three zebrafish embryos. Horizontal square bracket shows the statistical analysis of the comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=5; PBS VSV, n=13; AKT2 VSV, n=7; AKT2-T309A/S474A VSV, n=9. H&E staining (middle panel) and survival rates (Kaplan-Meier curve) (right panel) were collected from zebrafish larvae after VSV micro-injection for 18 hours or longer. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test or log-rank (Mantel-Cox) test Data are from three independent biological replicates Error bars mean ± SD).


D WT and Akt2 KO PEMs were infected with or without VSV for 6 hours . Then , cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti - AKT2 antibody , followed by immunoblotting using anti - IRF3 antibody ( left panel ) . Alternatively , cell lysates were prepared for immunoprecipitation using anti - IRF3 antibody , followed by immunoblotting using anti - AKT2 antibody ( right panel ) to detect the endogenous interaction between AKT2 and IRF3 . Data information : * Data are representative of two or three independent biological

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D WT and Akt2 KO PEMs were infected with or without VSV for 6 hours. Then, cell lysates of WT or Akt2 KO PEMs were prepared for immunoprecipitation using anti-AKT2 antibody, followed by immunoblotting using anti-IRF3 antibody (left panel). Alternatively, cell lysates were prepared for immunoprecipitation using anti-IRF3 antibody, followed by immunoblotting using anti-AKT2 antibody (right panel) to detect the endogenous interaction between AKT2 and IRF3. Data information: * Data are representative of two or three independent biological replicates


G Immunofluorescent microscopic imaging for ( left panel ) and statistics analysis ( right panel ) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post - VSV infection . IRF3 ( red ) , Nuclei ( Hoechst , green ) . The white arrows indicate the nuclei with IRF3 translocation . Mock , n = 2 ; VSV , n = 3 . Bar , 20 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using two - way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM ) .

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G Immunofluorescent microscopic imaging for (left panel) and statistics analysis (right panel) for IRF3 nuclear translocation in WT and Akt2 KO PEMs at 6 hour post-VSV infection. IRF3 (red), Nuclei (Hoechst, green). The white arrows indicate the nuclei with IRF3 translocation. Mock, n=2; VSV, n=3. Bar, 20 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using two-way ANOVA test Data are representative of two or three independent biological replicates Error bars mean ± SEM).


H Immunoassay of IRF3 , AKT2 , LaminB1 and GAPDH in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 hours . Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively . Data information : * Data are representative of two or three independent biological

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H Immunoassay of IRF3, AKT2, LaminB1 and GAPDH in the nuclear and cytoplasmic fractions from the WT and Akt2 KO PEMs after VSV treatment for 6 hours. Tubulin and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates


I Immunoassay of IRF3 , SP1 and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 hours and stimulated with VSV for 6 hours . Tubulin and SP1 served as cytoplasmic and nucleic protein loading control respectively . Data information : * Data are representative of two or three independent biological

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I Immunoassay of IRF3, SP1 and Tubulin in the nuclear and cytoplasmic fractions of the HEK293T cells overexpressed with indicated constructs for 24 hours and stimulated with VSV for 6 hours. Tubulin and SP1 served as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of two or three independent biological replicates


D Immunofluorescence microscopy ( top panel ) of AKT2 - , Flag - IRF3 - or Flag - IRF3 - T207A - overexpressed MEF cells after poly ( A : T ) stimulation for 6 hours . Flag ( IRF3 , red ) and Hoechst ( nuclei , blue ) . Immunoassay of nuclear - cytoplasm extractions ( bottom panel ) from HEK293T - IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours . GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively . Bar , 20 μm . Data information : * Data are representative of three independent

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D Immunofluorescence microscopy (top panel) of AKT2-, Flag-IRF3- or Flag-IRF3-T207A-overexpressed MEF cells after poly(A:T) stimulation for 6 hours. Flag (IRF3, red) and Hoechst (nuclei, blue). Immunoassay of nuclear-cytoplasm extractions (bottom panel) from HEK293T-IRF3 KO cells with overexpression of the indicated plasmids for 24 hours followed by VSV infection for 6 hours. GAPDH and SP1 were used as cytoplasmic and nucleic protein loading control respectively. Bar, 20 μm. Data information: * Data are representative of three independent experiments


F Survival rates ( left panel ) until 72 hours and H & E staining ( right panel ) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge . The arrows indicated the VSV - infected eye and skeletal muscle in zebrafish larvae . Bars , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent

