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 ) .

O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O

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 ×

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

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 .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 O O O O O O O O B-GENEPROD O O O O O 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 B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

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 .

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

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 .

O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O 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 I-GENEPROD I-GENEPROD 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

(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 .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O 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 B-EXP_ASSAY O

(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 .

O O O 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 O O O O O O

(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 .

O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O 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 O O O O O O O O O O O O O

(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 .

O O O B-CELL_TYPE 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 B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O 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) 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 .

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

(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 B-CELL_TYPE 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 B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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 .

O O O O O O O O O B-GENEPROD I-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 O O O

(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 .

O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 O O O O O O O O O O O

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

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

(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 .

O O O B-CELL_TYPE 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 B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(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 .

O O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_TYPE O

(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 B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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

(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 B-CELL_TYPE 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 B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY O O B-GENEPROD O O O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE 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 .

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 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

(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 .

O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-GENEPROD 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

(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 B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE 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 B-GENEPROD O O B-GENEPROD 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 B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O 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 O O O O O O O O O O O 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 B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O 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 I-GENEPROD I-GENEPROD 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

(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 .

O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O

(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

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

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 .

B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-ORGANISM O O O O O O

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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O 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 B-ORGANISM 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 .

B-TISSUE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O

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 .

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

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 .

B-TISSUE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O B-SMALL_MOLECULE O O O O O O O O O O O B-ORGANISM O O O O O O

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 ) .

O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O

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 ) .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O

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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

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 ) .

B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O 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

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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD 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 .

B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD 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 O O O O O O B-GENEPROD 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY 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 B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-CELL_LINE 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 B-GENEPROD O B-GENEPROD O B-GENEPROD 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD 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 .

B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE 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 .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-CELL_TYPE 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 ) .

B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O 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 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 B-GENEPROD O O B-TISSUE O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O 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 O O O 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 B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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 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 B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O B-GENEPROD O B-CELL_TYPE O O O O B-SMALL_MOLECULE 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O 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 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-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 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 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR 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 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O B-EXP_ASSAY 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 O O O O O O O O O O O O O O

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 ) .

B-GENEPROD O O B-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 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 ) .

B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-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 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 ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD 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 .

B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O O B-CELL_TYPE 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 .

B-TISSUE B-GENEPROD O O O O B-ORGANISM O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O B-ORGANISM 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-TISSUE O O O O O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O 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 I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O B-SUBCELLULAR O O B-SUBCELLULAR 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 O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD 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 .

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


Electron micrographs showing ultrastructures of cotyledon cells of germinatedV . mungo seeds ( A - G ) , immunogold localization of α - amylase in cotyledon cells ( H ) , and ultrastructure of cells of detached cotyledons ( I ) . Toluidine blue ( TB ) staining of sections from cotyledons of day 3 V . mungo seedlings . Conversion from TB - stained cells ( region I ) to TB - stainless cells ( region III ) was accompanied with that of the PSV to LV . ( B ) SGs and PSVs in TB - stained cells ( A , region I ) . The PSV was filled with storage proteins . Arrowheads indicate border between SGs and the cytoplasm . ( C ) SGs and PSVs in cotyledons at region II in A . Electron density of PSVs became low . SGs were surrounded with membranous structure ( arrows ) . LED areas were found between SGs and the cytoplasm . ( D ) SG and LV in TB - stainless cells ( region III in A ) . The PSV was converted to the LV in the cells . The areas around SG with LED were enlarged . ( E ) Ultrastructure of TB - stainless cells . SGs wrapped with a LED area contacted with LVs ( arrowheads ) . Vesicles with similar density to LVs were observed . ( F ) LED membranes around the SGs fused with the LV membranes ( arrow ) . ( G ) SGs were observed in LVs . The shape of SGs were largely different from those in B - E . ( H ) An Immunogold image representing degradation of SGs by α - amylase localized in LVs . Gold particles from anti - α - amylase antibody were densely detected in the peripheral region of SGs , which is inserted into the inside of the LV . ( I ) PSVs were not converted to LVs , and SGs were not taken up into PSVs in the cells of detached cotyledons . Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells . LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars : ( A ) 50 μm ; ( E and G ) 2 μm ; ( D , F , and I ) 1 μm ; ( B , C , and H ) 200 nm .

