Representative TEM images of autophagosomes in control and TCHP knock - down HUVECs in the complete medium after 3h incubation with BafA1 ( Scale bars , 600nm ) : i , quantification of mean autophagic vesicles ( AVs ) and autophagosome ( APs ) area and ii , distribution of the cross - section areas of the analysed vesicles expressed in percentage . ( n = 7 cells ; > 100 vesicles per sample ; AVs : unpaired t - test ; * * p < 0 . 0001 vs control ; APs : unpaired t - test * * p < 0 . 0001 vs control ) .

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Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).


Left panels : Representative single - channel and merged images of HUVECs expressing GFP - LC3 and immunostained for STX17 . Scale bars , 25 µm and 2 µm in the inset . Right panels : Quantification of LC3 / STX17 positive cells in the presence or absence of BafA1 ( one - way ANOVA ; * * p = 0 . 0060 vs control ; # p = 0 . 0136 vs control BafA1 ) and quantification LC3 / STX17 positive puncta per cells ( n = 50 cells ; one - way ANOVA ; * * p < 0 . 0001 vs shTCHP BafA1 ) .

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Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).


Expression of TCHP , p62 , IL - 1β , IL - 8 , and IL - 6 in ECs from vessels wall from patients with CAD ( n = 8 patients per group , unpaired t - test ; TCHP * * p < 0 . 0001 ; p62 * * p = 0 . 0007 ; IL - 1β * * p < 0 . 0001 ; IL - 8 * * p < 0 . 0001 ; and IL - 6 * * p < 0 . 0001 vs . Healthy subject ) .

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

Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject).


Staining , and quantification of p62 in ECs from healthy subject and patients either treated with vehicle ( DMSO ) or BAY . Scale bars , 50 µm ; ( n = 3 patients per group ; n = 80 cells ; one - way ANOVA ; * * p < 0 . 0001 vs healthy DMSO ; # # p = 0 . 003 vs patients DMSO ) .

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Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).


Expression of IL - 6 , IL - 8 and IL - 1β ( n = 3 patients per group ; one - way ANOVA ; IL - 6 : * p = 0 . 0135 vs healthy DMSO ; # p = 0 . 0180 vs patients DMSO ; IL - 8 : * p = 0 . 0139 vs healthy DMSO ; # # p = 0 . 0024 vs patients DMSO ; IL - 1β : * p = 0 . 0104 vs healthy DMSO ; # # p = 0 . 0069 vs patients DMSO ) .

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

Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO).


migration speed , in ECs from healthy subject and patients either treated with vehicle ( DMSO ) or BAY . ( n = 3 patients per group ; one - way ANOVA ; * p = 0 . 0325 vs healthy DMSO ; # p = 0 . 0336 vs patients DMSO ) .

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migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).


Left panels : representative images of the heart of wild - type and Tchp knock - out mice stained for CD31 ( green ) and p62 ( magenta ) . Scale bars , 100µm , and 50µm for the inset . Right panels : Quantification of the percentage of cardiac area ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 083 vs wild - type ) or vessels positive for p62 ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 058 vs wild - type ) and vessel density ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 0097 vs wild - type ) .

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Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).


Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM . Representative of three samples examined . Scale bar 100

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Electron micrograph of fibrils formed by a synthetic peptide encompassing the M45 RHIM. Representative of three samples examined. Scale bar 100 nm


HT29 cell survival 18h after treatment with TNF + BV - 6 + z - VAD - fmk to induce necroptosis . Cells expressed lentivirus constructs encoding 1 - 90 or 1 - 277 fragments of M45 and were cultured + / - the addition of coumermycin A1 to induce dimerisation . N = 2 for M45 - 90 gyrase + / - coumermycin A1 and 4 for M45 - 277 gyrase and for empty vector , + / - coumermycin

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HT29 cell survival 18h after treatment with TNF+BV-6+z-VAD-fmk to induce necroptosis. Cells expressed lentivirus constructs encoding 1-90 or 1-277 fragments of M45 and were cultured +/- the addition of coumermycin A1 to induce dimerisation. N=2 for M45-90 gyrase +/- coumermycin A1 and 4 for M45-277 gyrase and for empty vector, +/- coumermycin A1


Thioflavin T fluorescence as a function of time , following dilution of WT or AAAA His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , and His - Ub - RIPK3387 ‑ 518 from 8 M to 200 mM urea - containing buffer . Buffer sample contains ThT but no protein . Samples were tested in triplicate and solid line is the average of the triplicates and the error bars ( in grey ) represent + / - s .