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F Survival rates (left panel) until 72 hours and H&E staining (right panel) at 18 hours of the zebrafish larvae with indicated protein expression and VSV challenge. The arrows indicated the VSV-infected eye and skeletal muscle in zebrafish larvae. Bars, 100 μm. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


G qRT - PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later . Every dot represents three zebrafish embryos . Horizontal vertical square bracket shows the statistical analysis of comparison with ' PBS mock ' , the rest shows the comparison with ' PBS VSV ' . PBS mock , n = 15 ; PBS VSV , n = 9 ; IRF3 VSV , n = 16 ; IRF3 - T207A VSV , n = 11 ; AKT2 VSV , n = 14 ; AKT2 - T309A / S474A VSV , n = 9 ; IRF3 + AKT2 VSV , n = 9 ; IRF3 + AKT2 - T309A / S474A VSV , n = 13 ; IRF3 - T207A + AKT2 VSV , n = 10 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a one - way ANOVA test Data are representative of three independent experiments Error bars mean ± SD ) .

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G qRT-PCR analysis of the VSV copies in overexpressed zebrafish embryos with the VSV challenge at 18 hours later. Every dot represents three zebrafish embryos. Horizontal vertical square bracket shows the statistical analysis of comparison with 'PBS mock', the rest shows the comparison with 'PBS VSV'. PBS mock, n=15; PBS VSV, n=9; IRF3 VSV, n=16; IRF3-T207A VSV, n=11; AKT2 VSV, n=14; AKT2-T309A/S474A VSV, n=9; IRF3 + AKT2 VSV, n=9; IRF3 + AKT2-T309A/S474A VSV, n=13; IRF3-T207A + AKT2 VSV, n=10. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a one-way ANOVA test Data are representative of three independent experiments Error bars mean ± SD).


H The survival rates of over - expressed zebrafish larvae as long as 72 hours after the VSV challenge . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 and ns , not significant ( P > 0 . 05 ) ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent

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H The survival rates of over-expressed zebrafish larvae as long as 72 hours after the VSV challenge. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and ns, not significant (P > 0.05); using a log-rank (Mantel-Cox) test Data are representative of three independent experiments


B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent experiments

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B Survival rates of WT and Akt2 KO mice after lethal dose injection of VSV during 120 hours (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


E WT and Akt2 KO mice were injected with VSV for 24 hours ( 1 × 106 PFU / g , i . v . ) . Representative images of the VSV infected cells ( GFP ) were collected by fluorescence microscope ( left panel ) and the VSV copies ( WT , n = 6 ; Akt2 KO n = 7 ) were measured by qRT - PCR ( right panel ) in the liver of mice . Bar , 100 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a unpaired t - test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD ) .

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E WT and Akt2 KO mice were injected with VSV for 24 hours (1 × 106 PFU/g, i.v.). Representative images of the VSV infected cells (GFP) were collected by fluorescence microscope (left panel) and the VSV copies (WT, n=6; Akt2 KO n=7) were measured by qRT-PCR (right panel) in the liver of mice. Bar, 100 μm. Data information: *P < 0.05, **P < 0.01; using a unpaired t-test Data are representative of three independent experiments respectively in which the different groups of mice were sacrificed and quantified for indicated experimental purposes Error bars mean ± SD).


Bone marrow cells from WT and Akt2 KO mice ( CD45 . 2 ) were adoptively transferred to lethally irradiated CD45 . 1 mice and infected with VSV . representative images were analyzed Data information : * Data are representative of three independent experiments

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Bone marrow cells from WT and Akt2 KO mice (CD45.2) were adoptively transferred to lethally irradiated CD45.1 mice and infected with VSV. representative images were analyzed Data information: * Data are representative of three independent experiments respectively


I Survival rates of WT mice that were intraperitoneal injected with CCT128930 ( 20 mg / kg ) twice every other day before VSV infection ( 1 × 107 PFU / g , i . v . ) . Data information : * P < 0 . 05 , * * P < 0 . 01 ; using a log - rank ( Mantel - Cox ) test Data are representative of three independent experiments

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I Survival rates of WT mice that were intraperitoneal injected with CCT128930 (20 mg/kg) twice every other day before VSV infection (1 × 107 PFU/g, i.v.). Data information: *P < 0.05, **P < 0.01; using a log-rank (Mantel-Cox) test Data are representative of three independent experiments respectively


immunoblot analysis of IRF3 , LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions ( C ) from WT and Akt2 KO mice treated with TMPD for 2 weeks . GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively . Data information : * Data are representative of three independent

