B Wild - type or Cuedc2 - / - mice were subjected to a heart ischemia for 30 - minute in vivo , followed by reperfusion for indicated times . was extracted from at risk of left ventricle subjected to immunoblot .

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B Wild-type or Cuedc2-/- mice were subjected to a heart ischemia for 30-minute in vivo, followed by reperfusion for the indicated times. Protein was extracted from the area at risk of left ventricle and subjected to immunoblot analysis.


C GPX1 level was plotted using immunohistochemical scores as described in methods . ( left ) representative from immunohistochemistry for GPX1 in tissues from who suffered acute myocardial infarctions . ( right ) plot of GPX1 scores in each BZ DZ ( n = 9 ) . DZ , distant ; BZ , border zone . Scale bars , 250 μm ( left ) . * p = 0 . 0039 .

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C GPX1 protein level was plotted using the immunohistochemical scores as described in methods. (left) representative images from immunohistochemistry staining for GPX1 in tissues from patients who suffered acute myocardial infarctions. (right) plot of GPX1 scores in each BZ and DZ (n = 9 patients). DZ, distant area; BZ, border zone. Scale bars, 250 μm (left). * p = 0.0039.


D neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 ( 1 mM ) for 3 hours . viability was detected by FACS . * p = 0 . 0001 ; * * p = 0 . 0275 , n = 3 wells per group . NS , not significant .

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D Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were treated with H2O2 (1 mM) for 3 hours. Cell viability was detected by FACS. * p = 0.0001; ** p = 0.0275, n = 3 wells per group. NS, not significant.


E neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA , efficiency of GPX1 knockdown was tested by immunoblotting .

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E Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA, and the efficiency of GPX1 knockdown was tested by immunoblotting.


F neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 ( 1 mM ) for 30 minutes then stained with 5 μM CellROX ® Deep Red Reagent analyzed by flow cytometry . * p = 0 . 0056 ; * * p = 0 . 0218 , n = 3 wells per group , n = 3 wells per group . NS , not significant .

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F Primary neonatal mousecardiomyocytes stably expressing control or GPX1 shRNA by lentivirus infection were subjected to H2O2 (1 mM) for 30 minutes and then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. * p = 0.0056; ** p = 0.0218, n = 3 wells per group, n = 3 wells per group. NS, not significant.


A Lysates from MEF - WT or MEF - Cuedc2 - / - treated with 20μM cycloheximide ( CHX ) for indicated times were subjected to immunoblotting ( left ) . Relative GPX1 were quantified by densitometry ( right ) .

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A Lysates from MEF-WT or MEF-Cuedc2-/- cells treated with 20μM cycloheximide (CHX) for the indicated times were subjected to immunoblotting (left). Relative GPX1 levels were quantified by densitometry (right).


B MEF - Cuedc2 - / - were transfected with increasing amounts of FLAG - CUEDC2 ( mouse ) plasmids ( 1 μg 2 μg ) . At 24 hours after transfection , were treated with proteasome inhibitor MG - 132 ( 10 μM ) . were cultured for additional 6 hours subjected to immunoblotting .

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B MEF-Cuedc2-/- cells were transfected with increasing amounts of FLAG-CUEDC2 (mouse) plasmids (1 μg and 2 μg). At 24 hours after transfection, the cells were treated with the proteasome inhibitor MG-132 (10 μM). Cells were cultured for additional 6 hours and subjected to immunoblotting.


C HEK293T were transfected with FLAG - GPX1 , HA - CUEDC2 , Myc - ubiquitin plasmids as indicated treated with MG - 132 ( 10 µM ) for 6 hours before harvest . lysates were immunoprecipitated ( IP ) with anti - FLAG ( M2 ) . immunoprecipitates were analyzed by western blot using an anti - GPX1 antibody . Whole - lysates ( WCL ) were analyzed by Western blots with anti - FLAG or anti - HA antibody to determine of Flag - GPX1 HA - CUEDC2 .

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C HEK293T cells were transfected with the FLAG-GPX1, HA-CUEDC2, and Myc-ubiquitin plasmids as indicated and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-FLAG (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-FLAG or anti-HA antibody to determine the protein of Flag-GPX1 and HA-CUEDC2.


D heart tissue lysates from wide - type Cuedc2 - / - mice were incubated with IgG or anti - GPX1 antibody , then captured on G - Sepharose beads . immunoprecipitates were analyzed by immunoblotting with an anti - ubiquitin antibody .