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Thioflavin T fluorescence as a function of time, following dilution of WT or AAAA His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 from 8 M to 200 mM urea-containing buffer. Buffer sample contains ThT but no protein. Samples were tested in triplicate and solid line is the average of the triplicates and the error bars (in grey) represent +/- s.d


Absorbance spectra from solutions containing Congo red with WT ( black lines ) or AAAA mutant ( dark grey lines ) His ‑ Ub ‑ M451 ‑ 90 , His - Ub - RIPK1497 ‑ 583 , and His - Ub - RIPK3387 ‑ 518 that have been dialysed against Assembly Buffer . Congo red sample alone with no protein ( light grey line ) served as a control for the experiment . Experiment conducted

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Absorbance spectra from solutions containing Congo red with WT (black lines) or AAAA mutant (dark grey lines) His‑Ub‑M451‑90, His-Ub-RIPK1497‑583, and His-Ub-RIPK3387‑518 that have been dialysed against Assembly Buffer. Congo red sample alone with no protein (light grey line) served as a control for the experiment. Experiment conducted once


Electron micrograph of material eluted in peak 1 from the three MBP fusion proteins , as labelled . These are representative micrographs of the material observed . Scale bar 100 nm in all

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Electron micrograph of material eluted in peak 1 from the three MBP fusion proteins, as labelled. These are representative micrographs of the material observed. Scale bar 100 nm in all panels


Representative micrographs of insoluble material generated after treatment with thrombin removed the MBP . Experiment repeated three times . Scale bar 100 nm in all

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Representative micrographs of insoluble material generated after treatment with thrombin removed the MBP. Experiment repeated three times. Scale bar 100 nm in all panel


Fluorescence polarisation profiles from incorporation of FITC - M451 ‑ 90 into excess unlabeled M451 ‑ 90 ( green ) , RIPK1497 ‑ 583 ( red ) , RIPK3387 ‑ 518 ( blue ) or ZBP1170 - 355 ( purple ) . Average polarisation profile from smoothed triplicate samples plotted , with s . d . indicated . Experiment performed twice and representative results shown

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Fluorescence polarisation profiles from incorporation of FITC-M451‑90 into excess unlabeled M451‑90 (green), RIPK1497‑583 (red), RIPK3387‑518 (blue) or ZBP1170-355 (purple). Average polarisation profile from smoothed triplicate samples plotted, with s.d. indicated. Experiment performed twice and representative results shown here


Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of ( C ) mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK1497 ‑ 583 and ( D ) mCHERRY - RIPK3387 ‑ 518 and YPet - RIPK3387 ‑ 518 . E , F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of ( E ) mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑ 518 ( inset is expansion of region indicated by * ) and ( F ) mCHERRY - RIPK1497 ‑ 583 - AAAA and YPet - RIPK3387 ‑ 518 . All mixtures examined in at least three different experiments on three separate

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Representative fluorescence intensity trace showing homomeric assembly over 40 s in mixtures of (C) mCHERRY-RIPK1497‑583 and YPet-RIPK1497‑583 and (D) mCHERRY-RIPK3387‑518 and YPet-RIPK3387‑518. E, F Representative fluorescence intensity traces showing heteromeric assembly over 40 s in mixtures of (E) mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 (inset is expansion of region indicated by *) and (F) mCHERRY-RIPK1497‑583-AAAA and YPet-RIPK3387‑518. All mixtures examined in at least three different experiments on three separate occasions


Representative fluorescence intensity traces collected over 50 s for mixtures of ( A ) mCHERRY - M451 - 90 and YPet ‑ only ; ( B ) mCHERRY - M451 - 90 and YPet ‑ RIPK1497 ‑ 583 ; ( C ) mCHERRY - M451 - 90 and YPet - RIPK3387 ‑ 518 ; ( D ) mCHERRY - M451 - 90 and YPet - ZBP1170 - 355 ; ( E ) mCHERRY - M451 - 90AAAA and YPet ‑ only ; ( F ) mCHERRY - M451 - 90AAAA and YPet - RIPK1497 - 583 ; ( G ) mCHERRY - M451 - 90AAAA and YPet - RIPK3387 ‑ 518 and ( H ) mCHERRY - M451 - 90 and YPet - ZBP1170 - 355 . Independent replicates performed at least

