G - I Splenicdendritic cells were treated with OxPAPC or DPPC ( 40 μg / ml ) before co - culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61 . ( G ) After 4 days of cell culture , T - cell polarization was assessed by intracellular staining for the cytokines IL - 4 ( Th2 ) and IFN - γ ( Th1 ) . ( H ) Bar graphs represent the frequencies of IL - 4 - and IFN - γ - producing T cells after 4 days of co - culture with OxPAPC - treated and DPPC - treated splenicdendritic cells . Bars represent mean ± SD of duplicate experiments . * P ≤ 0 . 05 by unpaired two - tailed t - test . ( I ) IFN - γ production in supernatants of SMARTA CD4T cells ( stimulated with 1 , 000 nM gp61 ) and co - cultured with OxPAPC - and DPPC - treated splenicdendritic cells for 4 days . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Bars represent mean ± SD .

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

G-I Splenicdendritic cells were treated with OxPAPC or DPPC (40 μg/ml) before co-culturing with naïve transgenic SMARTA CD4T cells in the presence of the specific peptide gp61. (G) After 4 days of cell culture, T-cell polarization was assessed by intracellular staining for the cytokines IL-4 (Th2) and IFN-γ (Th1). (H) Bar graphs represent the frequencies of IL-4- and IFN-γ-producing T cells after 4 days of co-culture with OxPAPC-treated and DPPC-treated splenicdendritic cells. Bars represent mean ± SD of duplicate experiments. *P ≤ 0.05 by unpaired two-tailed t-test. (I) IFN-γ production in supernatants of SMARTA CD4T cells (stimulated with 1,000 nM gp61) and co-cultured with OxPAPC- and DPPC-treated splenicdendritic cells for 4 days. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Bars represent mean ± SD.


Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4 - catalyzed oxidation of PAPC for 2 and 24 h . Mean ± SEM of duplicate determinations are shown .

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

Mass spectrometric quantification of a variety of OxPL species obtained by CuSO4-catalyzed oxidation of PAPC for 2 and 24 h. Mean ± SEM of duplicate determinations are shown.


Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL - 12 secretion of thioglycollate - elicited macrophages . Dots represent data of individual OxPAPC preparations .

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Correlation between the abundance of individual OxPL species detected in mixtures of differentially oxidized OxPL preparations and the capacity of the respective overall OxPL mixtures to suppress the IL-12 secretion of thioglycollate-elicited macrophages. Dots represent data of individual OxPAPC preparations.


Selected candidate lipids were tested for their inhibitory activity on R837 - induced ( 5 μg / ml ; 18 h ) cytokine secretion in BMDCs . Concentrations of indicated lipids : PECPC ( 10 μM ) , PEIPC ( 10 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , and KOdiAPC ( 40 μM ) . Representative data ( mean ± SD of triplicate determinations ) from one of three independent experiments are shown . * * * * P < 0 . 0001 ; ns , not significant ; as determined by one - way ANOVA adjusted by Sidak ' s multiple comparisons test .

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

Selected candidate lipids were tested for their inhibitory activity on R837-induced (5 μg/ml; 18 h) cytokine secretion in BMDCs. Concentrations of indicated lipids: PECPC (10 μM), PEIPC (10 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), and KOdiAPC (40 μM). Representative data (mean ± SD of triplicate determinations) from one of three independent experiments are shown. ****P < 0.0001; ns, not significant; as determined by one-way ANOVA adjusted by Sidak's multiple comparisons test.


Dose - response curves of IL - 12 secretion in BMDCs pulsed for 60 min with the indicated lipids , followed by stimulation with R837 ( 5 μg / ml ) for 18 h . Toxic concentrations of lipids were excluded from analysis . Mean ± SEM of triplicate determinations are shown .

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Dose-response curves of IL-12 secretion in BMDCs pulsed for 60 min with the indicated lipids, followed by stimulation with R837 (5 μg/ml) for 18 h. Toxic concentrations of lipids were excluded from analysis. Mean ± SEM of triplicate determinations are shown.


EC50 values of anti - inflammatory lipid products normalized to the capacity of 15d - PGJ2 to suppress IL - 12 production in BMDCs ( left panel ) and their respective bioactivities ( right panel ) depicted as the fold increase relative to 15d - PGJ2 . Mean ± SEM of triplicate determinations are shown .

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EC50 values of anti-inflammatory lipid products normalized to the capacity of 15d-PGJ2 to suppress IL-12 production in BMDCs (left panel) and their respective bioactivities (right panel) depicted as the fold increase relative to 15d-PGJ2. Mean ± SEM of triplicate determinations are shown.


IL - 12 production of BMDC stimulated via TLR 4 ( LPS ; 100 ng / ml ) , TLR 9 ( CpG ; 100 nM ) , and TLR 7 ( R837 ; 5 μg / ml ) after pretreatment with the indicated free and esterified versions of EC and 15d - PGJ2 . Lipids were used at 1 μM ( EC ) , 10 μM ( PECPC ) , or 20 μM ( 15d - PGJ2 and 15d - PGJ2PC ) . Data ( mean ± SEM ) are representative of 3 independent experiments . * * P ≤ 0 . 01 ; * * * P ≤ 0 . 001 ; * * * * P ≤ 0 . 0001 ; determined by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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

IL-12 production of BMDC stimulated via TLR 4 (LPS; 100 ng/ml), TLR 9 (CpG; 100 nM), and TLR 7 (R837; 5 μg/ml) after pretreatment with the indicated free and esterified versions of EC and 15d-PGJ2. Lipids were used at 1 μM (EC), 10 μM (PECPC), or 20 μM (15d-PGJ2 and 15d-PGJ2PC). Data (mean ± SEM) are representative of 3 independent experiments. **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; determined by one-way ANOVA adjusted by Dunnett's multiple comparisons test.