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immunoblot analysis of IRF3, LaminB1 and GAPDH in the PIC nuclear and cytoplasmic fractions (C) from WT and Akt2 KO mice treated with TMPD for 2 weeks. GAPDH and LaminB1 were used as cytoplasmic and nucleic protein loading control respectively. Data information: * Data are representative of three independent experiments


H & E - staining ( E left panel ) and immunofluorescence of IgG deposits ( E right panel ) in the kidney sections were collected from WT and Akt2 KO bone marrow - reconstituted CD45 . 1 mice Bar , 50 μm . Data information : * Data are representative of three independent

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H&E-staining (E left panel) and immunofluorescence of IgG deposits (E right panel) in the kidney sections were collected from WT and Akt2 KO bone marrow-reconstituted CD45.1 mice Bar, 50 μm. Data information: * Data are representative of three independent experiments


J . qRT - PCR analysis of the relative mRNA expression levels of AKT2 ( left panel ) and IFNA ( middle panel ) or IFNβ1 ( right panel ) in CD14 positive monocytes isolated from SLE patients ( n = 9 ) or healthy donors ( n = 9 ) . Data information : * P < 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 ; using unpaired t - test or correlation analyses Data are pooled from different individuals Error bars mean ± SD ) .

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J. qRT-PCR analysis of the relative mRNA expression levels of AKT2 (left panel) and IFNA (middle panel) or IFNβ1 (right panel) in CD14 positive monocytes isolated from SLE patients (n=9) or healthy donors (n=9). Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using unpaired t-test or correlation analyses Data are pooled from different individuals Error bars mean ± SD).


K Immunofluorescence of IRF3 ( Red ) , AKT2 ( Green ) , DAPI ( Blue , nucleus ) in CD14 + cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software . Cells ( n = 39 ) were collected 3 patients . Bar , 2 μm . Data information : * P < 0 . 05 , * * P < 0 . 01 and * * * * P < 0 . 0001 ; using correlation analyses Data are pooled from different individuals or representative of three independent

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K Immunofluorescence of IRF3 (Red), AKT2 (Green), DAPI (Blue, nucleus) in CD14+ cells from SLE patients followed by analysis of the cells about mean amount of AKT2 in this cell as well as mean amount of IRF3 in the same nucleus with Image J software. Cells (n=39) were collected 3 patients. Bar, 2 μm. Data information: *P < 0.05, **P < 0.01 and ****P < 0.0001; using correlation analyses Data are pooled from different individuals or representative of three independent experiments


Ex vivo generation of CAR T cells . Activated human PBMC were left untransduced or incubated with CD8 - LVCD19CAR at an MOI of 2 . Five days later , expression of CD19 - CAR and CD8 were determined on CD3 + cells . Numbers indicate the percentage of cells in the respective gate .

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Ex vivo generation of CAR T cells. Activated human PBMC were left untransduced or incubated with CD8-LVCD19CAR at an MOI of 2. Five days later, expression of CD19-CAR and CD8 were determined on CD3+ cells. Numbers indicate the percentage of cells in the respective gate.


Detection of CAR T cells by vector copy numbers ( VCN ) . Genomic DNA was isolated from peritoneal cavity , spleen and blood cells . VCN were determined in technical duplicates by qPCR for two individual mice of each group . Presence of B cells in the transplanted PBMC is indicated below .

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Detection of CAR T cells by vector copy numbers (VCN). Genomic DNA was isolated from peritoneal cavity, spleen and blood cells. VCN were determined in technical duplicates by qPCR for two individual mice of each group. Presence of B cells in the transplanted PBMC is indicated below.


Cells isolated from the peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for the percentages of human CD8 + in CD3 + cells ( D ) , within the fraction of human CD45 + cells . Representative density plots are shown for the peritoneal cells .

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Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD8+ in CD3+ cells (D), within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.


Cells isolated from the peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for the percentages of CAR + or RFP + cells in the CD8 + and CD8 - fractions , respectively ( E ) within the fraction of human CD45 + cells . Representative density plots are shown for the peritoneal cells .

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Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of CAR+ or RFP+ cells in the CD8+ and CD8- fractions, respectively (E) within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.


Cells isolated from the peritoneal cavity ( peritoneum ) , spleen , or blood were evaluated by flow cytometry for the percentages of human CD19 + cells within the fraction of human CD45 + cells . Representative density plots are shown for the peritoneal cells .