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Representative fluorescence intensity traces collected over 50 s for mixtures of (A) mCHERRY-M451-90 and YPet‑only; (B) mCHERRY-M451-90 and YPet‑RIPK1497‑583; (C) mCHERRY-M451-90 and YPet-RIPK3387‑518; (D) mCHERRY-M451-90 and YPet-ZBP1170-355; (E) mCHERRY-M451-90AAAA and YPet‑only; (F) mCHERRY-M451-90AAAA and YPet-RIPK1497-583; (G) mCHERRY-M451-90AAAA and YPet-RIPK3387‑518 and (H) mCHERRY-M451-90 and YPet-ZBP1170-355. Independent replicates performed at least twice


Representative electron micrographs of homofibrils formed by M451 - 90 , RIPK1497 ‑ 583 , His - Ub - RIPK3387 ‑ 518 or ZBP1170 - 355 individually , and heterofibrils formed by indicated mixtures . Samples prepared twice from independent protein

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Representative electron micrographs of homofibrils formed by M451-90, RIPK1497‑583, His-Ub-RIPK3387‑518 or ZBP1170-355 individually, and heterofibrils formed by indicated mixtures.Samples prepared twice from independent protein preparations


Heterofibrils containing mCHERRY - M451 - 90 and YPet ‑ RIPK1497 ‑ 583 C Heterofibrils containing mCHERRY - M451 - 90 and YPet - RIPK3387 ‑ 518 D Heterofibrils containing mCHERRY - M451 - 90 and YPet ‑ ZBP1170 ‑ 355 E Heterofibrils containing mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑

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Heterofibrils containing mCHERRY-M451-90 and YPet‑RIPK1497‑583 C Heterofibrils containing mCHERRY-M451-90 and YPet-RIPK3387‑518 D Heterofibrils containing mCHERRY-M451-90 and YPet‑ZBP1170‑355 E Heterofibrils containing mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑51


Heterofibrils containing mCHERRY - RIPK3387 ‑ 518 and YPet ‑ ZBP1170 ‑ 355 Pearson ' s correlation coefficients measured from two confocal images of the indicated protein mixtures .

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Heterofibrils containing mCHERRY- RIPK3387‑518 and YPet‑ZBP1170‑355 Pearson's correlation coefficients measured from two confocal images of the indicated protein mixtures.


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Heterofibrils containing ECFP-M451-90 with mCHERRY- RIPK1497‑583 and YPet-RIPK3387‑518


STED images of heteromeric fibrils formed by co - assembly of mCHERRY - M451 - 90 and YPet - RIPK3387 ‑

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STED images of heteromeric fibrils formed by co-assembly of mCHERRY-M451-90 and YPet-RIPK3387‑518


SDS AGE analysis of monomeric or assembled forms of YPet - RIPK3387 ‑ 518 and mCHERRY - M451 - 90WT or mCHERRY - M451 - 90AAAA . Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis , as indicated . Assembled samples incubated with 0 or 2 % SDS at RT for 10 min before electrophoresis . Protein components as indicated above each lane .

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SDS AGE analysis of monomeric or assembled forms of YPet-RIPK3387‑518 and mCHERRY-M451-90WT or mCHERRY-M451-90AAAA. Monomeric forms of the proteins maintained in 8 M urea prior to electrophoresis, as indicated. Assembled samples incubated with 0 or 2% SDS at RT for 10 min before electrophoresis. Protein components as indicated above each lane.


SDS AGE analysis of assemblies of mCHERRY - RIPK1497 ‑ 583 and YPet - RIPK3387 ‑ 518 with or without four times molar equivalents of His - Ub - M45 .

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SDS AGE analysis of assemblies of mCHERRY-RIPK1497‑583 and YPet-RIPK3387‑518 with or without four times molar equivalents of His-Ub-M45.


SDS AGE analysis of assemblies of YPet - RIPK3387 ‑ 518 and mCHERRY - ZBP1170 - 355 with or without four times molar equivalents of His - Ub - M45 .

SDS AGE analysis of assemblies of YPet-RIPK3387‑518 and mCHERRY-ZBP1170-355 with or without four times molar equivalents of His-Ub-M45.