Analysis of the capacity of various OxPL - derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset . Concentrations of indicated lipids : EC ( 1 μM ) , PECPC ( 10 μM ) , 15d - PGJ2 ( 20 μM ) , OxPAPC ( 40 μg / ml ) , DPPC ( 40 μg / ml ) , POVPC ( 40 μM ) , PGPC ( 40 μM ) , KOdiAPC ( 40 μM ) .

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

Analysis of the capacity of various OxPL-derived species to license splenicdendritic cells to polarize naïve CD4T cells toward the Th2 subset. Concentrations of indicated lipids: EC (1 μM), PECPC (10 μM), 15d-PGJ2 (20 μM), OxPAPC (40 μg/ml), DPPC (40 μg/ml), POVPC (40 μM), PGPC (40 μM), KOdiAPC (40 μM).


A IL - 12 production of BMDC from wild - type , Nrf2 − / − , Pparg − / − , and Pparg litter mate control mice normalized to medium control ( open bars ) . Cells were treated with the indicated lipids ( filled bars ) for 60 min prior TLR 7 ligation with R837 ( 5 μg / ml ) for 18 h . Lipids were used at starting concentrations of 40 μM ( POVPC , PGPC , and KOdiAPC ) , 40 μg / ml ( OxPAPC ) , 20 μM ( 15d - PGJ2 ) , and 1 . 25 μM ( EC ) , depicted as black bars , and 2 - fold serial dilutions thereof ( gray bars ) . Data represent mean ± SEM of triplicates from one of three independent experiments .

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

A IL-12 production of BMDC from wild-type, Nrf2−/−, Pparg−/−, and Pparg litter mate control mice normalized to medium control (open bars). Cells were treated with the indicated lipids (filled bars) for 60 min prior TLR 7 ligation with R837 (5 μg/ml) for 18 h. Lipids were used at starting concentrations of 40 μM (POVPC, PGPC, and KOdiAPC), 40 μg/ml (OxPAPC), 20 μM (15d-PGJ2), and 1.25 μM (EC), depicted as black bars, and 2-fold serial dilutions thereof (gray bars). Data represent mean ± SEM of triplicates from one of three independent experiments.


B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild - type and Nrf2 − / − BMDM stimulated with EC ( 2 μM ) for 60 min . Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx . Data ( mean ± SEM ) are representative of two independent experiments .

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

B Expression of Nrf2 target genes Hmox1 and Nqo1 in wild-type and Nrf2−/−BMDM stimulated with EC (2 μM) for 60 min. Gene expression levels are presented relative to that of untreated cells after normalization to G6pdx. Data (mean ± SEM) are representative of two independent experiments.


C , D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 ( C ) and of the pro - inflammatory cytokines IL - 6 and IL - 12 ( D ) in wild - type , PPAR - γ - deficient , and Nrf2 - deficient BMDM after treatment with EC or 15d - PGJ2 for 60 min followed by LPS treatment for 3 h . Expression levels are normalized to G6pdx . Data represent mean ± SEM of triplicate cultures from one of two independent experiments .

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

C, D mRNA expression levels of the Nrf2 targets Gclc and Gsta3 (C) and of the pro-inflammatory cytokines IL-6 and IL-12 (D) in wild-type, PPAR-γ-deficient, and Nrf2-deficient BMDM after treatment with EC or 15d-PGJ2 for 60 min followed by LPS treatment for 3 h. Expression levels are normalized to G6pdx. Data represent mean ± SEM of triplicate cultures from one of two independent experiments.


E mRNA expression of the indicated chemokines as determined by qPCR . Wild - type BMDCs were treated with EC ( 1 μM ) or 15d - PGJ2 ( 20 μM ) for 60 min followed by R837 stimulation ( 5 μg / ml ) for 3 h . Expression levels are shown normalized to G6pdx . Data ( mean ± SEM , n = 2 ) are representative of three independent experiments .

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E mRNA expression of the indicated chemokines as determined by qPCR. Wild-type BMDCs were treated with EC (1 μM) or 15d-PGJ2 (20 μM) for 60 min followed by R837 stimulation (5 μg/ml) for 3 h. Expression levels are shown normalized to G6pdx. Data (mean ± SEM, n = 2) are representative of three independent experiments.


A C57BL / 6mice were treated ( i . v . ) with 500 μg EC or DPPC control 2 h prior to i . p injection of 150 ng / g LPS together with 800 μg / g D - galactosamine . 4 h after LPS application , lungs were perfused with PBS and embedded in paraffin . Tissue sections were hematoxylin - stained to visualize adherent cells . Bars represent 100 µm . B , C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in ( B ) and as averages of single mice in ( C ) . Pooled data of two independent experiments are shown ( n = 10 for EC , n = 14 for DPPC ) . Unpaired two - tailed t - test .

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

A C57BL/6mice were treated (i.v.) with 500 μg EC or DPPC control 2 h prior to i.p injection of 150 ng/g LPS together with 800 μg/g D-galactosamine. 4 h after LPS application, lungs were perfused with PBS and embedded in paraffin. Tissue sections were hematoxylin-stained to visualize adherent cells. Bars represent 100 µm.B, C Leukocyte adhesion to lung microvascular endothelium as determined by morphometric image analysis of lung tissue sections is presented for individual vessels in (B) and as averages of single mice in (C). Pooled data of two independent experiments are shown (n = 10 for EC, n = 14 for DPPC). Unpaired two-tailed t-test.


D , E C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) and 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS and 800 μg / g D - galactosamine . Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils ( D ) . Unpaired two - tailed t - test . Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL cells of EC - / LPS - treated and DPPC - / LPS - treated mice .

