B ) Representative images showing full length control and selected Miro1 lysine mutants ( Miro15R , Miro1allR and Miro1R572K ) expressing in MiroDKO MEF cells . All constructs ( myc - tag , green ) localise to mitochondria ( Tom20 , red ) .

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B) Representative images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red).


C ) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH - SY5Y cells .

C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells.


D ) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment ( 1h , 10 μM ) in FlagParkin overexpressing SH - SY5Y . The effect is quantified in ( E ) ( n = 4 independent experiments for Miro1WT and Miro15R and n = 3 for Miro1allR and Miro1R572K , ANOVA with Sidak ' s post hoc test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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D) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


F ) Representative western blot showing a degradation assay in FlagParkin overexpressing SH - SY5Y cells transfected with Miro1WT , Miro15R or Miro1allR constructs and treated with FCCP ( 10 μM ) for 3 or 6 hours . Quantification of Miro1 levels ( G ) the matrix protein PDH E1α in ( H ) and Parkin levels ( I ) ( n = 4 independent experiments ; ANOVA with Sidak ' s post hoc test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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F) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y cells transfected with Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative images at 3 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO cells co - transfected with the specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal the mitochondrial network . A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition .

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(A) Representative images at 3 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition.


( B and C ) Quantification of the fraction of cells showing advanced stages of the mitophagic process ( cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network ) at 1 hour ( B ) or 3 hours ( C ) after FCCP treatment ( data collected from at least 3 independent experiments : WT n = 7 , MiroDKO n = 8 , + mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; mycMiro2 = 3 ; one way ANOVA with Dunnett post test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

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(B and C) Quantification of the fraction of cells showing advanced stages of the mitophagic process (cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network) at 1 hour (B) or 3 hours (C) after FCCP treatment (data collected from at least 3 independent experiments: WT n=7, MiroDKO n=8, +mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; mycMiro2=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


D ) Western blots and quantification of Miro1 degradation in MiroDKO cells co - expressing YFPParkin and the indicated myc - tagged Miro1 constructs and treated with FCCP ( 10 μM ) for the indicated times ( n = 3 different experiments ; 2 - way - ANOVA with Dunnett post test ; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs , comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black ) .

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D) Western blots and quantification of Miro1 degradation in MiroDKO cells co-expressing YFPParkin and the indicated myc-tagged Miro1 constructs and treated with FCCP (10 μM) for the indicated times (n=3 different experiments; 2-way-ANOVA with Dunnett post test; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs, comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black).


E ) . Representative images at 24 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO cells co - transfected with the specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal the mitochondrial network . Expression of both ubiquitin mutants ( Miro15R and Miro1allR - arrowheads ) protects from the mitophagic clearance of mitochondria ( arrows ) after damage . ( F ) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment ( data collected from at least 3 independent experiments : MiroDKO n = 8 , mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; one way ANOVA with Dunnett post test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001

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E). Representative images at 24 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. Expression of both ubiquitin mutants (Miro15R and Miro1allR - arrowheads) protects from the mitophagic clearance of mitochondria (arrows) after damage. (F) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment (data collected from at least 3 independent experiments: MiroDKO n=8, mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points ( scale bars = 5 µm ) .

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(A) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points (scale bars = 5 µm).


( B ) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes ( scale bars = 2 . 5 µm ) .

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(B) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes (scale bars = 2.5 µm).


( C ) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment ( lines shown in A ) .

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(C) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment (lines shown in A).


( D ) Quantification of Parkin recruitment to mitochondria ( YFPParkin signal overlapping MtDsRed signal - intensity - adjusted and normalised to t = 0 ) in WT and Miro1KO neurons following valinomycin treatment ( n = 15 cells all conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

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(D) Quantification of Parkin recruitment to mitochondria (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted and normalised to t=0) in WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( E ) Quantification of mitochondrial occupancy ( area of MtDsRed signal in soma / entire area of soma ) in the somas of WT and Miro1KO neurons following valinomycin treatment ( n = 15 cells all conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) .

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(E) Quantification of mitochondrial occupancy (area of MtDsRed signal in soma / entire area of soma) in the somas of WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA).


( F ) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment ( n = 15 cells all conditions per genotype over 3 neuronal preparations ; Unpaired t - test ) .

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(F) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; Unpaired t-test).


( G ) Representative confocal images of WT , Miro1KO and PINK1KO cortical neurons immunostained with MAP2 ( cyan ) and s65 - phospho - ubiquitin ( green ) after valinomycin treatment ( Scale bars = 20µm ) . ( H ) Quantification of s65 - phospho - ubiquitin signal intensity within MAP2 signal ( normalised to MAP2 area and t = 0 ) in Miro1WT , Miro1KO and PINK1KO neurons following valinomycin treatment n = 3 , 4 and 3 embryos from WT , Miro1KO and PINK1KO embryos , respectively ( 6 ROIs per condition ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

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(G) Representative confocal images of WT, Miro1KO and PINK1KO cortical neurons immunostained with MAP2 (cyan) and s65-phospho-ubiquitin (green) after valinomycin treatment (Scale bars = 20µm). (H) Quantification of s65-phospho-ubiquitin signal intensity within MAP2 signal (normalised to MAP2 area and t=0) in Miro1WT, Miro1KO and PINK1KO neurons following valinomycin treatment n= 3, 4 and 3 embryos from WT, Miro1KO and PINK1KO embryos, respectively (6 ROIs per condition; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( I ) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1 , YFPParkin and MtDsRed without valinomycin treatment ( Scale bars = 10 µm ) . Quantification of parkin colocalization with MtDsRed ( YFPParkin signal overlapping MtDsRed signal - intensity - adjusted ) in the soma of Miro1KO and PINK1KO cortical neurons ( n = 12 cells all conditions per genotype over 3 neuronal preparations ; one - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 .

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(I) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1, YFPParkin and MtDsRed without valinomycin treatment (Scale bars = 10 µm). Quantification of parkin colocalization with MtDsRed (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted) in the soma of Miro1KO and PINK1KO cortical neurons (n=12 cells all conditions per genotype over 3 neuronal preparations; one-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( J ) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1 , YFPParkin and MtDsRed after 5 hours of valinomycin treatment ( Scale bars = 10 µm ) . Quantification of Parkin recruitment to mitochondria ( Normalised to t = 0 ) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment ( n = 12 cells all conditions per genotype over 3 neuronal preparations , two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001

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(J) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1, YFPParkin and MtDsRed after 5 hours of valinomycin treatment (Scale bars = 10 µm). Quantification of Parkin recruitment to mitochondria (Normalised to t=0) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment (n=12 cells all conditions per genotype over 3 neuronal preparations, two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative blots of 4 and 12 months old hippocampal lysates from control , Miro1CKO and Miro2KO . B ) Quantification of protein levels in 4 month lysates and 12 month lysates ( n = 3 animals / genotype at 4 months and n = 4 animals / genotype at 12 months ; one - way ANOVA with post - hoc Dunnett ' s test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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(A) Representative blots of 4 and 12 months old hippocampal lysates from control, Miro1CKO and Miro2KO. B) Quantification of protein levels in 4 month lysates and 12 month lysates (n=3 animals / genotype at 4 months and n=4 animals / genotype at 12 months; one-way ANOVA with post-hoc Dunnett's test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


C ) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals .

C) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals.


( D ) Details from cortical regions of control , Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals .

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(D) Details from cortical regions of control, Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals.


( E ) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment . The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age . This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age .

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(E) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment. The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age. This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age.


Representative images ( F ) and quantification ( G ) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice ( n = 4 animals / genotype ; student ' s t - test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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Representative images (F) and quantification (G) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice (n=4 animals / genotype; student's t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative western blot images of brain lysates from 12 - month WT , Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated . ( B ) Quantification of eIF2α and P - eIF2α band intensity ( normalised to actin band intensity ) from 12 - month WT , Miro1CKO and Miro2KO brain lysates . Phosphorylation of eIF2α is defined as P - eIF2α / eIF2α ( n = 4 mice per genotype , unpaired t - test ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 .