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

Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A-G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B-E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti-α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.


LysoTracker red staining of partially broken cotyledon cells , SGs , or cotyledon sections . Cotyledon cells , SGs , and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods . ( A ) Differential interface - contrast image of a broken cell . ( B ) Fluorescent image of A . ( C ) Differential interface - contrast image of a broken cell . ( D ) Fluorescent image of C . ( E ) Differential interface - contrast image of cells of a section from attached cotyledons . ( F ) Fluorescent image of E . ( G ) Magnified image of E . ( H ) Magnified image of F . ( I ) Differential interface - contrast image of an SG . ( J ) Fluorescent image of I . ( K ) Differential interface - contrast image of an SG . ( L ) Fluorescent image of K . ( M ) Differential interface - contrast image of cells of a section from detached cotyledons . ( N ) Fluorescent image of M . ( O ) Magnified image of M . ( P ) Magnified image of O . Bars : ( A - D , G - L , O , and P ) 20 μm ; ( E , F , M , and N ) 50 μm .

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

LysoTracker red staining of partially broken cotyledon cells, SGs, or cotyledon sections. Cotyledon cells, SGs, and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods. (A) Differential interface-contrast image of a broken cell. (B) Fluorescent image of A. (C) Differential interface-contrast image of a broken cell. (D) Fluorescent image of C. (E) Differential interface-contrast image of cells of a section from attached cotyledons. (F) Fluorescent image of E. (G) Magnified image of E. (H) Magnified image of F. (I) Differential interface-contrast image of an SG. (J) Fluorescent image of I. (K) Differential interface-contrast image of an SG. (L) Fluorescent image of K. (M) Differential interface-contrast image of cells of a section from detached cotyledons. (N) Fluorescent image of M. (O) Magnified image of M. (P) Magnified image of O. Bars: (A-D, G-L, O, and P) 20 μm; (E, F, M, and N) 50 μm.


Electron micrographs showing autophagosome - mediated autophagy in attached ( A - C ) and detached ( D and E ) cotyledon cells . ( A ) Two autophagosomes containing cytoplasm were observed . Mitochondria and the cytoplasm appeared to be enclosed with an ER - like double membrane . ( B ) An autophagosome containing mitochondria and cytoplasm was fused with LV ( arrowheads ) . ( C ) Autophagic body containing mitochondria was found in LV . ( D ) An autophagosome containing mitochondria and cytoplasm was observed . The PSV was not converted into the LV . ( E ) Autophagic body was observed in the PSV of detached cotyledon cells . AB , autophagic body ; AP , autophagosome ; ER , endoplasmic reticulum ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars , 200 nm .

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

Electron micrographs showing autophagosome-mediated autophagy in attached (A-C) and detached (D and E) cotyledon cells. (A) Two autophagosomes containing cytoplasm were observed. Mitochondria and the cytoplasm appeared to be enclosed with an ER-like double membrane. (B) An autophagosome containing mitochondria and cytoplasm was fused with LV (arrowheads). (C) Autophagic body containing mitochondria was found in LV. (D) An autophagosome containing mitochondria and cytoplasm was observed. The PSV was not converted into the LV. (E) Autophagic body was observed in the PSV of detached cotyledon cells. AB, autophagic body; AP, autophagosome; ER, endoplasmic reticulum; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


( B , C ) HEK293T cells were co ‐ transfected with the indicated constructs . Cell lysates were subjected to IP using anti ‐ FLAG antibodies . The resulting precipitates were examined by immunoblot analysis with anti ‐ FLAG and anti ‐ HA antibodies .

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

(B,C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were subjected to IP using anti‐FLAG antibodies. The resulting precipitates were examined by immunoblot analysis with anti‐FLAG and anti‐HA antibodies.


( D ) HEK293T cells were cultured in regular or starvation medium for 2 h . Cells were harvested and treated with DSP .

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(D) HEK293T cells were cultured in regular or starvation medium for 2 h. Cells were harvested and treated with DSP.


( E ) HEK293T cells were harvested and treated with DSP .

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(E) HEK293T cells were harvested and treated with DSP.


( F ) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h . After treatment with DSP , cell lysates were subjected to IP analysis .