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

D, E C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. Bar graphs represent absolute numbers of total infiltrating cells and of neutrophils (D). Unpaired two-tailed t-test. Data represent mean ± SEM from one of two independent experiments with at least 6 mice per group. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


F , G The concentrations of IL - 6 ( F ) and IL - 12 ( G ) in the BAL of mice treated as in ( D ) were quantified by ELISA . Data ( mean ± SEM ) are representative of two independent experiments with 3 mice per group . * * * * P < 0 . 0001 ; unpaired two - tailed t - test .

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F, G The concentrations of IL-6 (F) and IL-12 (G) in the BAL of mice treated as in (D) were quantified by ELISA. Data (mean ± SEM) are representative of two independent experiments with 3 mice per group. ****P < 0.0001; unpaired two-tailed t-test.


C57BL / 6mice were treated with EC or DPPC by intra - tracheal instillation at 18 h ( 50 μg ) and 1 . 5 h ( 100 μg ) prior to i . p . injection of 150 ng / g LPS and 800 μg / g D - galactosamine . ( E ) Dot plots depict exemplary gating of lungneutrophils on pregated CD45 + CD11c − SiglecF − BAL cells of EC - / LPS - treated and DPPC - / LPS - treated mice .

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C57BL/6mice were treated with EC or DPPC by intra-tracheal instillation at 18 h (50 μg) and 1.5 h (100 μg) prior to i.p. injection of 150 ng/g LPS and 800 μg/g D-galactosamine. (E) Dot plots depict exemplary gating of lungneutrophils on pregated CD45+CD11c−SiglecF− BAL cells of EC-/LPS-treated and DPPC-/LPS-treated mice.


Dose - response curves showing the modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h . Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in ( A ) , presented relative to EC .

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

Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion by prior treatment of BMDCs with the indicated synthetic lipids for 1 h.Chemical structures and ΔEC50 values of the synthetic OxPL variant lipids analyzed in (A), presented relative to EC.


Quantification of IL - 6 and IL - 12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 ( 5 μg / ml ; 18 h ) . Bars represent mean ± SEM from one of three independent experiments . * * P < 0 . 01 ; * * * P < 0 . 001 ; one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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

Quantification of IL-6 and IL-12 secretion by BMDC treated with 250 nM cEC or EC for 60 min before stimulation with R837 (5 μg/ml; 18 h). Bars represent mean ± SEM from one of three independent experiments. **P < 0.01; ***P < 0.001; one-way ANOVA adjusted by Dunnett's multiple comparisons test.


mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression . BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed t - test .

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

mRNA expression of the Nrf2 targets Hmox1 and Nqo1 normalized to G6pdx expression. BMDCs were treated for 60 min with 500 nM cEC or DPPC followed by R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.


ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in ( E ) . Dose - response curves showing the modulation of R837 - induced ( 5 μg / ml ; 18 h ) IL - 12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h .

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

ΔEC50 values of the indicated lipids and OxPL shown relative to that of EC as determined in (E).Dose-response curves showing the modulation of R837-induced (5 μg/ml; 18 h) IL-12 secretion in BMDCs by prior treatment with the indicated OxPL derivatives for 1 h.


mRNA quantification of the indicated chemokines relative to G6pdx expression . BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation ( 5 μg / ml ) for 3 h . Data ( mean ± SD ) are representative of three independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; unpaired two - tailed t - test .

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

mRNA quantification of the indicated chemokines relative to G6pdx expression. BMDC were pretreated for 60 min with 500 nM cEC or the variant BisRed prior to R837 stimulation (5 μg/ml) for 3 h. Data (mean ± SD) are representative of three independent experiments. *P < 0.05; **P < 0.01; unpaired two-tailed t-test.


Quantification and characterization of cellular infiltrates in BAL . Groups of six C57BL / 6mice were pretreated i . t . with 50 μg cEC , EC , or BisRed 24 and 2 h before challenge with 150 ng / g LPS in the presence of 800 μg / g D - galactosamine . BAL was harvested 4 h after LPS injection , and inflammatory cells were characterized by FACS analysis . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; one - way ANOVA with Sidak ' s multiple comparisons test .

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

Quantification and characterization of cellular infiltrates in BAL. Groups of six C57BL/6mice were pretreated i.t. with 50 μg cEC, EC, or BisRed 24 and 2 h before challenge with 150 ng/g LPS in the presence of 800 μg/g D-galactosamine. BAL was harvested 4 h after LPS injection, and inflammatory cells were characterized by FACS analysis. **P < 0.01; ****P < 0.0001; one-way ANOVA with Sidak's multiple comparisons test.


Comparison of the capacity of cEC , EC , and PECPC ( 1 μM ) to license splenicdendritic cells to polarize naïve CD4T cells into IFN - γ - producing ( Th1 ) and IL - 4 - producing ( Th2 ) effector cells . Data ( mean ± SD ) are representative of two independent experiments . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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Comparison of the capacity of cEC, EC, and PECPC (1 μM) to license splenicdendritic cells to polarize naïve CD4T cells into IFN-γ-producing (Th1) and IL-4-producing (Th2) effector cells. Data (mean ± SD) are representative of two independent experiments. One-way ANOVA adjusted by Dunnett's multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.


( C ) Representative confocal image of long propriospinal neurons expressing FGF22 ( magenta ) in cervical spinal cord .

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(C) Representative confocal image of long propriospinal neurons expressing FGF22 (magenta) in cervical spinal cord.