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(A) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( C ) Representative confocal images of cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice stained with MAP2 and P - eIF2α . ( D ) Quantification of P - eIF2α signal intensity from cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice ( n = 4 mice per genotype ; unpaired t - test ) .

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(C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test).


( E ) Representative confocal images ( 63x ) of cortical regions of 12 - month mitoDendra - crossed WT and Miro1CKO mice stained with MAP2 and P - eIF2α . Arrows indicate the presence of megamitochondria ( > 2 . 5 µm2 ) , stars indicate neuronal somas without megamitochondria .

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(E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria.


( F ) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO mice stained with P - eIF2α . ( G ) Quantification P - eIF2α signal intensity ( a . u . ) from neuronal somas with and without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO ( n = 16 sections from 4 mice ; paired t - test ) .

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(F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).


A Mpc1 and Mpc2 are present under fermentative conditions , while Mpc1 and Mpc3 are present under respiratory conditions . MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose ( YPD ) or glycerol ( YPG ) . Mitochondrial matrix protein aconitase ( Aco1 ) , 23 ‐ kDa translocase of the inner membrane subunit ( Tim23 ) , and 70 ‐ kDa translocase of the outer membrane subunit ( Tom70 ) were used as loading controls .

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A Mpc1 and Mpc2 are present under fermentative conditions, while Mpc1 and Mpc3 are present under respiratory conditions. MPC subunits were detected by Western blot in mitochondria isolated from yeast cells grown in glucose (YPD) or glycerol (YPG). Mitochondrial matrix protein aconitase (Aco1), 23‐kDa translocase of the inner membrane subunit (Tim23), and 70‐kDa translocase of the outer membrane subunit (Tom70) were used as loading controls.


B Kinetics of the switch from Mpc2 to Mpc3 . Yeast cells were grown in YPD until mid ‐ log phase and then shifted to YPG medium . Whole cell extracts were taken at the indicated time points after the shift , and HA ‐ tagged Mpc2 and Mpc3 detected by Western blotting . Mpc3 is induced very rapidly by switch from YPD to YPG , while Mpc2 levels decrease only gradually .

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B Kinetics of the switch from Mpc2 to Mpc3. Yeast cells were grown in YPD until mid‐log phase and then shifted to YPG medium. Whole cell extracts were taken at the indicated time points after the shift, and HA‐tagged Mpc2 and Mpc3 detected by Western blotting. Mpc3 is induced very rapidly by switch from YPD to YPG, while Mpc2 levels decrease only gradually.


C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN ‐ PAGE . Isolated mitochondria expressing HA ‐ tagged MPC proteins were run on blue native gels , and MPC complexes detected with an HA ‐ specific antibody . MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions , and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions . In addition , a complex of ˜ 300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3 . Non ‐ specific reactivity of the anti ‐ HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk ( * ) .

C Alternative pyruvate carrier complexes MPCFERM and MPCOX are detected by BN‐PAGE. Isolated mitochondria expressing HA‐tagged MPC proteins were run on blue native gels, and MPC complexes detected with an HA‐specific antibody. MPCFERM consisting of Mpc1 and Mpc2 is present under fermentative conditions, and MPCOX consisting of Mpc1 and Mpc3 is present under respiratory conditions. In addition, a complex of ˜300 kDa is observed under respiratory conditions that contains Mpc1 and Mpc3. Non‐specific reactivity of the anti‐HA antibody toward respiratory chain complexes that are highly abundant in oxidative growth conditions is denoted by an asterisk (*).


A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant . Growth tests were performed with cells transformed with MPC1 , MPC2 , or MPC3 expression plasmids or with empty vectors in indicated combinations . A serial dilution of yeast cells was spotted on agar plates with glucose ‐ containing synthetic minimal medium with ( SD + AA ) or without ( SD − AA ) amino acids .

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A Constitutive expression of either MPCFERM or MPCOX can rescue the slow growth phenotype of the mpc1Δmpc2Δmpc3Δ triple deletion mutant. Growth tests were performed with cells transformed with MPC1, MPC2, or MPC3 expression plasmids or with empty vectors in indicated combinations. A serial dilution of yeast cells was spotted on agar plates with glucose‐containing synthetic minimal medium with (SD+AA) or without (SD−AA) amino acids.


B Plasmid ‐ encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co ‐ expressed with either Mpc2 or Mpc3 . Cells were grown in selective medium containing glycerol as a carbon source . MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti ‐ HA or anti ‐ Flag antibodies . Mitochondrial enzymes aconitase ( Aco1 ) and malate dehydrogenase ( Mdh1 ) were used as loading controls .

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B Plasmid‐encoded MPC proteins can be detected in isolated mitochondria when Mpc1 is co‐expressed with either Mpc2 or Mpc3. Cells were grown in selective medium containing glycerol as a carbon source. MPC subunits were constitutively expressed from plasmids in indicated combinations in the mpc1Δmpc2Δmpc3Δ background and detected by Western blot using anti‐HA or anti‐Flag antibodies. Mitochondrial enzymes aconitase (Aco1) and malate dehydrogenase (Mdh1) were used as loading controls.


C BN ‐ PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid ‐ based protein expression . Non ‐ specific reactivity of the antibody is denoted by an asterisk ( * ) . It has to be noted that all complexes migrate slightly faster than in Fig C , most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA ‐ tag in Fig C .

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C BN‐PAGE confirms the formation of MPCFERM and MPCOX complexes after constitutive plasmid‐based protein expression. Non‐specific reactivity of the antibody is denoted by an asterisk (*). It has to be noted that all complexes migrate slightly faster than in Fig C, most likely because MPC proteins are tagged with a single epitope tag as opposed to the 3HA‐tag in Fig C.


D Mpc1 is co ‐ immunoprecipitated with Mpc2 or Mpc3 . Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol ‐ containing medium were lysed in 1 % digitonin , and either Mpc2 ‐ Flag or Mpc3 ‐ Flag was immunoprecipitated . Mpc1 ‐ HA was detected by Western blot with an antibody directed against the epitope tag . The matrix protein aconitase ( Aco1 ) and 40 ‐ kDa translocase of the outer membrane subunit ( Tom40 ) were not co ‐ immunoprecipitated .

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D Mpc1 is co‐immunoprecipitated with Mpc2 or Mpc3. Mitochondria isolated from cells harboring appropriate expression plasmids and grown in glycerol‐containing medium were lysed in 1% digitonin, and either Mpc2‐Flag or Mpc3‐Flag was immunoprecipitated. Mpc1‐HA was detected by Western blot with an antibody directed against the epitope tag. The matrix protein aconitase (Aco1) and 40‐kDa translocase of the outer membrane subunit (Tom40) were not co‐immunoprecipitated.


E , F Stoichiometry of Mpc1 complexes . Chemical cross ‐ linking indicates that Mpc1 / Mpc2 and Mpc1 / Mpc3 form heterodimers of the predicted molecular weight . DSG , disuccimidyl glutarate . Cross ‐ linking products with unknown partners are labeled with a question mark ( ? ) .

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E, F Stoichiometry of Mpc1 complexes. Chemical cross‐linking indicates that Mpc1/Mpc2 and Mpc1/Mpc3 form heterodimers of the predicted molecular weight. DSG, disuccimidyl glutarate. Cross‐linking products with unknown partners are labeled with a question mark (?).


A , B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 ( A ) and Mpc3 ( B ) . Mitochondria were treated with IASD as indicated . Negative [ quenching by pretreatment with DTT ( pre ‐ DTT ) ] and positive [ lysis with 0 . 5 % Triton X ‐ 100 ( T ‐ X100 ) ] controls for the labeling reaction are shown . As a further control , also translocase of the inner membrane subunit Tim23 is detected by Western blot , which contains three endogenous cysteines , all of which are located in membrane ‐ embedded regions . Labeling of an IMS ‐ exposed cysteine with IASD is indicated by a mobility shift on SDS - PAGE corresponding to the molecular weight of IASD ( ˜ 500 Da ) .