O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O

(F) HEK293T cells were cultured in regular DMEM or starvation medium in the presence or absence of 200 nM wortmannin for 2 h. After treatment with DSP, cell lysates were subjected to IP analysis.


( G ) Atg14F / F ( undeleted ) or Atg14Δ / Δ ( deleted ) MEFs were harvested and treated with DSP . * ( E ) and * * ( G ) indicate the positions of the immunoglobulin light and heavy chains , respectively . aa , amino acid ; DSP , dithiobis ( succinimidyl propionate ) ; IP , immunoprecipitation ; MEFs , mouse embryonic fibroblasts .

O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O B-EXP_ASSAY O B-CELL_LINE O O O O O

(G) Atg14F/F (undeleted) or Atg14Δ/Δ (deleted) MEFs were harvested and treated with DSP. * (E) and ** (G) indicate the positions of the immunoglobulin light and heavy chains, respectively. aa, amino acid; DSP, dithiobis(succinimidyl propionate); IP, immunoprecipitation; MEFs, mouse embryonic fibroblasts.


( A ) Immunoblot analysis of Atg proteins in the indicated MEFs .

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(A) Immunoblot analysis of Atg proteins in the indicated MEFs.


( B ) m5 ‐ 7 cells were cultured in regular medium containing 10 ng / ml Dox for 8 days , and then cultured in the absence of Dox for a further 2 days .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O

(B) m5‐7 cells were cultured in regular medium containing 10 ng/ml Dox for 8 days, and then cultured in the absence of Dox for a further 2 days.


( C , D ) m5 ‐ 7 cells were cultured in regular medium with or without 10 ng / ml Dox for 4 days , and then cultured in the presence of 50 μg / ml CHX for the indicated time periods ( C ) . mRNA levels of the indicated genes were measured by quantitative PCR following the 4 ‐ day Dox treatment . Data represent mean ± s . e . ( * P0 . 05 ) ( D ) . CHX , cycloheximide ; Dox , doxycycline ; MEFs , mouse embryonic fibroblasts ; mRNA , messenger RNA ; WT , wild ‐ type .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O

(C, D) m5‐7 cells were cultured in regular medium with or without 10 ng/ml Dox for 4 days, and then cultured in the presence of 50 μg/ml CHX for the indicated time periods (C). mRNA levels of the indicated genes were measured by quantitative PCR following the 4‐day Dox treatment. Data represent mean±s.e. (*P0.05) (D). CHX, cycloheximide; Dox, doxycycline; MEFs, mouse embryonic fibroblasts; mRNA, messenger RNA; WT, wild‐type.


( B , C ) HEK293T cells were co ‐ transfected with the indicated constructs . Cell lysates were analysed by IP . IP , immunoprecipitation ; ND , not determined .

O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O

(B, C) HEK293T cells were co‐transfected with the indicated constructs. Cell lysates were analysed by IP. IP, immunoprecipitation; ND, not determined.


( A ) WT MEFs , Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h . * indicates nonspecific immunoreactive bands .

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

(A) WT MEFs, Atg16L1 KO MEFs or Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium in the presence or absence of 100 nM BafA1 for 2 h. * indicates nonspecific immunoreactive bands.


( B , C ) Atg16L1 KO MEFs stably expressing GFP - ULK1 and either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM ( C ) or starvation medium ( B , C ) for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP , anti ‐ Atg16L1 and anti ‐ LC3 antibodies . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) . The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods . Data represent mean ± s . e . ( * P0 . 001 , analysis of variance followed by Bonferroni / Dunn post hoc test ) . Scale bar , 10 μm , and 2 μm in inset .

O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Atg16L1 KO MEFs stably expressing GFP-ULK1 and either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM (C) or starvation medium (B,C) for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP, anti‐Atg16L1 and anti‐LC3 antibodies. Arrowheads indicate the perinuclear localization of Atg16L1(1-230). The number of dots was quantified from more than 30 randomly selected cells from three independent samples as described in the Methods. Data represent mean±s.e. (*P0.001, analysis of variance followed by Bonferroni/Dunn post hoc test). Scale bar, 10 μm, and 2 μm in inset.