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Cells isolated from the peritoneal cavity (peritoneum), spleen, or blood were evaluated by flow cytometry for the percentages of human CD19+ cells within the fraction of human CD45+ cells. Representative density plots are shown for the peritoneal cells.


Mice were transplanted with B - cell depleted human PBMC and then received CD8 - LVCD19CAR ( filled circle ) or PBS ( open circle ) . As control , CD8 - LVCD19CAR or PBS were injected into mice transplanted with non - depleted PBMC . Data represent mean ± SD for all groups ( CD8 - LVCD19CAR : n = 6 in D , n = 4 in G ; CD8 - LVRFP : n = 4 ; PBS : n = 4 in G , all others n = 3 ) .

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Mice were transplanted with B-cell depleted human PBMC and then received CD8-LVCD19CAR (filled circle) or PBS (open circle). As control, CD8-LVCD19CAR or PBS were injected into mice transplanted with non-depleted PBMC. Data represent mean ± SD for all groups (CD8-LVCD19CAR: n=6 in D, n=4 in G; CD8-LVRFP: n=4; PBS: n=4 in G, all others n=3).


CAR T cell levels in the CD8 + and CD8 - T cells harvested from blood , spleen and bone marrow of PBS ( PBS ) or vector - injected ( CAR ) mice determined by flow cytometry .

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CAR T cell levels in the CD8+ and CD8- T cells harvested from blood, spleen and bone marrow of PBS (PBS) or vector-injected (CAR) mice determined by flow cytometry.


Detection of CAR T cells by determining vector copy numbers ( VCN ) in genomic DNA isolated from CD8 + enriched cells harvested from bone marrow .

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Detection of CAR T cells by determining vector copy numbers (VCN) in genomic DNA isolated from CD8+ enriched cells harvested from bone marrow.


Detection of CAR T cells in bone marrow quantified by flow cytometry .

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Detection of CAR T cells in bone marrow quantified by flow cytometry.


CD19 + B cell levels in blood , spleen and bone marrow determined by flow cytometry .

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CD19+ B cell levels in blood, spleen and bone marrow determined by flow cytometry.


Relative human CD19 + B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration .

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Relative human CD19+ B cell level in blood calculated by normalizing the levels at the day the animals were sacrificed to those before vector administration.


Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection . The distinct symbols used for each individual mouse are identical to the ones used in Fig . 2 . Mean values ± SD of n = 2 technical replicates .

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Cytokine levels in plasma of individual mice obtained from blood at 7 weeks after vector injection. The distinct symbols used for each individual mouse are identical to the ones used in Fig. 2. Mean values ± SD of n=2 technical replicates.


Immunohistochemistry of paraffin embedded sections from the lungs , brain meninges , brain striatum , spleen and liver of the vector - injected mouse M16 ( CAR ) and a control mouse injected with PBS ( PBS ) stained against CD8 ( αCD8 ) , the CAR ( αCAR ) , CD4 ( αCD4 ) , or CD19 ( αCD19 ) . Black arrowheads point at infiltrated lymphocytes , red arrowheads at histiocytes , and the yellow line indicates B - lymphocyte rich zones .

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Immunohistochemistry of paraffin embedded sections from the lungs, brain meninges, brain striatum, spleen and liver of the vector-injected mouse M16 (CAR) and a control mouse injected with PBS (PBS) stained against CD8 (αCD8), the CAR (αCAR), CD4 (αCD4), or CD19 (αCD19). Black arrowheads point at infiltrated lymphocytes, red arrowheads at histiocytes, and the yellow line indicates B-lymphocyte rich zones.


( A ) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc - tagged PINK1 genomic construct and a UAS - RNAi construct driven by elav - GAL4 . The RNAi constructs used were as follows : the non - specific control mCherry - RNAi ( Control - R ) , Lon - RNAi1 ( Lon - R1 ) , and Lon - RNAi2 ( Lon - R2 ) . An anti - Myc antibody was used to detect PINK1 and an anti - Actin antibody was used as a protein loading control . ( B ) Densitometry of the PINK1 - Myc bands from the indicated genotypes was performed using Fiji software . The PINK1 - Myc band intensities were then normalized to their respective Actin loading controls , and these ratios were in turn normalized to the Control - R PINK1 / Actin ratio .