( E ) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals ( green ) , stained for vGlut ( red ) and vGAT ( blue ) 21 days following spinal cord injury . Insets represent 3D views generated in Imaris of the deconvolved confocal image . ( F ) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice ( n = 4 - 5 mice per group ) . (

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(E) Schematic illustration of the hCST collateral into the cervical cord and confocal images of hCST collaterals (green), stained for vGlut (red) and vGAT (blue) 21 days following spinal cord injury. Insets represent 3D views generated in Imaris of the deconvolved confocal image. (F) Quantification of bouton density and characterization of bouton between control and FGF22 overexpressing mice (n = 4-5 mice per group). (


( G ) Schematic illustration and representative confocal image of LPSN viral labeling ( magenta ) and CST collaterals ( green ) . Insets represent 3D views generated in Imaris of the deconvolved confocal image . ( H ) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs ( n = 4 - 5 mice per group ) .

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(G) Schematic illustration and representative confocal image of LPSN viral labeling (magenta) and CST collaterals (green). Insets represent 3D views generated in Imaris of the deconvolved confocal image. (H) Quantification of hCST contacts onto LPSNs and characterization of vGlut intensity onto individual LPSNs (n = 4-5 mice per group).


( C ) Schematic illustration and representative confocal image for vGlut2 - Cre neuronal labeling ( cyan ) and hCST collaterals ( white ) . Insets represent 3D views generated in Imaris of the confocal image . Quantification of hCST contacts onto vGlut neurons ( n = 4 - 6 mice per group ) .

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(C) Schematic illustration and representative confocal image for vGlut2-Cre neuronal labeling (cyan) and hCST collaterals (white). Insets represent 3D views generated in Imaris of the confocal image. Quantification of hCST contacts onto vGlut neurons (n = 4-6 mice per group).


( C ) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control ( left panel ) and FGF22 treated ( right panel ) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury . ( n = 6 mice per group ) .

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(C) Schematic illustration of the hCST collateral exiting into the cervical cord and confocal images showing exiting hCST collaterals in control (left panel) and FGF22 treated (right panel) mice and quantification of the number of hCST collaterals that enter the cervical grey matter 3 weeks following spinal cord injury. (n = 6 mice per group).


( E ) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons ( arrows ) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 ( right ) and a control ( left ) mice . Quantification of bouton density in control and FGF22 treated mice . ( n = 6 mice per

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(E) Schematic illustration of the boutons on cervical hCST collateral and confocal images showing putative synaptic boutons (arrows) on newly formed cervical hCST collaterals at 3 weeks following spinal cord injury in a FGF22 (right) and a control (left) mice. Quantification of bouton density in control and FGF22 treated mice. (n = 6 mice per group


( F ) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals ( red ) and cervical interneurons ( cyan ) . Insets represent 3D views generated in Imaris of the confocal image . Quantification of the contacts between the hCST collaterals and cervical interneurons neurons . ( n = 6 per group ) .

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(F) Schematic illustration of the hCST contacts onto relay neurons in the cervical cord and confocal images showing contacts between CST collaterals (red) and cervical interneurons (cyan). Insets represent 3D views generated in Imaris of the confocal image. Quantification of the contacts between the hCST collaterals and cervical interneurons neurons. (n = 6 per group).


( G ) Confocal images showing contacts between CST collaterals ( red ) and long propriospinal neurons ( magenta ) transduced with FGF22 ( cyan ) . Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus ( DP refers to double positive cells ) . Insets on the right are magnification of the boxed area on the left . ( n = 6 mice per group ) .

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(G) Confocal images showing contacts between CST collaterals (red) and long propriospinal neurons (magenta) transduced with FGF22 (cyan). Quantification of the contacts between the hCST collaterals and long propriospinal transduced with FGF22 or control virus (DP refers to double positive cells). Insets on the right are magnification of the boxed area on the left. (n = 6 mice per group).


( C ) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in controls ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 mice per group ) . ( D ) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in control ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " or 2 days " 2dpi for the late treatment ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ( E ) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion ( top ) . Quantification of the functional recovery in the regular ( middle ) and irregular ( bottom ) ladder rung test in control ( grey ) and FGF22 ( orange ) treated mice at baseline , at 3 days post injury ( " 3dpi " ) , 14dpi and 21dpi after spinal cord injury . ( n = 9 - 10 per group ) . ) . (

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(C) Timeline of the experiment with FGF22 injection right after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in controls (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 mice per group). (D) Timeline of the experiment with FGF22 injection 24hrs after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi" or 2 days "2dpi for the late treatment), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group). (E) Timeline of the experiment with FGF22 injection 5 days after the onset of the spinal cord lesion (top). Quantification of the functional recovery in the regular (middle) and irregular (bottom) ladder rung test in control (grey) and FGF22 (orange) treated mice at baseline, at 3 days post injury ("3dpi"), 14dpi and 21dpi after spinal cord injury. (n = 9-10 per group).). (F


( F ) Representative confocal image of CST boutons ( green ) and synapsin staining ( red ) and quantification of the percentage of bouton synapsin positive ( n = 6 - 7 mice per group ) . ( G ) Representative confocal images of CST bouton ( green ) and bassoon staining ( magenta ) and quantification of the percentage of boutons that are bassoon positive ( n = 5 - 6 mice per group ) .

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(F) Representative confocal image of CST boutons (green) and synapsin staining (red) and quantification of the percentage of bouton synapsin positive (n = 6-7 mice per group). (G) Representative confocal images of CST bouton (green) and bassoon staining (magenta) and quantification of the percentage of boutons that are bassoon positive (n = 5-6 mice per group). D


( D ) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies . 300 assemblies ( 100 from each biological replicate ; 3 biological replicates ) were analyzed over at least 3 min at 5 s intervals . The central dash line indicates the median while the upper and lower dash lines indicate the quartiles . Unpaired t - test was performed ( ns : not significant ) .

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(D) Violin plots showing the distribution of sheath polymerization speed for both modes of T6SS assemblies. 300 assemblies (100 from each biological replicate; 3 biological replicates) were analyzed over at least 3 min at 5 s intervals. The central dash line indicates the median while the upper and lower dash lines indicate the quartiles. Unpaired t-test was performed (ns: not significant).