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A, B Determination of MPC membrane topology by IASD labeling of isolated mitochondria expressing the indicated single cysteine variants of Mpc1 (A) and Mpc3 (B). Mitochondria were treated with IASD as indicated. Negative [quenching by pretreatment with DTT (pre‐DTT)] and positive [lysis with 0.5% Triton X‐100 (T‐X100)] controls for the labeling reaction are shown. As a further control, also translocase of the inner membrane subunit Tim23 is detected by Western blot, which contains three endogenous cysteines, all of which are located in membrane‐embedded regions. Labeling of an IMS‐exposed cysteine with IASD is indicated by a mobility shift on SDS-PAGE corresponding to the molecular weight of IASD (˜500 Da).


C Protease protection assay in mitochondria expressing Mpc1 ‐ GFP , Mpc2 ‐ GFP , or Mpc3 ‐ GFP . Intact mitochondria ( M ) , mitoplasts with a ruptured outer membrane after hypo ‐ osmotic swelling ( Sw ) , or mitochondrial lysates with 0 . 5 % Triton X ‐ 100 ( Tr ) were treated with proteinase K ( PK ) . Loading controls are Tom70 ( outer membrane ) , Tim23 ( inner membrane ) , and Mdh1 ( matrix ) . GFP ‐ fused Mpc2 and Mpc3 are degraded in mitoplasts , whereas Mpc1 ‐ GFP is not .

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C Protease protection assay in mitochondria expressing Mpc1‐GFP, Mpc2‐GFP, or Mpc3‐GFP. Intact mitochondria (M), mitoplasts with a ruptured outer membrane after hypo‐osmotic swelling (Sw), or mitochondrial lysates with 0.5% Triton X‐100 (Tr) were treated with proteinase K (PK). Loading controls are Tom70 (outer membrane), Tim23 (inner membrane), and Mdh1 (matrix). GFP‐fused Mpc2 and Mpc3 are degraded in mitoplasts, whereas Mpc1‐GFP is not.


A MPCOX has higher transport activity than MPCFERM . The uptake of 14C ‐ labeled pyruvate into intact mitochondria was measured in vitro . Mitochondria had been isolated from cells grown in glycerol ‐ containing medium and expressing no subunit ( vectors ) , MPCFERM , MPCOX , Mpc2 , or Mpc3 . Imported pyruvate was quantified by re ‐ isolation of mitochondria and subsequent scintillation counting after 1 , 2 , or 5 min of incubation with [ 14C ] ‐ pyruvate . B Imported [ 14C ] ‐ pyruvate as in ( A ) after 5 ‐ min incubation . The difference between MPCOX and MPCFERM ( * * P = 0 . 0037 ) was significant ( unpaired t ‐ test ) .

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A MPCOX has higher transport activity than MPCFERM. The uptake of 14C‐labeled pyruvate into intact mitochondria was measured in vitro. Mitochondria had been isolated from cells grown in glycerol‐containing medium and expressing no subunit (vectors), MPCFERM, MPCOX, Mpc2, or Mpc3. Imported pyruvate was quantified by re‐isolation of mitochondria and subsequent scintillation counting after 1, 2, or 5 min of incubation with [14C]‐pyruvate.B Imported [14C]‐pyruvate as in (A) after 5‐min incubation. The difference between MPCOX and MPCFERM (**P = 0.0037) was significant (unpaired t‐test).


C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit ( vectors ) , MPCFERM , or MPCOX .

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C Pyruvate dehydrogenase activity was measured in lysates of mitochondria expressing no subunit (vectors), MPCFERM, or MPCOX.


The chimeric construct Mpc2C3 is expressed in isolated mitochondria , while Mpc3C2 is not . Mpc1 ‐ HA and the Flag ‐ tagged chimeric proteins were detected by Western blotting in isolated mitochondria .

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The chimeric construct Mpc2C3 is expressed in isolated mitochondria, while Mpc3C2 is not. Mpc1‐HA and the Flag‐tagged chimeric proteins were detected by Western blotting in isolated mitochondria.


Mpc2C3 forms a carrier complex with Mpc1 , as analyzed by BN ‐ PAGE . When co ‐ expressed with Mpc1 , Mpc2C3 shows the same complex pattern as Mpc3 , including the 300K complex .

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Mpc2C3 forms a carrier complex with Mpc1, as analyzed by BN‐PAGE. When co‐expressed with Mpc1, Mpc2C3 shows the same complex pattern as Mpc3, including the 300K complex.


Pyruvate transport activity of the Mpc1 / Mpc2C3 complex reaches ˜ 85 % of MPCOX activity . Uptake of [ 14C ] ‐ pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro . Difference between MPCFERM and Mpc1 / Mpc2C3 is significant ( unpaired t ‐ test ; * P = 0 . 02 ) . Means of n = 4 experiments are shown , with error bars representing SEM .

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Pyruvate transport activity of the Mpc1/Mpc2C3 complex reaches ˜85% of MPCOX activity. Uptake of [14C]‐pyruvate into mitochondria isolated from yeastcells expressing the indicated MPC protein combinations was measured in vitro. Difference between MPCFERM and Mpc1/Mpc2C3 is significant (unpaired t‐test; *P = 0.02). Means of n = 4 experiments are shown, with error bars representing SEM.


a , A TRPML1Va - expressing HEK293T cell showed a large inwardly rectifying whole - cell current elicited by voltage steps ( from - 140 mV to + 80 mV in increments of 20 mV ) in the standard extracellular ( Tyrode ' s ) bath solution . Step duration , 90 ms ; holding potential , 0 mV ; Vm , membrane potential .

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a, A TRPML1Va-expressing HEK293T cell showed a large inwardly rectifying whole-cell current elicited by voltage steps (from -140 mV to +80 mV in increments of 20 mV) in the standard extracellular (Tyrode's) bath solution. Step duration, 90 ms; holding potential, 0 mV; Vm, membrane potential.


b , No significant inward current was detected in NMDG + ( Na + - free , Ca2 + - free , pH 4 . 6 ) solution .

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b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution.


c , Inwardly rectifying step currents were evoked by 30 mM Fe2 + solution ( pH 4 . 6 ) in the same cell as shown in a and b .

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c, Inwardly rectifying step currents were evoked by 30 mM Fe2+ solution (pH 4.6) in the same cell as shown in a and b.


d , pH - dependence of IFe / TRPML1Va . Whole - cell currents were elicited by repeated voltage ramps ( - 100 to + 100 mV ; 400 ms ) with a 4 - s interval between ramps . Only a portion of the voltage protocol is shown . Holding potential , 0 mV .

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d, pH-dependence of IFe/TRPML1Va. Whole-cell currents were elicited by repeated voltage ramps (-100 to +100 mV; 400 ms) with a 4-s interval between ramps. Only a portion of the voltage protocol is shown. Holding potential, 0 mV.


e , Large IFe / TRPML2Va was seen in the presence of 30 and 105 mM Fe2 + ( pH 4 . 6 ) .

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e, Large IFe/TRPML2Va was seen in the presence of 30 and 105 mM Fe2+ (pH 4.6).


f , Little or no IFe was seen in a TRPML3Va - expressing cell .

f, Little or no IFe was seen in a TRPML3Va-expressing cell.


a , Current densities ( mean ± s . e . m . , n = 4 - 10 ) of IFe ( 30 mM Fe2 + , pH 4 . 6 ) for TRPML1Va and ML4 mutant TRPML1Va channels . Asterisk indicates statistical difference ( P < 0 . 01 ) compared to TRPML1Va .

a, Current densities (mean ± s.e.m., n = 4-10) of IFe (30 mM Fe2+, pH 4.6) for TRPML1Va and ML4 mutant TRPML1Va channels. Asterisk indicates statistical difference (P < 0.01) compared to TRPML1Va.


b , 55Fe2 + uptake ( normalized ) in HEK293T cells transfected with vector control , with TRPML1Va , with F408D - TRPML1Va and with T232P - TRPML1Va constructs . Error bars indicate the standard deviation on the basis of two independent triplicate experiments .