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(A) Western blot analysis of whole head homogenates from transgenic flies expressing a Myc-tagged PINK1 genomic construct and a UAS-RNAi construct driven by elav-GAL4. The RNAi constructs used were as follows: the non-specific control mCherry-RNAi (Control-R), Lon-RNAi1 (Lon-R1), and Lon-RNAi2 (Lon-R2). An anti-Myc antibody was used to detect PINK1 and an anti-Actin antibody was used as a protein loading control. (B) Densitometry of the PINK1-Myc bands from the indicated genotypes was performed using Fiji software. The PINK1-Myc band intensities were then normalized to their respective Actin loading controls, and these ratios were in turn normalized to the Control-R PINK1/Actin ratio.


( C ) Western blot analysis of Lon protease ( Lon ) , Mitochondrial transcription factor A ( TFAM ) , Heat shock protein 60 ( Hsp60 ) and Actin in whole head homogenate from control and Lon deficient animals . ( D ) Quantification of the Lon band intensities from the indicated genotypes performed as described in B . ( E ) Quantification of the TFAM bands from the indicated genotypes performed as described in B . ( F ) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(C) Western blot analysis of Lon protease (Lon), Mitochondrial transcription factor A (TFAM), Heat shock protein 60 (Hsp60) and Actin in whole head homogenate from control and Lon deficient animals. (D) Quantification of the Lon band intensities from the indicated genotypes performed as described in B. (E) Quantification of the TFAM bands from the indicated genotypes performed as described in B. (F) Quantification of the Hsp60 bands from the indicated genotypes performed as described in B.


( G ) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs . The graph shows the normalized fluorescence intensity of TMRE , an indicator of mitochondrial membrane potential , relative to age - matched control animals . Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples . The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity . All experiments described in this figure were repeated at least three times per genotype . Error bars represent the standard error of the mean ( s . e . m . ) . * p < 0 . 05 , * * p < 0 . 005 by Student t test .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Flow cytometry analysis of dissociated cells from the brains of flies expressing the indicated RNAi constructs. The graph shows the normalized fluorescence intensity of TMRE, an indicator of mitochondrial membrane potential, relative to age-matched control animals. Fluorescence intensity was normalized to control samples prepared and analyzed on the same day as experimental samples. The mitochondrial membrane potential uncoupling agent CCCP was added to samples of the indicated genotypes to illustrate the effect of mitochondrial depolarization on TMRE signal intensity. All experiments described in this figure were repeated at least three times per genotype. Error bars represent the standard error of the mean (s.e.m.). *p<0.05, **p<0.005 by Student t test.


PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon . ( A ) Mitochondrial ( Mito ) and postmitochondrial supernatant ( Sup ) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc , Complex V β ( Comp V β ) , NADH dehydrogenase ( ubiquinone ) Fe - S protein 3 ( NDUFS3 ) , voltage - dependent anion channel ( VDAC ) and Actin . elav - GAL4 was used to drive expression of the RNAi constructs used in these experiments . ( B ) Densitometry of the MPP - PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software . PINK1 band intensities were normalized to VDAC intensity , and this ratio was in turn normalized to the MPP - PINK1 / VDAC ratio in Control - R animals . ( C ) Quantification of the Rho - PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B . ( D ) Quantification of the AFG - PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B . ( E ) Quantification of the MPP - PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control . ( F ) Quantification of the Rho - PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E . ( G ) Quantification of the AFG - PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E . Results shown are representative of at least three independent subcellular fractionation experiments per genotype . Error bars represent s . e . m . * p < 0 . 05 by Student t test .

B-GENEPROD O O O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-TISSUE O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

PINK1 accumulates in the mitochondrial and postmitochondrial supernatant fractions upon knockdown of Lon.(A) Mitochondrial (Mito) and postmitochondrial supernatant (Sup) protein fractions isolated from the heads of flies of the indicated genotypes were analyzed by western blot with antibodies to Myc, Complex V β (Comp V β), NADH dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), voltage-dependent anion channel (VDAC) and Actin. elav-GAL4 was used to drive expression of the RNAi constructs used in these experiments. (B) Densitometry of the MPP-PINK1 bands in the mitochondrial fractions from the indicated genotypes was performed using Fiji software. PINK1 band intensities were normalized to VDAC intensity, and this ratio was in turn normalized to the MPP-PINK1/VDAC ratio in Control-R animals. (C) Quantification of the Rho-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (D) Quantification of the AFG-PINK1 band intensities in mitochondrial fractions from the indicated genotypes was performed as described in B. (E) Quantification of the MPP-PINK1 band intensities in the supernatant fractions from the indicated genotypes was performed as described in B except that Actin rather than VDAC was used as the loading control. (F) Quantification of the Rho-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. (G) Quantification of the AFG-PINK1 band intensities in supernatant fractions from the indicated genotypes was performed as described in E. Results shown are representative of at least three independent subcellular fractionation experiments per genotype. Error bars represent s.e.m. *p<0.05 by Student t test.