( D ) The frequency of contact - dependent assembly ( left panel ) and number of assemblies per cell per min ( right panel ) in cells from different strains as indicated on x - axis . Three biological replicates were performed for each strain . At least 500 cells were analyzed per replicate on 3D - SIM time - lapse imaging of sheath dynamics ( TssB - sfGFP ) in 3 min period . Bar graph ( left panel ) and scatter dot plot ( right panel ) show mean ± SD . Unpaired t - test was performed .

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(D) The frequency of contact-dependent assembly (left panel) and number of assemblies per cell per min (right panel) in cells from different strains as indicated on x-axis. Three biological replicates were performed for each strain. At least 500 cells were analyzed per replicate on 3D-SIM time-lapse imaging of sheath dynamics (TssB-sfGFP) in 3 min period. Bar graph (left panel) and scatter dot plot (right panel) show mean ± SD. Unpaired t-test was performed.


C . Western blot analysis of CTRL # 1 or BRD9 - KO # 1 lysates from cells treated for the indicated times with 1000 IU / mL of IFN - α2 . The indicated proteins were detected with specific antibodies . Data are representative of at least two biological replicates .

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C. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.


D . Immunofluorescence analysis of CTRL # 1 or BRD9 - KO # 1 cells treated for the indicated times with 1000 IU / mL of IFN - α2 . The indicated proteins were detected with specific antibodies . Data are representative of at least two biological replicates . Scale bars indicate 10μm .

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D. Immunofluorescence analysis of CTRL#1 or BRD9-KO#1 cells treated for the indicated times with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates. Scale bars indicate 10μm.


F . Western blot analysis of CTRL # 1 or BRD9 - KO # 1 lysates from cells treated or not for 16h with 1000 IU / mL of IFN - α2 . The indicated proteins were detected with specific antibodies . Data are representative of at least two biological replicates .

F. Western blot analysis of CTRL#1 or BRD9-KO#1 lysates from cells treated or not for 16h with 1000 IU/mL of IFN-α2. The indicated proteins were detected with specific antibodies. Data are representative of at least two biological replicates.


I . CTRL # 1 or BRD9 - KO # 1 cells were stably - transduced with BRD9 - expressing , or control ( EV , empty vector ) , lentiviruses and treated with 1000 IU / mL of IFN - α2 for 16h prior to infection with IAV ( WSN / 33 ) at an MOI of 0 . 01 PFU / cell . Viral titers were determined after 24h by plaque assay . Data information : data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Statistical significance was determined by 1 - way ANOVA on log - transformed plaque counts ( * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ) . Dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN - α2 , respectively . Numbers above IFN - α2 - treated bars indicate their approximate difference to the respective untreated conditions .

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

I. CTRL#1 or BRD9-KO#1 cells were stably-transduced with BRD9-expressing, or control (EV, empty vector), lentiviruses and treated with 1000 IU/mL of IFN-α2 for 16h prior to infection with IAV (WSN/33) at an MOI of 0.01 PFU/cell. Viral titers were determined after 24h by plaque assay. Data information: data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed plaque counts (**p-value < 0.001; ****p-value < 0.0001). Dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.


D . A549 cells were treated with 125nM dBRD9 - A ( + ) or DMSO ( - ) for 6h prior stimulation with 1000 IU / mL of IFN - α2 for 16h . Following lysis , western blot was used to detect levels of the indicated proteins . Data are representative of at least two biological replicates .

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D. A549 cells were treated with 125nM dBRD9-A (+) or DMSO (-) for 6h prior stimulation with 1000 IU/mL of IFN-α2 for 16h. Following lysis, western blot was used to detect levels of the indicated proteins. Data are representative of at least two biological replicates.


I - L . Hep2 ( I ) , Huh - 7 ( J ) , MRC - 5 ( K ) , and undifferentiated HTBE ( L ) were treated and infected with VSV - GFP Data information : Total Integrated Green Fluorescent Intensities were determined using the Incucyte live - cell analysis system after 24h Data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Statistical significance was determined by 1 - way ANOVA on log - transformed intensity values ( * p - value < 0 . 05 ; * * p - value < 0 . 01 ; * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ) . , dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN - α2 , respectively . Numbers above IFN - α2 - treated bars indicate their approximate difference to the respective untreated conditions .

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I-L. Hep2 (I), Huh-7 (J), MRC-5 (K), and undifferentiated HTBE (L) were treated and infected with VSV-GFP Data information: Total Integrated Green Fluorescent Intensities were determined using the Incucyte live-cell analysis system after 24h Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (*p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001). , dotted lines are a visual guide for maximum and minimum virus replication in control cells in the absence and presence of IFN-α2, respectively. Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions.


C . Impact of dBRD9 - A pre - treatment on the induction of ISGs identified in ( B ) . Colors indicate IFN - induced transcripts significantly affected ( blue ) or unaffected ( gray ) by dBRD9 - A pretreatment ( FC > 2 , p - value ≤ 0 . 001 ) .

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C. Impact of dBRD9-A pre-treatment on the induction of ISGs identified in (B). Colors indicate IFN-induced transcripts significantly affected (blue) or unaffected (gray) by dBRD9-A pretreatment (FC>2, p-value ≤ 0.001).


F . RT - qPCR confirmation of transcriptomics results for selected transcripts ( MX1 , MX2 , IFITM1 , ISG15 and IFIT1 ) in A549 cells treated as outlined in ( A ) . GAPDH transcript levels were used for normalization . Data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Statistical significance was determined by unpaired 2 - tailed t - test on ΔCt values ( * p - value < 0 . 05 ; n . s . not significant ) .