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b, 55Fe2+uptake (normalized) in HEK293T cells transfected with vector control, with TRPML1Va, with F408D-TRPML1Va and with T232P-TRPML1Va constructs. Error bars indicate the standard deviation on the basis of two independent triplicate experiments.


c , [ Fe2 + ] o - dependent quenching of Fura - 2 fluorescence in TRPML1Va - transfected cells ( arrows ) , but not in non - transfected control cells ( arrowheads ) or T232P - TRPML1Va - transfected cells ( bottom row ) . The fluorescence intensity was measured at an excitation wavelength of 360 nm ( F360 ) . The original magnification used for all micrographs was × 200 .

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c, [Fe2+]o-dependent quenching of Fura-2 fluorescence in TRPML1Va-transfected cells (arrows), but not in non-transfected control cells (arrowheads) or T232P-TRPML1Va-transfected cells (bottom row). The fluorescence intensity was measured at an excitation wavelength of 360 nm (F360). The original magnification used for all micrographs was ×200.


d , Average normalized responses of EGFP - positive TRPML1Va - transfected cells ( typically n = 20 - 40 cells ) to 1 or 10 mM Fe2 + ( pH 4 . 6 ) .

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d, Average normalized responses of EGFP-positive TRPML1Va-transfected cells (typically n = 20-40 cells) to 1 or 10 mM Fe2+ (pH 4.6).


e , No significant quenching was seen in T232P - TRPML1Va - transfected cells .

e, No significant quenching was seen in T232P-TRPML1Va-transfected cells.


f , Slightly less quenching was observed for the F408Dgr ; - TRPML1Va - expressing cells .

f, Slightly less quenching was observed for the F408Dgr;-TRPML1Va-expressing cells.


g , A small but significant quenching reaction was detected ( with 10 mM Fe2 + ) in R403C - TRPML1Va - expressing cells .

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g, A small but significant quenching reaction was detected (with 10 mM Fe2+) in R403C-TRPML1Va-expressing cells.


a , Co - localization of mCherry - TRPML1 and EGFP - LAMP1 at the membrane of an isolated enlarged LEL ( see Methods ) . The patch pipette was filled with red rhodamine B dye ( shown in blue for the purpose of illustration ) .

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a, Co-localization of mCherry-TRPML1 and EGFP-LAMP1 at the membrane of an isolated enlarged LEL (see Methods). The patch pipette was filled with red rhodamine B dye (shown in blue for the purpose of illustration).


c , Lysosomal ITRPML1Va . Switching from lysosome - attached to ( lysosome ) luminal - side - out configuration significantly reduced the amplitude of the current . The luminal - side - out patch was exposed to the Tyrode ' s solution . A Cs + - based solution ( 147 mM Cs - methanesulphonate ( Cs - MSA ) ) was used as a pipette solution for both configurations .

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c, Lysosomal ITRPML1Va. Switching from lysosome-attached to (lysosome) luminal-side-out configuration significantly reduced the amplitude of the current. The luminal-side-out patch was exposed to the Tyrode's solution. A Cs+-based solution (147 mM Cs-methanesulphonate (Cs-MSA)) was used as a pipette solution for both configurations.


d , NMDG + - impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations ( nominal divalent - free ) . e , Lowering pH potentiated lysosomal ITRPML1Va . f , IFe / TRPML1Va induced by 30 mM and 105 mM Fe2 + .

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d, NMDG+-impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations (nominal divalent-free). e, Lowering pH potentiated lysosomal ITRPML1Va. f, IFe/TRPML1Va induced by 30 mM and 105 mM Fe2+.


g , Whole - lysosome current in an enlarged lysosome expressing wild - type TRPML1 . The pipette ( lumen ) solution contained nominal divalent - free Tyrode solution . A Cs + - based bath solution ( 147 mM Cs - MSA ) was used . h , Whole - lysosome ITRPML1Va . i , Whole - lysosome IFe / TRPML1 . The pipette ( lumen ) solution contained 105 mM Fe2 + ( pH 4 . 6 ) .

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g, Whole-lysosome current in an enlarged lysosome expressing wild-type TRPML1. The pipette (lumen) solution contained nominal divalent-free Tyrode solution. A Cs+-based bath solution (147 mM Cs-MSA) was used. h, Whole-lysosome ITRPML1Va. i, Whole-lysosome IFe/TRPML1. The pipette (lumen) solution contained 105 mM Fe2+ (pH 4.6).


a , Cultured TRPML1 - / - ( ML1 - / - ) skin fibroblasts showed less de - quenching of the iron - sensitive fluorescence than ML1 + / - cells did . De - quenching was achieved by preloading the fibroblasts with an iron - sensitive dye , Phen Green SK ( PG SK ) , and then adding the membrane - permeable transition metal chelator , 2 , 2 ′ - bipyridyl ( BPD ) . 2 , 2 ′ - BPD is predicted to chelate free cellular iron ( also referred to as chelatable or labile iron ) , which subsequently increases PG SK fluorescence . The original magnification used was ×

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a, Cultured TRPML1-/- (ML1-/-) skin fibroblasts showed less de-quenching of the iron-sensitive fluorescence than ML1+/- cells did. De-quenching was achieved by preloading the fibroblasts with an iron-sensitive dye, Phen Green SK (PG SK), and then adding the membrane-permeable transition metal chelator, 2,2′-bipyridyl (BPD). 2,2′-BPD is predicted to chelate free cellular iron (also referred to as chelatable or labile iron), which subsequently increases PG SK fluorescence. The original magnification used was ×200


b , The average 2 , 2 ′ - BPD induced normalized change of fluorescence ( Dgr ; F / F0 ) for ML4 cells ( ML1 - / - ; n = 6 experiments ) is significantly ( asterisk , P < 0 . 01 ) lower than for the parental ML1 + / - cells . Fibroblast cells ( n = 10 - 20 ) were analysed for each individual experiment . 4 , 4 ′ - BPD , a 2 , 2 ′ - BPD analogue that cannot bind Fe2 + , did not induce any significant restoration of PG SK fluorescence . Error bars , s . e . m .

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b, The average 2,2′-BPD induced normalized change of fluorescence (Dgr;F/F0) for ML4 cells (ML1-/-; n = 6 experiments) is significantly (asterisk, P < 0.01) lower than for the parental ML1+/- cells. Fibroblast cells (n = 10-20) were analysed for each individual experiment. 4,4′-BPD, a 2,2′-BPD analogue that cannot bind Fe2+, did not induce any significant restoration of PG SK fluorescence. Error bars, s.e.m.


c , An ML1 - / - skin fibroblast cell showed autofluorescence in LAMP1 - positive compartments . Autofluorescence ( green ) was detected within a range of excitation wavelengths ( shown with excitation at 480 nm ) . No significant autofluorescence was observed for a ML1 + / + cell . Lysosomes were stained with a LAMP1 antibody ( red ) . Differential interference contrast ( DIC ) images are shown for comparison .

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c, An ML1-/- skin fibroblast cell showed autofluorescence in LAMP1-positive compartments. Autofluorescence (green) was detected within a range of excitation wavelengths (shown with excitation at 480 nm). No significant autofluorescence was observed for a ML1+/+ cell. Lysosomes were stained with a LAMP1 antibody (red). Differential interference contrast (DIC) images are shown for comparison.


( A ) Atg5fl / fl Cre − and Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with the inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . Cell culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 .

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(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n≥3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − and Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase ‐ 1 and IL ‐ 1β was determined by immunoblotting .

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(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of active caspase‐1 and IL‐1β was determined by immunoblotting.


( C ) As in ( A ) , assayed for IL ‐ 18 . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 .

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(C) As in (A), assayed for IL‐18. Data represent mean values±s.d. (n≥3); *P0.05.