A ) Mitochondrial fractions isolated from the heads of flies expressing Control - R were divided in half . One of the two samples was treated with 0 . 5 µg / ml Proteinase K and both samples were incubated at 4 ° C for 20 minutes . Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin ( Mfn ) , the inner membrane - associated matrix protein Complex V β ( Comp V β ) , and the matrix luminal protein pyruvate dehydrogenase ( PDH ) . elav - GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments . ( B ) Densitometry of Mfn , Comp V β , and PDH bands from experiments represented by panel A were performed using Fiji software , and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown .

O O B-SUBCELLULAR O O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD 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-GENEPROD O O O O B-GENEPROD O O O

A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane-associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown.


( C ) Mitochondrial fractions isolated from the heads of flies expressing Control - R and PINK1 - Myc were divided in half . Each sample was treated with the indicated concentrations of ProK . Following incubation , samples were subjected to western blot analysis using antibodies to the indicated proteins . ( D ) Densitometry of the PINK1 - Myc , Comp V β , and PDH bands from experiments represented by panel C were performed as described in B . The Mfn quantification data from panel B is also included for comparison .

O O O B-SUBCELLULAR O O O O B-TISSUE O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

(C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison.


( E ) Mitochondrial fractions isolated from the heads of Lon - R2 and PINK1 - Myc expressing flies were divided in half . Each sample was treated with the indicated concentrations of ProK . Following incubation , samples were subjected to western blot analysis using antibodies to the indicated proteins . ( F ) Densitometry of the PINK1 - Myc , Comp V β , and PDH bands from experiments represented by panel E were performed as described in B . All experiments described were repeated at least three times . Error bars represent s . e . m .

O O O B-SUBCELLULAR O O O O B-TISSUE O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O 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) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.


PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon . Immunofluorescent staining of thoracic muscles expressing matrix - targeted YFP ( Mito - YFP ) under control of the sqh genomic promoter , together with either ( A ) Myc - tagged MitochondrialRho ( Miro - Myc ) , ( B ) FLAG - tagged Optic atrophy 1 ( Opa1 - FLAG ) , ( C ) endogenous Cytochrome C ( Cyto C ) , ( D ) Myc - tagged PINK1 ( PINK1 - Myc ) , or ( E ) Lon - RNAi1 ( Lon - R1 ) and PINK1 - Myc . The muscle - specific Mhc - GAL4 driver was used to drive expression of Miro - Myc in panel A and Opa1 - FLAG in panel B , and the muscle - specific Dmef2 - GAL4 driver was used to drive expression of Lon - R1 in panel E . ( F ) We determined the percent volume shared between green objects ( representing the matrix marker Mito - YFP ) and red objects ( representing the mitochondrial proteins analyzed in A - E ) . Red objects that shared at least 60 % of their volume with Mito - YFP were considered to co - localize . Bar represents 2 µm .

B-GENEPROD O O O B-SUBCELLULAR O O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon.Immunofluorescent staining of thoracic muscles expressing matrix-targeted YFP (Mito-YFP) under control of the sqh genomic promoter, together with either (A) Myc-tagged MitochondrialRho (Miro-Myc), (B) FLAG-tagged Optic atrophy 1 (Opa1-FLAG), (C) endogenous Cytochrome C (Cyto C), (D) Myc-tagged PINK1 (PINK1-Myc), or (E) Lon-RNAi1 (Lon-R1) and PINK1-Myc. The muscle-specific Mhc-GAL4 driver was used to drive expression of Miro-Myc in panel A and Opa1-FLAG in panel B, and the muscle-specific Dmef2-GAL4 driver was used to drive expression of Lon-R1 in panel E. (F) We determined the percent volume shared between green objects (representing the matrix marker Mito-YFP) and red objects (representing the mitochondrial proteins analyzed in A-E). Red objects that shared at least 60% of their volume with Mito-YFP were considered to co-localize. Bar represents 2 µm.