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F. RT-qPCR confirmation of transcriptomics results for selected transcripts (MX1, MX2, IFITM1, ISG15 and IFIT1) in A549 cells treated as outlined in (A). GAPDH transcript levels were used for normalization. Data represent means and standard deviations from n=3 biological experiments (individual data points shown). Statistical significance was determined by unpaired 2-tailed t-test on ΔCt values (*p-value < 0.05; n.s. not significant).


G . Western blot analysis of CTRL # 3 or BRD9 - KO # 3 cells stably transduced with lentiviruses expressing empty vector ( EV ) , or wt and chimeric

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G. Western blot analysis of CTRL#3 or BRD9-KO#3 cells stably transduced with lentiviruses expressing empty vector (EV), or wt and chimeric constructs


H . Western blot analysis of input lysates and anti - BRD9 immunoprecipitations ( IPs ) from cells described in ( G ) expressing wt BRD9 or the chimeric BRD7DUF . The indicated proteins were detected with specific antibodies . Data are representative of two biological replicates .

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H. Western blot analysis of input lysates and anti-BRD9 immunoprecipitations (IPs) from cells described in (G) expressing wt BRD9 or the chimeric BRD7DUF. The indicated proteins were detected with specific antibodies. Data are representative of two biological replicates.


I . The various CTRL # 3 or BRD9 - KO # 3 cells described in ( G ) were stimulated and infected as described in ( B ) . Data information : data represent means and standard deviations from n = 3 biological experiments ( individual data points shown ) . Numbers above IFN - α2 - treated bars indicate their approximate difference to the respective untreated conditions . Dotted lines are a visual guide for minimum virus replication in control cells in the presence of IFN - α2 . Statistical significance was determined by 1 - way ANOVA on log - transformed intensity values ( * * * p - value < 0 . 001 ; * * * * p - value < 0 . 0001 ; n . s . not significant ) .

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I. The various CTRL#3 or BRD9-KO#3 cells described in (G) were stimulated and infected as described in (B). Data information: data represent means and standard deviations from n=3 biological experiments (individual data points shown). Numbers above IFN-α2-treated bars indicate their approximate difference to the respective untreated conditions. Dotted lines are a visual guide for minimum virus replication in control cells in the presence of IFN-α2. Statistical significance was determined by 1-way ANOVA on log-transformed intensity values (***p-value < 0.001; ****p-value < 0.0001; n.s. not significant).


F . Western blot analysis of samples from independent small - scale streptavidin purifications ( termed ' Eluates ' ) following the proximity labeling approach outlined in ( B ) . The indicated proteins were detected with specific antibodies . M indicates the marker lane . Data are representative of at least two biological replicates .

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F. Western blot analysis of samples from independent small-scale streptavidin purifications (termed 'Eluates') following the proximity labeling approach outlined in (B). The indicated proteins were detected with specific antibodies. M indicates the marker lane. Data are representative of at least two biological replicates.


siRNA depletion of COPI increases autophagy . ( A ) siRNA depletion of β ′ - , β - , and α - COPI ( top ) and Sec23A and B ( bottom ) in 293 / GFP - LC3 cells for 48 h . Decreased protein levels of the corresponding subunits is shown by immunoblots .

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siRNA depletion of COPI increases autophagy. (A) siRNA depletion of β′-, β-, and α-COPI (top) and Sec23A and B (bottom) in 293/GFP-LC3 cells for 48 h. Decreased protein levels of the corresponding subunits is shown by immunoblots.


( B ) Loss of β ′ - , α - , and β - COP but not subunits of COPII ( Sec 23A and B ) increase GFP - LC3 - II . In A and B , tubulin was the loading control .

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(B) Loss of β′-, α-, and β-COP but not subunits of COPII (Sec 23A and B) increase GFP-LC3-II. In A and B, tubulin was the loading control.


( C ) GFP - LC3 - positive vesicles were detectable in 293 / GFP - LC3 cells after siRNA depletion of β ′ - , α - , and β - COP but only basal levels of GFP - LC3 - positive vesicles are observed in control siRNA - depleted cells .

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(C) GFP-LC3-positive vesicles were detectable in 293/GFP-LC3 cells after siRNA depletion of β′-, α-, and β-COP but only basal levels of GFP-LC3-positive vesicles are observed in control siRNA-depleted cells.


Time course of COPI depletion shows that Golgi disperses before AVs are formed , but Golgi dispersal does not cause AV formation . ( A ) COPI subunits were depleted in 293 / GFP - LC3 cells and analyzed 24 , 36 , and 48 h after addition of siRNA . Immunoblotting for β ′ - and α - COP confirms loss of COP subunits .

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Time course of COPI depletion shows that Golgi disperses before AVs are formed, but Golgi dispersal does not cause AV formation. (A) COPI subunits were depleted in 293/GFP-LC3 cells and analyzed 24, 36, and 48 h after addition of siRNA. Immunoblotting for β′- and α-COP confirms loss of COP subunits.


( B ) GFP - LC3 - I and - II were monitored by immunoblots in parallel lysates using anti - GFP antibodies at the indicated times .

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(B) GFP-LC3-I and -II were monitored by immunoblots in parallel lysates using anti-GFP antibodies at the indicated times.


( C ) After 24 h siRNA treatment , depletion of β ′ - COP caused morphological changes and a reduction in perinuclear population of GM130 . Box indicates enlarged area .

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(C) After 24 h siRNA treatment, depletion of β′-COP caused morphological changes and a reduction in perinuclear population of GM130. Box indicates enlarged area.


( D ) Rab1a / b was depleted using siRNAs for 48 h . Lysates were probed with anti - Rab1 to confirm depletion .

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(D) Rab1a/b was depleted using siRNAs for 48 h. Lysates were probed with anti-Rab1 to confirm depletion.