( D , E ) Cells stably expressing GFP - LC3 were cultured in regular DMEM , starvation medium or starvation medium in the presence of 0 . 2 μM WM or 100 nM BafA1 for 6 h . Total cellular GFP - LC3 signals were analysed by flow cytometry . Representative FACS data were shown ( D ) . The geometric mean of fluorescence intensity was determined . Values are expressed as a percentage of the mean of control cells cultured in regular DMEM . Data represent mean ± s . e . ( * P0 . 05 ) ( E ) . BafA1 , bafilomycin A1 ; GFP , green fluorescent protein ; KO , knockout ; MEFs , mouse embryonic fibroblasts ; NS , not significant ; WM , wortmannin ; WT , wild ‐ type .

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 B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY 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 B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O

(D, E) Cells stably expressing GFP-LC3 were cultured in regular DMEM, starvation medium or starvation medium in the presence of 0.2 μM WM or 100 nM BafA1 for 6 h. Total cellular GFP-LC3 signals were analysed by flow cytometry. Representative FACS data were shown (D). The geometric mean of fluorescence intensity was determined. Values are expressed as a percentage of the mean of control cells cultured in regular DMEM. Data represent mean±s.e. (*P0.05) (E). BafA1, bafilomycin A1; GFP, green fluorescent protein; KO, knockout; MEFs, mouse embryonic fibroblasts; NS, not significant; WM, wortmannin; WT, wild‐type.


( A ) Atg16L1 KO MEFs stably expressing either full ‐ length Atg16L1 ( 1 - 588 ) , Atg16L1 ( 1 - 230 ) or Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and subjected to immunofluorescence microscopy using anti ‐ Atg16L1 antibody . Note that full ‐ length Atg16L1 and Atg16L1 ( 1 - 230 ) , but not Atg16L1Δ ( 230 - 300 ) , showed punctate structures under starvation conditions ( inset ) .

O O O B-GENEPROD O B-CELL_LINE O O O O O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD 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

(A) Atg16L1 KO MEFs stably expressing either full‐length Atg16L1(1-588), Atg16L1(1-230) or Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and subjected to immunofluorescence microscopy using anti‐Atg16L1 antibody. Note that full‐length Atg16L1 and Atg16L1(1-230), but not Atg16L1Δ(230-300), showed punctate structures under starvation conditions (inset).


( B ) FIP200 KO MEFs stably expressing either CFP - Atg16L1 ( 1 - 588 ) , CFP - Atg16L1 ( 1 - 230 ) or CFP - Atg16L1Δ ( 230 - 300 ) were cultured in regular DMEM or starvation medium for 1 h . Cells were fixed and analysed by immunofluorescence microscopy using anti ‐ GFP antibody . Arrowheads indicate the perinuclear localization of Atg16L1 ( 1 - 230 ) .

O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O

(B) FIP200 KO MEFs stably expressing either CFP-Atg16L1(1-588), CFP-Atg16L1(1-230) or CFP-Atg16L1Δ(230-300) were cultured in regular DMEM or starvation medium for 1 h. Cells were fixed and analysed by immunofluorescence microscopy using anti‐GFP antibody. Arrowheads indicate the perinuclear localization of Atg16L1(1-230).


( C , D ) FIP200 KO MEFs stably expressing CFP - Atg16L1 ( 1 - 230 ) were cultured in regular DMEM . Scale bar , 10 μm , and 2 μm in inset .

O O O O O B-GENEPROD O B-CELL_LINE 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

(C, D) FIP200 KO MEFs stably expressing CFP-Atg16L1(1-230) were cultured in regular DMEM. Scale bar, 10 μm, and 2 μm in inset.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant reduction in survival of these animals ( Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom , Log Rank , p < 0 . 001 , n ≥ 10 ) .

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

(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant reduction in survival of these animals (Vil Apc vs Vil Apc Huwe1het / Vil Apc Huwe1hom, Log Rank, p < 0.001, n ≥ 10).


( b ) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint . Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine ( red arrows ) .

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

(b) Wholemount isolation of small intestines from Vil Apc and Vil Apc Huwe1hom mice culled at clinical endpoint. Note the huge numbers of tiny macroscopic adenomas visible in the Vil Apc Huwe1hom intestine (red arrows).


( c ) H & amp ; amp ; E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion . Black arrows indicate individual adenomas . Scale bars = 200 µm .