A ) The eye severity score of flies co - overexpressing PINK1 with Control - R ( n = 74 ) , Lon - R1 ( n = 163 ) , or Lon - R2 ( n = 90 ) using the ey - GAL4 driver .

O O O B-TISSUE O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O

A) The eye severity score of flies co-overexpressing PINK1 with Control-R (n = 74), Lon-R1 (n = 163), or Lon-R2 (n = 90) using the ey-GAL4 driver.


( B ) Comparison of the frequency of thoracic indentations in flies expressing the PINK1 - RNAi construct ( PINK1 - R ) in an otherwise wild - type background to the frequency in animals that also bear heterozygous mutations in Lon : PINK1 - RNAi + / + ( n = 306 ) , PINK1 - RNAi LonEP / + ( n = 74 ) , and PINK1 - RNAi LonDf / + ( n = 83 ) . LonEP refers to the LonG3998 P - element allele of Lon , and LonDf refers to the Df ( 3L ) Exel9011 deletion that removes the Lon gene along with several other genes . The Dmef2 - GAL4 driver was used to drive expression of the PINK1 - R construct in experiments described in panel B . Error bars represent s . e . m . * p < 0 . 05 , * * p < 0 . 005 by Student t test .

O O O O O O O O B-TISSUE O O B-ORGANISM O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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) Comparison of the frequency of thoracic indentations in flies expressing the PINK1-RNAi construct (PINK1-R) in an otherwise wild-type background to the frequency in animals that also bear heterozygous mutations in Lon: PINK1-RNAi +/+ (n = 306), PINK1-RNAi LonEP/+ (n = 74), and PINK1-RNAi LonDf/+ (n = 83). LonEP refers to the LonG3998 P-element allele of Lon, and LonDf refers to the Df(3L)Exel9011 deletion that removes the Lon gene along with several other genes. The Dmef2-GAL4 driver was used to drive expression of the PINK1-R construct in experiments described in panel B. Error bars represent s.e.m. *p<0.05, **p<0.005 by Student t test.


A . Viability of primary human PMNs following treatment with purified recombinant bi - component leukocidins using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 5 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O

A. Viability of primary human PMNs following treatment with purified recombinant bi-component leukocidins using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 5 donors.


B . Half maximal effective concentrations ( EC50 ) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter . E = LukE , D = LukD , A = HlgA , B = HlgB , C = HlgC , S = LukS - PV , F = LukF - PV . Bars indicate Mean ± SEM , with n = 6 donors .

O O O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

B. Half maximal effective concentrations (EC50) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter. E = LukE, D = LukD, A = HlgA, B = HlgB, C = HlgC, S = LukS-PV, F = LukF-PV. Bars indicate Mean ± SEM, with n = 6 donors.


D . Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter . Insert : 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12 % SDS - PAGE gel . E = LukE , D = LukD , Emut1 = LukEmut1 , Dmut = LukDmut . Bars indicate Mean ± SEM , with n = 4 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

D. Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12% SDS-PAGE gel. E = LukE, D = LukD, Emut1 = LukEmut1, Dmut = LukDmut. Bars indicate Mean ± SEM, with n = 4 donors.


E . Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED ( 78 . 12 nM per subunit ) and a dose response of WT or dominant - negative ( DN ) toxin subunits using the viability dye LukE . E = LukE , D = LukD , Emut1 = LukEmut1 , Emut2 = LukEmut2 , Emut1Dmut = LukEmut1LukDmut , Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM , with n = 6 donors .

O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O B-GENEPROD O 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-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O

E. Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED (78.12 nM per subunit) and a dose response of WT or dominant-negative (DN) toxin subunits using the viability dye LukE. E = LukE, D = LukD, Emut1 = LukEmut1, Emut2 = LukEmut2, Emut1Dmut = LukEmut1LukDmut, Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM, with n = 6 donors.


C . Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 3 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O

C. Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 3 donors.


D - F . Viability of primary humanPMNs following incubation with WT or mutant toxin pairings . Insert : 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12 % SDS - PAGE gel . Amut1 = HlgAmut1 , Bmut = HlgBmut , Cmut1 = HlgCmut1 , Smut1 = LukS - PVmut1 , Fmut = LukF - PVmut . Bars indicate Mean ± SEM , with n = 4 donors ( D ) , n = 4 donors ( E ) , and n = 5 donors ( F ) .