Confocal microscopy image of a single fibril composed of YPet - M451 -

Confocal microscopy image of a single fibril composed of YPet-M451-90


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( A ) Supernatants were harvested after 18 h , and concentrations of IL - 6 and IL - 12 were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Mean ± SD of triplicate determinations from > 3 independent experiments are shown .

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A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (A) Supernatants were harvested after 18 h, and concentrations of IL-6 and IL-12 were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Mean ± SD of triplicate determinations from > 3 independent experiments are shown.


A , B BMDCs were treated with OxPAPC or DPPC ( 40 μg / ml ) for 60 min followed by R837 stimulation ( 5 μg / ml ) . ( B ) mRNA was harvested after 2 h , and expression of IL - 6 and IL - 12 was measured by real - time PCR and normalized to G6pdx . Unpaired two - tailed t - test . Data ( mean ± SD ) are representative of three independent experiments .

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A, B BMDCs were treated with OxPAPC or DPPC (40 μg/ml) for 60 min followed by R837 stimulation (5 μg/ml). (B) mRNA was harvested after 2 h, and expression of IL-6 and IL-12 was measured by real-time PCR and normalized to G6pdx. Unpaired two-tailed t-test. Data (mean ± SD) are representative of three independent experiments.


C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC . Periods of oxidation were 24 h for CuSO4 ( 10 μM ) , 48 h for FeSO4 ( 10 μM ) , and 72 h for air . BMDCs were treated for 60 min with lipids prior stimulation with R837 ( 5 μg / ml ) for 18 h . Cytokine concentrations in supernatants were quantified by ELISA . One - way ANOVA adjusted by Dunnett ' s multiple comparisons test . Data represent mean ± SEM of triplicate determinations .

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C Bioactive OxPAPC mixtures were obtained by various oxidation protocols from highly pure PAPC. Periods of oxidation were 24 h for CuSO4 (10 μM), 48 h for FeSO4 (10 μM), and 72 h for air. BMDCs were treated for 60 min with lipids prior stimulation with R837 (5 μg/ml) for 18 h. Cytokine concentrations in supernatants were quantified by ELISA. One-way ANOVA adjusted by Dunnett's multiple comparisons test. Data represent mean ± SEM of triplicate determinations.


D PAPC ( 40 μg / ml ) oxidized with CuSO4 ( 10 μM ) for the indicated times was used for treatment of BMDCs prior to R837 ( 5 μg / ml ) stimulation and measurement of IL - 12 secretion . Mean ± SEM of triplicate determinations from three different oxidation series are shown .

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D PAPC (40 μg/ml) oxidized with CuSO4 (10 μM) for the indicated times was used for treatment of BMDCs prior to R837 (5 μg/ml) stimulation and measurement of IL-12 secretion. Mean ± SEM of triplicate determinations from three different oxidation series are shown.


E , F Treatment of BMDCs with the indicated concentrations of CuSO4 - oxidized PAPC suppressed IL - 6 ( E ) and IL - 12 ( F ) secretion triggered by a variety of different TLR agonists . After OxPAPC treatment for 60 min , BMDCs were stimulated for 18 h with LTA ( 500 ng / ml ) , Poly I : C ( 50 μg / ml ) , LPS ( 10 ng / ml ) , R837 ( 5 μg / ml ) , and CpG ( 100 nM ) . Cytokine concentrations in the supernatant were quantified by ELISA . Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one - way ANOVA adjusted by Dunnett ' s multiple comparisons test .

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E, F Treatment of BMDCs with the indicated concentrations of CuSO4-oxidized PAPC suppressed IL-6 (E) and IL-12 (F) secretion triggered by a variety of different TLR agonists. After OxPAPC treatment for 60 min, BMDCs were stimulated for 18 h with LTA (500 ng/ml), Poly I:C (50 μg/ml), LPS (10 ng/ml), R837 (5 μg/ml), and CpG (100 nM). Cytokine concentrations in the supernatant were quantified by ELISA. Data are shown as mean ± SD of triplicate determinations from three independent experiments and were analyzed by one-way ANOVA adjusted by Dunnett's multiple comparisons test.


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 .

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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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 .

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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 .

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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 .

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

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 ) .

(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 .

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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 .

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.