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

(c) H&amp;E staining of intestinal cross sections from Vil Apc and Vil Apc Huwe1hom mice demonstrating the increased adenoma burden following Huwe1 deletion. Black arrows indicate individual adenomas. Scale bars = 200 µm.


( d ) Quantification of total tumour numbers per gut in sacrificed Vil Apc , Vil Apc Huwe1het and Vil Apc Huwe1hom mice . Deletion of Huwe1 led to a significant increase in the number of tumours per gut ( Mann Whitney , n ≥ 5 ) .

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

(d) Quantification of total tumour numbers per gut in sacrificed Vil Apc, Vil Apc Huwe1het and Vil Apc Huwe1hom mice. Deletion of Huwe1 led to a significant increase in the number of tumours per gut (Mann Whitney, n ≥ 5).


( a ) H & amp ; amp ; E staining of control and Huwe1 deleted intestinal epithelium . Shortened villi in Huwe1 deficient tissue are indicated . Scale bars = 100 µm . ( b ) BrdU IHC of control and Huwe1 deleted intestinal epithelium . Scale bars = 100 µm . ( c ) PAS staining identifying Goblet cells . No gross changes were observed . Scale bars = 100 µm . ( d ) Lysozyme staining ( Paneth cell marker ) of control and Huwe1 deleted small intestine . Note the occurrence of lysozyme positive cells away from the crypt base ( black arrows ) . Scale bars = 100 µm .

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

(a) H&amp;E staining of control and Huwe1 deleted intestinal epithelium. Shortened villi in Huwe1 deficient tissue are indicated. Scale bars = 100 µm. (b) BrdU IHC of control and Huwe1 deleted intestinal epithelium. Scale bars = 100 µm. (c) PAS staining identifying Goblet cells. No gross changes were observed. Scale bars = 100 µm. (d) Lysozyme staining (Paneth cell marker) of control and Huwe1 deleted small intestine. Note the occurrence of lysozyme positive cells away from the crypt base (black arrows). Scale bars = 100 µm.


( e ) Dual Periodic acid - Schiff / Alcian blue staining to identify Paneth cell secretory vesicles ( light blue / pink - marked with black arrows ) and goblet cells ( dark blue / purple - marked with red arrows ) . Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines ( inset - black arrows ) . Scale bars = 100µm .

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

(e) Dual Periodic acid-Schiff / Alcian blue staining to identify Paneth cell secretory vesicles (light blue / pink - marked with black arrows) and goblet cells (dark blue / purple - marked with red arrows). Note Paneth cell secretory vesicles are restricted to crypt base in both control and Huwe1 deficient small intestines (inset - black arrows). Scale bars = 100µm.


( f ) MMP7 staining of control and Huwe1 deleted small intestine . Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines . Scale bars = 100µm .

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

(f) MMP7 staining of control and Huwe1 deleted small intestine. Note MMP7 staining is restricted to crypt base in Huwe1 deficient intestines. Scale bars = 100µm.


( a ) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines . Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

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

(a) HUWE1 and MYC Western blot analysis of epithelial cell extractions from control and Huwe1 deficient intestines. Levels of MYC protein are significantly increased in normal intestines lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( b ) QRT - PCR analysis of Myc expression in control and Huwe1 deficient intestines .

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

(b) QRT-PCR analysis of Myc expression in control and Huwe1 deficient intestines.


( c ) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice . Scale bars = 50 µm .

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

(c) MYC IHC in tumours from Vil Apc and Vil Apc Huwe1hom mice. Scale bars = 50 µm.


( d ) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours . Levels of MYC protein are significantly increased in tumours lacking HUWE1 ( Mann Whitney , p = 0 . 04 , n = 3 ) .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-DISEASE O O O B-GENEPROD O O O O O B-DISEASE O B-GENEPROD O O O O O O O O O O O O O O O

(d) MYC Western blot in protein extracts from Vil Apc and Vil Apc Huwe1hom tumours. Levels of MYC protein are significantly increased in tumours lacking HUWE1 (Mann Whitney, p = 0.04, n = 3).


( e ) QRT - PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice .

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

(e) QRT-PCR analysis of Myc expression in tumours from Vil Apc and Vil Apc Huwe1hom mice.