( D ) LPS ‐ pretreated BMMs were exposed to alum ( 250 μg / ml ) for 1 h with or without autophagic induction by starvation . Secreted IL ‐ 1β was measured as in ( A ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(D) LPS‐pretreated BMMs were exposed to alum (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.


( E ) LPS ‐ pretreated BMMs were exposed to silica ( 250 μg / ml ) for 1 h with or without autophagic induction by starvation . Secreted IL ‐ 1β was measured as in ( A ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

(E) LPS‐pretreated BMMs were exposed to silica (250 μg/ml) for 1 h with or without autophagic induction by starvation. Secreted IL‐1β was measured as in (A). Data represent mean values±s.d. (n⩾3); *P0.05.


( F ) BMMs were transfected with scramble ( Scr ) control siRNA or siRNAs against ASC and NLRP3 . After 48 h following transfection , cells were treated overnight with LPS and subjected to nigericin ( 20 μM ) and starvation for 1 h . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(F) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to nigericin (20 μM) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.


( G ) Immunoblot analysis of ASC and NLRP3 knockdowns .

(G) Immunoblot analysis of ASC and NLRP3 knockdowns.


( H ) BMMs were transfected with scramble ( Scr ) control siRNA or siRNAs against ASC and NLRP3 . After 48 h following transfection , cells were treated overnight with LPS and subjected to silica ( 250 μg / ml ) and starvation for 1 h . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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

(H) BMMs were transfected with scramble (Scr) control siRNA or siRNAs against ASC and NLRP3. After 48 h following transfection, cells were treated overnight with LPS and subjected to silica (250 μg/ml) and starvation for 1 h. Data represent mean values±s.d. (n⩾3); *P0.05.


( I ) Colocalization of IL ‐ 1β with the basal autophagic machinery factor LC3 . Fluorescence : LC3 ( green , Alexa488 ) ; IL ‐ 1β ( red , Alexa568 ) . BMMs were from GFP - LC3 knock ‐ in mice , treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL ‐ 1β .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of IL‐1β with the basal autophagic machinery factor LC3. Fluorescence: LC3 (green, Alexa488); IL‐1β (red, Alexa568). BMMs were from GFP-LC3 knock‐in mice, treated with LPS then prepared for immunofluorescence microscopy using fluorescently labelled antibodies against GFP and IL‐1β.


( J , K ) A line fluorescence tracing from images in ( I ) .

(J, K) A line fluorescence tracing from images in (I).


( L ) Pearson ' s colocalization coefficient for IL ‐ 1β and LC3 . Pearson ' s coefficient was derived from three independent experiments with five fields per experiment , for a total of 15 fields contributing to the cumulative result . Figure source data can be found in Supplementary data .

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(L) Pearson's colocalization coefficient for IL‐1β and LC3. Pearson's coefficient was derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.


( A , B ) LPS ‐ pretreated BMMs were treated with 20 μM nigericin ( Nig ) and 100 nM bafilomycin A1 ( Baf ) with ( Starvation ) or without ( Full ) autophagic induction for 1 h and secreted IL ‐ 1β ( A ) and IL ‐ 18 ( B ) were measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A, B) LPS‐pretreated BMMs were treated with 20 μM nigericin (Nig) and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β (A) and IL‐18 (B) were measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( C ) LPS ‐ pretreated BMMs were treated with 250 μg / ml of silica and 100 nM bafilomycin A1 ( Baf ) with ( Starvation ) or without ( Full ) autophagic induction for 1 h and secreted IL ‐ 1β were measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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 O O O O O O O O O O O O O O O O O O O O O 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) LPS‐pretreated BMMs were treated with 250 μg/ml of silica and 100 nM bafilomycin A1 (Baf) with (Starvation) or without (Full) autophagic induction for 1 h and secreted IL‐1β were measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( D ) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL ‐ 1β . Fluorescence ; LC3 ( green , Alexa488 ) , IL ‐ 1β ( red , Alexa568 ) , and cathepsin B ( blue , Alexa633 ) . BMMs from GFP - LC3 knock ‐ in mice were treated with LPS and then analysed for immunofluorescence .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of cathepsin B with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), and cathepsin B (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were treated with LPS and then analysed for immunofluorescence.


( E ) Colocalization line tracing analysis from images in ( D ) .

(E) Colocalization line tracing analysis from images in (D).


( F ) LPS ‐ pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA ‐ 074 Me ( 10 μM ) , with ( Starvation ) or without ( Full ) autophagic induction , for 1 h and secreted IL ‐ 1β was measured . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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

(F) LPS‐pretreated BMMs were treated with 20 μM nigericin and cathepsin B inhibitor CA‐074 Me (10 μM), with (Starvation) or without (Full) autophagic induction, for 1 h and secreted IL‐1β was measured. Data represent mean values±s.d. (n⩾3); *P0.05.


( G ) LPS ‐ pretreated Atg5fl / fl Cre − and Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting . Figure source data can be found in Supplementary data .

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

(G) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and release of cathepsin B was determined by immunoblotting. Figure source data can be found in Supplementary data.


( A ) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL ‐ 1β . Fluorescence ; LC3 ( green , Alexa488 ) , IL ‐ 1β ( red , Alexa568 ) , Rab8a ( blue , Alexa633 ) . BMMs from GFP - LC3 knock ‐ in mice were pretreated with LPS and analysed by immunofluorescence microscopy . Arrows indicate triple colocalization .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of Rab8a with the basal autophagic machinery factor LC3 and IL‐1β. Fluorescence; LC3 (green, Alexa488), IL‐1β (red, Alexa568), Rab8a (blue, Alexa633). BMMs from GFP-LC3 knock‐in mice were pretreated with LPS and analysed by immunofluorescence microscopy. Arrows indicate triple colocalization.


( B ) Line tracing analysis of fluorescence signal intensity .

(B) Line tracing analysis of fluorescence signal intensity.


( C ) Pearson ' s colocalization coefficient for IL ‐ 1β and Rab8a . Pearson ' s coefficients were derived from three completely independent experiments with > 5 fields per experiment , for a total of ⩾ 15 fields contributing to the cumulative result .

O O O O O O O O O O O O O O O O O O O O O O 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) Pearson's colocalization coefficient for IL‐1β and Rab8a. Pearson's coefficients were derived from three completely independent experiments with >5 fields per experiment, for a total of ⩾15 fields contributing to the cumulative result.


( D ) BMMs were transfected with siRNAs against Rab8a or scramble ( Scr ) control . At 24 h after the first transfection , cells were transfected again with siRNA , treated with LPS and the day after subjected to nigericin in full medium for 1 h , and IL ‐ 1β secretion measured .

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(D) BMMs were transfected with siRNAs against Rab8a or scramble (Scr) control. At 24 h after the first transfection, cells were transfected again with siRNA, treated with LPS and the day after subjected to nigericin in full medium for 1 h, and IL‐1β secretion measured.


( E ) Immunoblot analysis of Rab8a knockdown in BMMs .

(E) Immunoblot analysis of Rab8a knockdown in BMMs.


( F ) RAW 264 . 7 macrophages were transfected with GFP ‐ tagged Rab8a constructs ( WT , wild type ; S22N , dominant ‐ negative mutant ) , treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation . IL ‐ 1β secretion was measured by ELISA . Data represent mean values ± s . d . ( n ≥ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RAW 264.7 macrophages were transfected with GFP‐tagged Rab8a constructs (WT, wild type; S22N, dominant‐negative mutant), treated overnight with LPS and stimulated for 1 h with 20 μM nigericin along with induction of autophagy by starvation. IL‐1β secretion was measured by ELISA. Data represent mean values±s.d. (n≥3); *P0.05. Figure source data can be found in Supplementary data.


( A ) BMM cells were transfected with scramble ( Scr ) control siRNA or siRNA against GRASP55 . After 48 h of transfection , cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS , and secreted IL ‐ 1β was measured by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Inset : Immunoblot analysis of GRASP55 knockdown .