O O O O B-EXP_ASSAY O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O 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. Viability of primary humanPMNs following incubation with WT or mutant toxin pairings. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12% SDS-PAGE gel. Amut1 = HlgAmut1, Bmut = HlgBmut, Cmut1 = HlgCmut1, Smut1 = LukS-PVmut1, Fmut = LukF-PVmut. Bars indicate Mean ± SEM, with n = 4 donors (D), n = 4 donors (E), and n = 5 donors (F).


A - D . Viability of primary human PMNs following incubation with a LD90 of LukED ( 78 . 12 nM ) , HlgAB ( 78 . 12 nM ) , HlgCB ( 39 . 1 nM ) , or LukSF - PV ( 19 . 53 nM ) and the indicated dominant - negative toxins using the viability dye CellTiter . Open white symbols indicate no treatment . Filled black symbols indicate treatment with dominant - negative toxins . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 5 donors .

O O O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

A-D. Viability of primary human PMNs following incubation with a LD90 of LukED (78.12 nM), HlgAB (78.12 nM), HlgCB (39.1 nM), or LukSF-PV (19.53 nM) and the indicated dominant-negative toxins using the viability dye CellTiter. Open white symbols indicate no treatment. Filled black symbols indicate treatment with dominant-negative toxins. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 5 donors.


E . Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA - MRSA USA300 strain LAC using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 6 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O B-ORGANISM I-ORGANISM O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O

E. Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA-MRSA USA300 strain LAC using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 6 donors.


F . Viability of primary human PMNs following incubation with 1 . 25 % filtered culture supernatants and a dose response of the indicated dominant - negative ( DN ) toxins using the viability dye CellTiter . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 3 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O 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-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

F. Viability of primary human PMNs following incubation with 1.25% filtered culture supernatants and a dose response of the indicated dominant-negative (DN) toxins using the viability dye CellTiter. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.


G . Viability of primary human PMNs during a 3 - hour ex vivo infection with the S . aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± SEM , with n = 3 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O

G. Viability of primary human PMNs during a 3-hour ex vivo infection with the S. aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3 donors.


H . Viability of primary human PMNs during a 4 - hr ex vivo infection with a MOI of 50 of S . aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 3 donors .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

H. Viability of primary human PMNs during a 4-hr ex vivo infection with a MOI of 50 of S. aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.


B . Exoproteome analysis of LukAB mutants produced by S . aureus strain Newman in a toxinless background . Samples were electrophoresed on a 12 % SDS - PAGE gel and visualized by staining with Coomassie blue . A representative of two experiments is shown .

O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O

B. Exoproteome analysis of LukAB mutants produced by S. aureus strain Newman in a toxinless background. Samples were electrophoresed on a 12% SDS-PAGE gel and visualized by staining with Coomassie blue. A representative of two experiments is shown.


C . Exoproteome and immunoblot analyses of wildtype S . aureus strain Newman transformed with empty plasmid ( p - ) or with plasmids containing the mutated lukAB loci . A representative of two independent experiments is shown .

O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O

C. Exoproteome and immunoblot analyses of wildtype S. aureus strain Newman transformed with empty plasmid (p-) or with plasmids containing the mutated lukAB loci. A representative of two independent experiments is shown.


D . Viability of primary human PMNs during a 2 - hour ex vivo infection with the indicated S . aureus strains using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± SEM , with n = 3 . Statistical significance is indicated as follows : MOI of 100 : * * p = 0 . 0026 for p - vs LukABmut1 and * * * p = 0 . 0003 for p - vs LukABmut2 ; MOI of 50 : * * * * p < 0 . 0001 for p - vs LukABmut1 and * * * * p < 0 . 0001 for p - vs LukABmut2 ; MOI of 25 : * * * p = 0 . 0007 for p - vs LukABmut1 and * * * * p < 0 . 0001 for p - vs LukABmut2 using two - way ANOVA with Tukey ' s post hoc test correction for multiple comparisons .

O O B-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability of primary human PMNs during a 2-hour ex vivo infection with the indicated S. aureus strains using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3. Statistical significance is indicated as follows: MOI of 100: **p = 0.0026 for p- vs LukABmut1 and ***p = 0.0003 for p- vs LukABmut2; MOI of 50: ****p <0.0001 for p- vs LukABmut1 and ****p <0.0001 for p- vs LukABmut2; MOI of 25: ***p = 0.0007 for p- vs LukABmut1 and ****p < 0.0001 for p- vs LukABmut2 using two-way ANOVA with Tukey's post hoc test correction for multiple comparisons.