( E ) β ′ - COP or Rab1a / b were depleted as in D , and analyzed by indirect immunofluorescence using anti - GM130 and GFP fluorescence . In β ′ - COP panel , the asterisk indicates cells that did not show an accumulation of GFP - LC3 - positive AVs .

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(E) β′-COP or Rab1a/b were depleted as in D, and analyzed by indirect immunofluorescence using anti-GM130 and GFP fluorescence. In β′-COP panel, the asterisk indicates cells that did not show an accumulation of GFP-LC3-positive AVs.


AV formation after siRNA depletion of COPI requires Atg5 and Atg7 . 293 / GFP - LC3 cells were incubated with siRNA for β ′ - , α - COP , or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h , then ( A ) lysed and analyzed by immunoblot for Atg5 , Atg7 , GFP - LC3 , and tubulin ( loading control ) ,

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AV formation after siRNA depletion of COPI requires Atg5 and Atg7. 293/GFP-LC3 cells were incubated with siRNA for β′-, α-COP, or control siRNA alone or combined with siRNA against Atg5 or Atg7 for 48 h, then (A) lysed and analyzed by immunoblot for Atg5, Atg7, GFP-LC3, and tubulin (loading control),


( B ) fixed for immunofluorescence analysis using anti - ERGIC - 53 antibodies , or GFP fluorescence . In A , the asterisk indicates nonspecific band ; in B and C , asterisk indicates cells that did not show a fragmented ERGIC - 53 .

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(B) fixed for immunofluorescence analysis using anti-ERGIC-53 antibodies, or GFP fluorescence. In A, the asterisk indicates nonspecific band; in B and C, asterisk indicates cells that did not show a fragmented ERGIC-53.


Correlative light - electron microscopy analysis of GFP - LC3 puncta demonstrates they are AVs . ( A ) Phase , ( B ) fluorescent , and ( C ) TEM of the same field of 293 / GFP - LC3 cells 48 h after siRNA depletion of α - COP . In B the image was inverted to allow better visualization of the GFP - LC3 - positive structures . ( D ) Higher magnification of cell shown with asterisk in A and C . Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B . ( F and G ) Higher magnification of AVs shown by arrows in E .

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Correlative light-electron microscopy analysis of GFP-LC3 puncta demonstrates they are AVs. (A) Phase, (B) fluorescent, and (C) TEM of the same field of 293/GFP-LC3 cells 48 h after siRNA depletion of α-COP. In B the image was inverted to allow better visualization of the GFP-LC3-positive structures. (D) Higher magnification of cell shown with asterisk in A and C. Top region of cell of interest is enlarged in E to show the two AVs structures indicated by arrows in B. (F and G) Higher magnification of AVs shown by arrows in E.


Ub and p62 accumulate in α - and β ′ - COP - depleted 293 / GFP - LC3 cells . ( A ) 293 / GFP - LC3 cells were treated with control or β ′ - COP siRNA for 48 h , then fixed and labeled with anti - p62 and anti - ubiquitin antibodies for colocalization with GFP - LC3 .

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Ub and p62 accumulate in α- and β′-COP-depleted 293/GFP-LC3 cells. (A) 293/GFP-LC3 cells were treated with control or β′-COP siRNA for 48 h, then fixed and labeled with anti-p62 and anti-ubiquitin antibodies for colocalization with GFP-LC3.


( B ) HEK293 cells treated as in A and labeled with anti - ubiquitin and anti - p62 antibodies .

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(B) HEK293 cells treated as in A and labeled with anti-ubiquitin and anti-p62 antibodies.


( C ) Cryosections from β ′ - COP - depleted cells were labeled with anti - Ub antibodies followed by 10 - nm protein A - gold . AV , autophagosomes .

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(C) Cryosections from β′-COP-depleted cells were labeled with anti-Ub antibodies followed by 10-nm protein A-gold. AV, autophagosomes.


AVs in COPI - depleted cells are not degradative . ( A ) Long - lived protein degradation was assessed 72 h after transfection with control or β ′ - COP siRNA in 293 / GFP - LC3 cells . Cells were either starved of amino acids for 2 h ( St ) or incubated in fresh growth medium ( Fed ) for 2 h . The amount of protein degradation is presented as mean ± SEM .

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AVs in COPI-depleted cells are not degradative. (A) Long-lived protein degradation was assessed 72 h after transfection with control or β′-COP siRNA in 293/GFP-LC3 cells. Cells were either starved of amino acids for 2 h (St) or incubated in fresh growth medium (Fed) for 2 h. The amount of protein degradation is presented as mean ± SEM.


( B ) siControl - treated cells and β ′ - and α - COP were depleted for 48 h , then incubated with Lysotracker red ( 50 nM ) for 30 min before fixation . Merged image shows very little colocalization of GFP - LC3 AV - positive structures . Asterisk shows cell enlarged in

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(B) siControl-treated cells and β′- and α-COP were depleted for 48 h, then incubated with Lysotracker red (50 nM) for 30 min before fixation. Merged image shows very little colocalization of GFP-LC3 AV-positive structures. Asterisk shows cell enlarged in inset


( C ) After β ′ - and α - COP siRNA depletion , Magic Red - RR2 ( MR ) was used to detect active cathepsin B in cells which were fed or starved ( St ) for 2 h . The intensity of MR labeling was measured in live cells by flow cytometry .

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(C) After β′- and α-COP siRNA depletion, Magic Red-RR2 (MR) was used to detect active cathepsin B in cells which were fed or starved (St) for 2 h. The intensity of MR labeling was measured in live cells by flow cytometry.


GFP - LC3 AVs are LAMP2 positive . ( A ) 293 / GFP - LC3 cells were incubated with control or β ′ - or α - COP siRNA for 48 h , fixed and labeled with anti - LAMP2 antibodies , followed by Alexa 555 anti - mouse antibodies . After COPI depletion many GFP - LC3 - positive AVs are positive for LAMP2 . Asterisk indicates cell enlarged in inset .