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

(A) BMM cells were transfected with scramble (Scr) control siRNA or siRNA against GRASP55. After 48 h of transfection, cells were treated with LPS and the day after subjected to 20 μM nigericin in EBSS, and secreted IL‐1β was measured by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05. Inset: Immunoblot analysis of GRASP55 knockdown.


( B ) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution . LC3 ( green , Alexa488 ) , GRASP55 ( red , Alexa568 ) . BMMs were pretreated overnight with 100 ng / ml LPS and either not stimulated ( Ctrl ) or stimulated ( Nig ) for 30 min with the inflammasome agonist nigericin ( 20 μM ) in full medium .

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

(B) Immunofluorescence confocal microscopy analysis of LC3 and GRASP55 distribution. LC3 (green, Alexa488), GRASP55 (red, Alexa568). BMMs were pretreated overnight with 100 ng/ml LPS and either not stimulated (Ctrl) or stimulated (Nig) for 30 min with the inflammasome agonist nigericin (20 μM) in full medium.


( C ) Line tracings , analysis of fluorescence signal intensity from images in ( B ) .

(C) Line tracings, analysis of fluorescence signal intensity from images in (B).


( D ) Pearson ' s coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles . Pearson ' s coefficients were derived from three independent experiments with five fields per experiment , for a total of 15 fields contributing to the cumulative result . Figure source data can be found in Supplementary data .

(D) Pearson's coefficients for LC3 and GRASP55 were quantified using SlideBook morphometric analysis software as a measure of adjacency between GRASP55 and LC3 profiles. Pearson's coefficients were derived from three independent experiments with five fields per experiment, for a total of 15 fields contributing to the cumulative result. Figure source data can be found in Supplementary data.


( A , B ) Effect of GRASP55 on autophagy induction by measuring LC3 ‐ II . BMM cells were transfected with GRASP55 siRNAs or scramble ( Scr ) control . At 72 h post transfection , cells were induced for autophagy , treated or not with Bafilomycin A1 ( Baf ) to inhibit autophagic degradation and LC3 ‐ II / actin ratios determined by immunoblotting ( A ) followed by densitometry ( B ) . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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(A, B) Effect of GRASP55 on autophagy induction by measuring LC3‐II. BMM cells were transfected with GRASP55 siRNAs or scramble (Scr) control. At 72 h post transfection, cells were induced for autophagy, treated or not with Bafilomycin A1 (Baf) to inhibit autophagic degradation and LC3‐II/actin ratios determined by immunoblotting (A) followed by densitometry (B). Data represent mean values±s.d. (n⩾3); *P0.05.


( C , D ) RAW 264 . 7 was transfected with GRASP55 siRNAs or scramble ( Scr ) siRNA control . Following 48 h of siRNA treatment , cells were transfected with RFP - GFP - LC3 plasmid ( GFP is sensitive to acidification , whereas RFP is not ) , after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified ( graph in D ) by determining the number of early autophagic organelles ( GFP + RFP + puncta ) and autolysosomal organelles ( GFP − RFP + puncta ) per cell as illustrated in fluorescent images ( yellow arrows , GFP + RFP + ; red arrows , GFP − RFP + ) . Total , yellow + red puncta per cell . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 . Figure source data can be found in Supplementary data .

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

(C, D) RAW 264.7 was transfected with GRASP55 siRNAs or scramble (Scr) siRNA control. Following 48 h of siRNA treatment, cells were transfected with RFP-GFP-LC3 plasmid (GFP is sensitive to acidification, whereas RFP is not), after 24 h induced for autophagy in EBSS for 1 h and autophagic induction and flux quantified (graph in D) by determining the number of early autophagic organelles (GFP+RFP+ puncta) and autolysosomal organelles (GFP−RFP+ puncta) per cell as illustrated in fluorescent images (yellow arrows, GFP+RFP+; red arrows, GFP−RFP+). Total, yellow+red puncta per cell. Data represent mean values±s.d. (n⩾3); *P0.05. Figure source data can be found in Supplementary data.


( A ) Atg5fl / fl Cre − and Atg5fl / fl Cre + bone marrow ‐ derived macrophages ( BMMs ) , pretreated overnight with 100 ng / ml LPS , were stimulated for 1 h with the inflammasome agonist nigericin ( 20 μM ) with ( Starvation ; EBSS ) or without ( Full ; full medium ) autophagic induction . Cell culture supernatants were assayed for murine IL ‐ 1β by ELISA . Data represent mean values ± s . d . ( n ⩾ 3 ) ; * P0 . 05 .

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

(A) Atg5fl/fl Cre− and Atg5fl/fl Cre+ bone marrow‐derived macrophages (BMMs), pretreated overnight with 100 ng/ml LPS, were stimulated for 1 h with the inflammasome agonist nigericin (20 μM) with (Starvation; EBSS) or without (Full; full medium) autophagic induction. Cell culture supernatants were assayed for murine IL‐1β by ELISA. Data represent mean values±s.d. (n⩾3); *P0.05.


( B ) LPS ‐ pretreated Atg5fl / fl Cre − and Atg5fl / fl Cre + BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting . Figure source data can be found in Supplementary data .

(B) LPS‐pretreated Atg5fl/fl Cre− and Atg5fl/fl Cre+ BMMs were stimulated with 20 μM nigericin for 1 h in OptiMEM and the release of HMGB1 was determined by immunoblotting. Figure source data can be found in Supplementary data.


Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS ( 4 . 5 mg / kg BW ) or PBS injection ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate

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Percent body weight of male Prdx4 WT and KO mice over the 72 h course of LPS (4.5 mg/kg BW) or PBS injection (i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM


Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection . ( B ) Cxcl1 levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( C ) TNF - α levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . ( D ) IL - 1β levels in serum ( left ) or peritoneal lavage ( right ) at indicated time points after LPS or PBS injection . Each dot ( B - D ) represents an individual mouse . Horizontal lines indicate mean .

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Cytokine concentration in Prdx4 WT and KO mice in response to LPS injection. (B) Cxcl1 levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (C) TNF-α levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. (D) IL-1β levels in serum (left) or peritoneal lavage (right) at indicated time points after LPS or PBS injection. Each dot (B-D) represents an individual mouse. Horizontal lines indicate mean.


Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS ( 4 . 5 mg / kg BW ) injection ( i . p . ) and treatment with IL - 1 receptor antagonist ( IL - 1RA ) Anakinra ( 200 µg / mouse ) or control . Arrows indicate time point of Anakinra injection . Each circle represents a mean of n = 5 mice , vertical lines indicate SEM .

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

Percent body weight of male Prdx4 WT and KO mice over the 48 h course of LPS (4.5 mg/kg BW) injection (i.p.) and treatment with IL-1 receptor antagonist (IL-1RA) Anakinra (200 µg/mouse) or control. Arrows indicate time point of Anakinra injection. Each circle represents a mean of n=5 mice, vertical lines indicate SEM.


Serum concentration of Cxcl1 , TNF - α and IL - 1β in Prdx4 WT and KO mice injected with LPS , LPS and IL - 1RA or control . Horizontal lines indicate mean .

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Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4 WT and KO mice injected with LPS, LPS and IL-1RA or control. Horizontal lines indicate mean.


Percent body weight of male Prdx4 - flox and Prdx4 - ∆ LysMCre mice over the 48 h course of 4 . 5 mg / kg BW LPS ( i . p . ) . Each circle represents a mean of n = 7 mice , vertical lines indicate SEM .

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Percent body weight of male Prdx4-flox and Prdx4-∆LysMCre mice over the 48 h course of 4.5 mg/kg BW LPS ( i.p.). Each circle represents a mean of n=7 mice, vertical lines indicate SEM.


Serum concentration of Cxcl1 , TNF - α and IL - 1β in Prdx4 - flox and Prdx4 - ∆ LysMCre mice injected with LPS . Each dot ( B , D ) represents an individual mouse . Horizontal lines indicate mean .