( a ) Kaplan - Meier survival plot of cohorts of induced Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc , Log Rank , p < 0 . 001 , n ≥ 16 ) .

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

(a) Kaplan-Meier survival plot of cohorts of induced Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc led to a specific and significant increase in survival of Huwe1 deleted animals (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc, Log Rank, p < 0.001, n ≥ 16).


( b ) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten , Vil Apc Pten Huwe1 , Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice . Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice ( Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc . Mann Whitney , n ≥ 10 ) .

O O O O O O B-TISSUE O O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O B-GENEPROD O O O O O O B-DISEASE O B-GENEPROD O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O

(b) Quantification of total tumour numbers per gut in sacrificed Vil Apc Pten, Vil Apc Pten Huwe1, Vil Apc Pten Myc and Vil Apc Pten Huwe1 Myc mice. Heterozygous deletion of Myc did not reduce the number of tumours in Huwe1 deleted mice (Vil Apc Pten Huwe1 vs Vil Apc Pten Huwe1 Myc. Mann Whitney, n ≥ 10).


( c ) Kaplan - Meier survival plot of cohorts of induced AhCre - ERT Apc Pten and AhCre - ERT Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC led to a significant reduction in survival ( Log Rank , p < 0 . 001 , n = 14 vs 21 ) .

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

(c) Kaplan-Meier survival plot of cohorts of induced AhCre-ERT Apc Pten and AhCre-ERT Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC led to a significant reduction in survival (Log Rank, p < 0.001, n = 14 vs 21).


( d ) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice . Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice ( Mann Whitney , n = 9 vs 19 ) .

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

(d) Quantification of total tumour numbers per gut in sacrificed Apc Pten and Apc Pten MycT58A mice. Overexpression of proteolytically stabilised MYC did not increase the number of tumours in Apc Pten mice (Mann Whitney, n = 9 vs 19).


( a ) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells . Band intensity relative to βactin displayed under each lane ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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

(a) H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells. Band intensity relative to βactin displayed under each lane (Mann-Whitney, p = 0.04, n = 3 vs 3).


( b ) γ - H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells . Scale bars = 50 µm .

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

(b) γ-H2AX IHC showing increased positivity in Huwe1 deleted intestinal cells. Scale bars = 50 µm.


( c ) γ - H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells ( Mann - Whitney , p = 0 . 04 , n = 3 vs 3 ) .

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

(c) γ-H2AX Western blot in protein extracts from control and Huwe1 deficient intestinal epithelial cells (Mann-Whitney, p = 0.04, n = 3 vs 3).


( d ) γ - H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours , note increased positivity in Huwe1 deficient tumours . Scale bars = 50 µm .

O O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-DISEASE O O O O O B-GENEPROD O B-DISEASE O O O O O O O

(d) γ-H2AX IHC of Vil Apc and Vil Apc Huwe1hom tumours, note increased positivity in Huwe1 deficient tumours. Scale bars = 50 µm.


( a ) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7 . 5mg / kg cisplatin . Arrows identify caspase 3 positive cells . Scale bars = 50 µm . ( b ) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells ( Mann - Whitney , n = 3 vs 5 ) .

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-DISEASE O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O

(a) Caspase 3 IHC of tumours from Lgr5 Apc and Lgr5 Apc Huwe1 mice either untreated or 6h post treatment with 7.5mg/kg cisplatin. Arrows identify caspase 3 positive cells. Scale bars = 50 µm. (b) Quantification of cisplatin treatment showing a significant increase in apoptosis in Huwe1 deficient tumour cells (Mann-Whitney, n = 3 vs 5).


( c ) Comparison of somatic mutation rate ( mutations / Mb ) between human tumours carrying HUWE1 mutations or not . Note the significant increase in mutational burden in HUWE1 mutated tumours ( Mann - Whitney , p = 0 . 0002 , n ≥ 15 ) .

O O O O O O O O O O O O O O B-ORGANISM B-DISEASE O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-DISEASE O O O O O O O O O O O O O O O O

(c) Comparison of somatic mutation rate (mutations / Mb) between human tumours carrying HUWE1 mutations or not. Note the significant increase in mutational burden in HUWE1 mutated tumours (Mann-Whitney, p = 0.0002, n ≥ 15).