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GFP-LC3 AVs are LAMP2 positive. (A) 293/GFP-LC3 cells were incubated with control or β′- or α-COP siRNA for 48 h, fixed and labeled with anti-LAMP2 antibodies, followed by Alexa 555 anti-mouse antibodies. After COPI depletion many GFP-LC3-positive AVs are positive for LAMP2. Asterisk indicates cell enlarged in inset.


( B ) Cryosections of β ′ - COP - depleted cells labeled with anti - GFP ( left ) or anti - GFP and anti - LAMP2 ( right ) followed by protein A - gold as indicated . AV , autophagosomes , M , mitochondria .

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(B) Cryosections of β′-COP-depleted cells labeled with anti-GFP (left) or anti-GFP and anti-LAMP2 (right) followed by protein A-gold as indicated. AV, autophagosomes, M, mitochondria.


GFP - LC3 - positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion . ( A ) 293 / GFP - LC3 cells were incubated with siRNA for β ′ - COP or control siRNA ( Cont ) for 48 h , processed for immunofluorescence , and labeled with antibodies for TGN46 .

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GFP-LC3-positive AVs colocalize TGN46 and early endocytotic markers after COPI depletion. (A) 293/GFP-LC3 cells were incubated with siRNA for β′-COP or control siRNA (Cont) for 48 h, processed for immunofluorescence, and labeled with antibodies for TGN46.


( B ) siRNA - treated cells were fixed and labeled , or starved for 2 h ( St ) , and labeled with antibodies to EEA1 . (

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(B) siRNA-treated cells were fixed and labeled, or starved for 2 h (St), and labeled with antibodies to EEA1. (


( C ) Control - starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation .

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(C) Control-starved HEK293 cells were labeled for endogenous LC3 and EEA1 by double labeling after methanol fixation.


( D ) After siRNA treatment , rhodamine - labeled dextran was internalized for 20 min , followed by a 40 - min chase after which the 293 / GFP - LC3 cells were fixed and analyzed by confocal microscopy .

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(D) After siRNA treatment, rhodamine-labeled dextran was internalized for 20 min, followed by a 40-min chase after which the 293/GFP-LC3 cells were fixed and analyzed by confocal microscopy.


Transferrin and EGF trafficking is inhibited by COPI depletion . 293 / GFP - LC3 cells were treated with β ′ - COP siRNA or control siRNA . After 48 h cells were incubated with Alexa 555 transferrin ( A ) for 2 min , ( B ) 30 min , or ( C ) 30 min followed by a 30 - min chase , then washed and fixed and analyzed by confocal microscopy .

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Transferrin and EGF trafficking is inhibited by COPI depletion. 293/GFP-LC3 cells were treated with β′-COP siRNA or control siRNA. After 48 h cells were incubated with Alexa 555 transferrin (A) for 2 min, (B) 30 min, or (C) 30 min followed by a 30-min chase, then washed and fixed and analyzed by confocal microscopy.


( D and E ) 125I - EGF was added to HeLa cells after 48 h of siRNA depletion of β ′ - COP , or control siRNA for 10 min . The cells were then washed as described in the Materials and methods , and chased for 30 , 60 , 120 , and 180 min ( including 10 - min incubation ) . ( D ) The amount of 125I - EGF internalized was shown as a percentage of the total 125I - EGF taken up in 10 min .

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(D and E) 125I-EGF was added to HeLa cells after 48 h of siRNA depletion of β′-COP, or control siRNA for 10 min. The cells were then washed as described in the Materials and methods, and chased for 30, 60, 120, and 180 min (including 10-min incubation). (D) The amount of 125I-EGF internalized was shown as a percentage of the total 125I-EGF taken up in 10 min.


( E ) The amount of degraded 125I - EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I - EGF internalized . The experiment was performed three times in duplicate and the data shown is the mean ± SEM . The significance was determined by Student ' s t test . * , P ≤ 0 . 05 .

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(E) The amount of degraded 125I-EGF was determined as a percentage of the total radioactivity present after TCA precipitation over the total 125I-EGF internalized. The experiment was performed three times in duplicate and the data shown is the mean ± SEM. The significance was determined by Student's t test. *, P ≤ 0.05.


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


( A ) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation . Cells expressing GFP - tagged Ykt6 under the control of the GAL1 promoter and tdTomato - tagged Sec63 were transformed with a centromeric plasmid expressing BFP - Ape1 . Cells were grown in SGC - LEU , shifted to SG - N for the indicated time , and analyzed by fluorescence microscopy . Images are shown as single planes . Scale bar , 5 µm . Arrows indicate Ykt6 dots colocalizing with Ape1 . ( B ) Percentage of Ykt6 puncta simultaneously co - localizing with both Ape1 and Sec63 . The data were quantified from ( A ) . Error bars represent standard deviation of three independent experiments . Cells ( n ≥ 250 ) and Ape1 dots ( n ≥ 50 ) were quantified .

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

(A) Localization of Ape1 relative to Ykt6 and Sec63 during nitrogen starvation. Cells expressing GFP-tagged Ykt6 under the control of the GAL1 promoter and tdTomato-tagged Sec63 were transformed with a centromeric plasmid expressing BFP-Ape1. Cells were grown in SGC-LEU, shifted to SG-N for the indicated time, and analyzed by fluorescence microscopy. Images are shown as single planes. Scale bar, 5 µm. Arrows indicate Ykt6 dots colocalizing with Ape1. (B) Percentage of Ykt6 puncta simultaneously co-localizing with both Ape1 and Sec63. The data were quantified from (A). Error bars represent standard deviation of three independent experiments. Cells (n≥250) and Ape1 dots (n≥50) were quantified.