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Serum concentration of Cxcl1, TNF-α and IL-1β in Prdx4-flox and Prdx4-∆LysMCre mice injected with LPS. Each dot (B, D) represents an individual mouse. Horizontal lines indicate mean.


Concentration of Cxcl1 , TNF - α and IL - 1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation ( 100 ng / ml LPS , time points indicated ) .

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Concentration of Cxcl1, TNF-α and IL-1β in the supernatants of Prdx4 WT and KO BMDMs in response to a time course of LPS stimulation (100 ng/ml LPS, time points indicated).


IL - 1β release of Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for indicated time points with ATP ( 5 mM ) .

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IL-1β release of Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for indicated time points with ATP (5 mM).


Western blot analysis of IL - 1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs , primed with LPS ( 100 ng / ml ) and pulsed with ATP ( 5 mM ) for 4 h or left untreated . Dashed line indicates vertical slice .

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Western blot analysis of IL-1β in cell lysates and supernatants of Prdx4 WT and KO BMDMs, primed with LPS (100 ng/ml) and pulsed with ATP (5 mM) for 4 h or left untreated. Dashed line indicates vertical slice.


Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 45 min of LPS - primed cells . Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI . Scale bar indicates 20 µm . ASC speck - positive cells were counted and expressed as percentage of total cells . Bars represent a mean of n = 4 mice , vertical lines indicate SD . n . s . not significant ( two - tailed t - test ) .

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Immunofluorescence microscopy of ASC speck formation in Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 45 min of LPS-primed cells. Cells were stained with an antibody to ASC and nuclei were counterstained using DAPI. Scale bar indicates 20 µm. ASC speck-positive cells were counted and expressed as percentage of total cells. Bars represent a mean of n=4 mice, vertical lines indicate SD. n.s. not significant (two-tailed t-test).


Western blot analysis of caspase - 1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin ( 10 µg / ml ) stimulation for 1 h after priming with LPS ( 100 ng / ml ) , LPS - priming alone or without stimulation . Whole cell lysates were analyzed for pro - caspase - 1 and Gapdh levels .

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 B-CONTROLLED_VAR 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

Western blot analysis of caspase-1 cleavage in the supernatant of Prdx4 WT and KO BMDMs in response to Nigericin (10 µg/ml) stimulation for 1 h after priming with LPS (100 ng/ml), LPS-priming alone or without stimulation. Whole cell lysates were analyzed for pro-caspase-1 and Gapdh levels.


IL - 1β release in Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or Flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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

IL-1β release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or Flagellin (1 µg/ml each) or treated with transfection agent only.


Quantification of cell death by LDH - release in Prdx4 WT and KO BMDMs , untreated or primed for 6 h with LPS ( 100 ng / ml ) and then pulsed for 3 h with ATP ( 5 mM ) or Nigericin ( 10 µg / ml ) or transfected for 3 h with poly ( dA : dT ) or flagellin ( 1 µg / ml each ) or treated with transfection agent only .

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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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

Quantification of cell death by LDH-release in Prdx4 WT and KO BMDMs, untreated or primed for 6 h with LPS (100 ng/ml) and then pulsed for 3 h with ATP (5 mM) or Nigericin (10 µg/ml) or transfected for 3 h with poly (dA:dT) or flagellin (1 µg/ml each) or treated with transfection agent only.


Western blot analysis of rPRDX4 , rCASP - 1 and co - incubated rPRDX4 and rCASP - 1 after non - reducing SDS - PAGE . Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP - 1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co - incubation with rCASP - 1 . Black arrow at approx . 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer .

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

Western blot analysis of rPRDX4, rCASP-1 and co-incubated rPRDX4 and rCASP-1 after non-reducing SDS-PAGE. Black arrows at 250 kDa indicate the corresponding molecular weights of rCASP-1 and rPRDX4 oligomers and decrease of the Prdx4 band intensity upon co-incubation with rCASP-1. Black arrow at approx. 50 kDa indicates the appearance of an additional rPRDX4 band which corresponds to the molecular weight of a Prdx4 dimer.


Western blot analysis of rPRDX4 ( = P ) and rCASP - 1 ( = C ) after alkylation ( = A ) and non - reducing SDS - PAGE . Black arrows indicate the termination of the integration of rCASP - 1 into the rPRDX4 decamer / HMWC upon alkylation .

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

Western blot analysis of rPRDX4 (=P) and rCASP-1 (=C) after alkylation (=A) and non-reducing SDS-PAGE. Black arrows indicate the termination of the integration of rCASP-1 into the rPRDX4 decamer/HMWC upon alkylation.


Rate of caspase - 1 activity in the presence of non - reduced decameric rPrdx4 , reduced dimeric and monomeric rPRDX4 , YVAD or control .

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

Rate of caspase-1 activity in the presence of non-reduced decameric rPrdx4, reduced dimeric and monomeric rPRDX4, YVAD or control.


Foldchange in IL - 1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3 , ASC , IL - 1β and caspase - 1 WT or Cys - to - Ser mutants C362S or C397S and co - transfected with Prdx4 or control . Cells were stimulated with 2 . 5 mM ATP for 30 min before analysis .

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

Foldchange in IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 WT or Cys-to-Ser mutants C362S or C397S and co-transfected with Prdx4 or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.


Western blot analysis of non - reducing SDS - PAGE of cell lysates from HEK293 cells transfected with caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants and co - transfected with Prdx4 - GFP or GFP as control .

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

Western blot analysis of non-reducing SDS-PAGE of cell lysates from HEK293 cells transfected with caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.


Western blot analysis of co - immunoprecipitation using HA - magnetic beads from cell lysates of HEK293 cells transfected with HA - tagged caspase - 1 WT , C362S , C397S , or C362S plus C397S mutants and co - transfected with Prdx4 - GFP or GFP as control .

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

Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT, C362S, C397S, or C362S plus C397S mutants and co-transfected with Prdx4-GFP or GFP as control.


IL - 1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3 , ASC , IL - 1β and caspase - 1 and co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A or control . Cells were stimulated with 2 . 5 mM ATP for 30 min before analysis .

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

IL-1β concentration in supernatants of HEK293 cells transfected with plasmids for NLRP3, ASC, IL-1β and caspase-1 and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A or control. Cells were stimulated with 2.5 mM ATP for 30 min before analysis.


Western blot analysis of co - immunoprecipitation using HA - magnetic beads from cell lysates of HEK293 cells transfected with HA - tagged caspase - 1 WT or control and co - transfected with Prdx4 WT or Cys - to - Ala mutants C51A , C124A , C245A or DM C124A / C245A .

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

Western blot analysis of co-immunoprecipitation using HA-magnetic beads from cell lysates of HEK293 cells transfected with HA-tagged caspase-1 WT or control and co-transfected with Prdx4 WT or Cys-to-Ala mutants C51A, C124A, C245A or DM C124A/C245A.


Western blot analysis of Prdx4 , pro - caspase ‑ 1 , Gapdh and E - Cadherin from the cytosolic and insoluble cell fraction of LPS and / or ATP - stimulated BMDMs or untreated controls .

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

Western blot analysis of Prdx4, pro-caspase‑1, Gapdh and E-Cadherin from the cytosolic and insoluble cell fraction of LPS and/or ATP-stimulated BMDMs or untreated controls.


Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs , primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP . Each circle represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) .

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 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

Prdx4 concentration in supernatants of Prdx4 WT or KO BMDMs, primed for 6 h with LPS and pulsed for indicated time points with 5 mM ATP. Each circle represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).


Concentration of Prdx4 in the serum of WT mice , injected with LPS ( 4 . 5 mg / kg BW ) for the time points indicated . Each dot represents an individual mouse . Horizontal lines indicate mean . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) .

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

Concentration of Prdx4 in the serum of WT mice, injected with LPS (4.5 mg/kg BW) for the time points indicated. Each dot represents an individual mouse. Horizontal lines indicate mean. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs . Cells were primed with LPS ( 100 ng / ml ) for 6 h , followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation . Each bar represents a mean of n = 3 mice , vertical lines indicate SD . * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) .

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 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

Concentration of Prdx4 in supernatants of Prdx4 WT and KO BMDMs. Cells were primed with LPS (100 ng/ml) for 6 h, followed by pretreatment with 20 µM YVAD or DMSO as control for 30 min and stimulated with 5 mM ATP for 4 h or no further stimulation. Each bar represents a mean of n=3 mice, vertical lines indicate SD. **, p < 0.01; n.s. not significant (two-tailed t-test).


Relative levels of Prdx4 secretion in response to LPS + ATP stimulation and pretreatment with either Glycine , NSA or GW4869 and relative levels of caspase - 1 secretion in response to LPS + ATP stimulation and pretreatment with in response to LPS + ATP stimulation and pretreatment with GW4869 . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; n . s . not significant ( two - tailed t - test ) .

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

Relative levels of Prdx4 secretion in response to LPS+ATP stimulation and pretreatment with either Glycine, NSA or GW4869 and relative levels of caspase-1 secretion in response to LPS+ATP stimulation and pretreatment with in response to LPS+ATP stimulation and pretreatment with GW4869. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; n.s. not significant (two-tailed t-test).


Western blot analysis of subcellular organelle fractions . OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS ( 100 ng / ml ) for 12 h and stimulated with 5 mM ATP for 4 h .

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

Western blot analysis of subcellular organelle fractions. OptiPrep density gradient ultracentrifugation was used to fractionate subcellular organelles from Prdx4 WT BMDMs that were primed with LPS (100 ng/ml) for 12 h and stimulated with 5 mM ATP for 4 h.


BCA analysis of EV protein concentration in EVs isolates . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) .

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

BCA analysis of EV protein concentration in EVs isolates. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Exocet quantification assay of EVs particle numbers . Each bar represents a mean of n = 3 technical replicates , vertical lines indicate SD . * , p < 0 . 05 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) .

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

Exocet quantification assay of EVs particle numbers. Each bar represents a mean of n=3 technical replicates, vertical lines indicate SD. *, p < 0.05; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Transmission electron microscopy ( TEM ) of EV isolates . Three representative pictures are displayed . Scale bar indicates 200 nm .

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

Transmission electron microscopy (TEM) of EV isolates. Three representative pictures are displayed. Scale bar indicates 200 nm.


Analysis of size distribution of EVs . Each dot indicates the diameter in nm of an individual vesicle . SD . * * * , p < 0 . 001 ; n . s . not significant ( one - way ANOVA , followed by a Tukey multiple comparison test ) .

Analysis of size distribution of EVs. Each dot indicates the diameter in nm of an individual vesicle. SD. ***, p < 0.001; n.s. not significant (one-way ANOVA, followed by a Tukey multiple comparison test).


Prdx4 concentration in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) .

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

Prdx4 concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Caspase - 1 and IL - 1β concentration in EV lysates . Each bar represents a mean of n = 2 technical replicates , vertical lines indicate SD . * * , p < 0 . 01 ; * * * , p < 0 . 001 ; n . s . not significant ( two - tailed t - test ) .

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

Caspase-1 and IL-1β concentration in EV lysates. Each bar represents a mean of n= 2 technical replicates, vertical lines indicate SD. **, p < 0.01; ***, p < 0.001; n.s. not significant (two-tailed t-test).


Western blot analysis of Prdx4 , NLRP3 , ASC , caspase ‑ 1 , IL - 1β and CD63 from EV lysates ( upper panel ) or caspase - 1 after immunoprecipitation against caspase - 1 ( lower panel ) .

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

Western blot analysis of Prdx4, NLRP3, ASC, caspase‑1, IL-1β and CD63 from EV lysates (upper panel) or caspase-1 after immunoprecipitation against caspase-1 (lower panel).


Cxcl1 concentration in supernatants of caspase - 1 - deficient BMDMs stimulated with EVs from LPS , LPS and ATP or control - treated Prdx4 WT or KO BMDMs , as well as caspase - 1 - deficient BMDMs pre - treated with Anakinra and stimulated with EVs from LPS and ATP - treated Prdx4 WT or KO BMDMs . Each bar represents a mean of n = 3 biological with 2 technical replicates , vertical lines indicate SD .

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

Cxcl1 concentration in supernatants of caspase-1-deficient BMDMs stimulated with EVs from LPS, LPS and ATP or control-treated Prdx4 WT or KO BMDMs, as well as caspase-1-deficient BMDMs pre-treated with Anakinra and stimulated with EVs from LPS and ATP-treated Prdx4 WT or KO BMDMs. Each bar represents a mean of n=3 biological with 2 technical replicates, vertical lines indicate SD.


Serum Cxcl1 in C57Bl6 / N mice injected with either PBS or EVs from LPS and ATP or control - treated Prdx4 WT or KO or ASC KO BMDMs . Each dot represents an individual mouse . Horizontal lines indicate mean .

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

Serum Cxcl1 in C57Bl6/N mice injected with either PBS or EVs from LPS and ATP or control-treated Prdx4 WT or KO or ASC KO BMDMs. Each dot represents an individual mouse. Horizontal lines indicate mean.


Changes with time in amounts of SH - EP , α - amylase , and VmPE - 1 in cotyledons of V . mungo seedlings ( Attached cotyledons ) or embryo axis - removed cotyledons of V . mungo seeds ( Detached cotyledons ) . Attached or detached cotyledons were prepared as described in Materials and methods . 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris - Cl ( pH 7 . 4 ) , and the homogenates were centrifuged at 15 , 000 g for 10 min . Supernatants ( 10 μl each ) were analyzed by SDS - PAGE immunoblotting with anti - SH - EP , α - amylase , or VmPE - 1 antibody .

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

Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis-removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti-SH-EP, α-amylase, or VmPE-1 antibody.


Subcellular distribution of α - amylase in V . mungo cotyledon cells . Microsome and PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials and methods . Extracts from dry seeds and day 3 cotyledons , microsomes , and PSV fractions ( 20 μg protein each ) were separated by SDS - PAGE . Proteins in the gel were analyzed by Coomassie Brilliant blue staining ( A ) , or by immunoblotting with anti - SH - EP antibody ( B ) or anti - α - amylase antibody ( C ) . Bracket indicates the polypeptides derived from storage globulins . Arrowhead indicates α - amylase .

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

Subcellular distribution of α-amylase in V. mungo cotyledon cells. Microsome and PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials and methods. Extracts from dry seeds and day 3 cotyledons, microsomes, and PSV fractions (20 μg protein each) were separated by SDS-PAGE. Proteins in the gel were analyzed by Coomassie Brilliant blue staining (A), or by immunoblotting with anti-SH-EP antibody (B) or anti- α-amylase antibody (C). Bracket indicates the polypeptides derived from storage globulins. Arrowhead indicates α-amylase.


Electron micrographs showing the immunogold localization of α - amylase in attached cotyledons cells ( A - E ) , detached cotyledons cells ( F ) , or control for immunogold labeling ( G ) . ( A ) Anti - α - amylase antibody immunogold - stained PSV , but not SG . ( B and C ) Golgi complex and PSVs were both immunogold - labeled with anti - α - amylase antibody . ( D ) Immunogold localization of α - amylase ( 15 - nm particles ) and SH - EP ( 10 - nm particles ) . α - Amylase and SH - EP were localized in PSVs and KVs , respectively . ( E ) Gold particles from anti - α - amylase antibody were detected in LVs as well as PSVs . ( F ) PSVs in detached cotyledons were immunogold labeled with anti - α - amylase antibody . ( G ) Immunogold staining of detached cotyledon cells without first antibody ( anti - α - amylase antibody ) . No gold particles were observed in the cell . CW , cell wall ; G , Golgi complex ; KV , KDEL - tailed cysteine proteinase - accumulating vesicle ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars , 200 nm .

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

Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A-E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti-α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti- α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti-α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti- α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti-α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.