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D The heart tissue lysates from wide-type and Cuedc2-/- mice were incubated with IgG or anti-GPX1 antibody, and then captured on protein G-Sepharose beads. The immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody.


E HEK293T were transfected with Flag - CUEDC2 or Flag - CUEDC2 △ CUE ( deletion of CUE domain of CUEDC2 ) , treated with MG - 132 ( 10 µM ) for 6 hours before harvest . lysates were immunoprecipitated ( IP ) with anti - Flag ( M2 ) . immunoprecipitates were analyzed by western blot using an anti - GPX1 antibody . Whole - lysates ( WCL ) were analyzed by Western blots with anti - Flag or anti - GPX1 antibody to determine of Flag - CUEDC2 GPX1 . v : vector .

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E HEK293T cells were transfected with the Flag-CUEDC2 or Flag-CUEDC2△CUE (deletion of the CUE domain of CUEDC2), and treated with MG-132 (10 µM) for 6 hours before harvest. Cell lysates were immunoprecipitated (IP) with anti-Flag (M2). The immunoprecipitates were analyzed by western blot using an anti-GPX1 antibody. Whole-cell lysates (WCL) were analyzed by Western blots with anti-Flag or anti-GPX1 antibody to determine the protein of Flag-CUEDC2 and GPX1. v: vector.


A HEK293 were transfected with increasing amounts of Flag - TRIM33 expression vectors 48 hours after transfection , lysates were subjected to immunoblotting ( IB ) using antibodies as indicated .

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A HEK293 cells were transfected with increasing amounts of Flag-TRIM33 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.


B HEK293 were transfected with increasing amounts of Flag - FBXW7 expression vectors 48 hours after transfection , lysates were subjected to immunoblotting ( IB ) .

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B HEK293 cells were transfected with increasing amounts of Flag-FBXW7 expression vectors and 48 hours after transfection, cell lysates were subjected to immunoblotting (IB).


C HEK293T were transfected as indicated , at 18 h after transfection , were treated with proteasome inhibitor MG132 ( 20μM ) for 6 hours then harvested . lysates were immunoprecipitated by Flag - antibody ( M2 beads ) Ubiquitin - conjugated GPX1 was detected by Western blotting with anti - Myc antibody .

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C HEK293T cells were transfected as indicated, at 18 h after transfection, the cells were treated with the proteasome inhibitor MG132 (20μM) for 6 hours and then harvested. Cell lysates were immunoprecipitated by Flag-antibody (M2 beads) and Ubiquitin-conjugated GPX1 was detected by Western blotting with anti-Myc antibody.


D HEK293 were transfected with either control or CUEDC2 siRNA increasing amounts of Flag - TRIM33 expression vectors , lysates were subjected to immunoblotting ( IB ) using antibodies as indicated .

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D HEK293 cells were transfected with either control or CUEDC2 siRNA and increasing amounts of Flag-TRIM33 expression vectors, cell lysates were subjected to immunoblotting (IB) using antibodies as indicated.


A CUEDC2 ablation reduced I / R injury , which was induced by 30 minutes of ischemia followed by 24 hours reperfusion . AAR , at risk ; LV , left ventricular , IRI , of I / R injury . ratios of AAR to LV , IRI to AAR IRI to LV are shown . * p < 0 . 0001 compared with WT , n = 15 mice per group . ( top panel ) Representative cross - sectional slices derived from hearts stained by TTC . of I / R injury is indicated in pink .

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A CUEDC2 ablation reduced I/R injury, which was induced by 30 minutes of ischemia followed by 24 hours reperfusion. AAR, area at risk; LV, left ventricular area, IRI, area of I/R injury. The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. * p < 0.0001 compared with WT, n = 15 mice per group. (top panel) Representative cross-sectional slices derived from the hearts stained by TTC. Area of I/R injury is indicated in light pink.


B Representative of ventricular myocardium sections from WT Cuedc2 - / - mice exposed to sham operation or I / R injury ( 2 hours after onset of reperfusion following 30 - min ischemia ) . Green , TUNEL - positive myocytenuclei ; red , Troponin I - stained cardiomyocytes ; blue , DAPI - stained nuclei . Scale bar , 50 μm .

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B Representative images of ventricular myocardium sections from WT and Cuedc2-/- mice exposed to sham operation or I/R injury (2 hours after the onset of reperfusion following 30-min ischemia). Green, TUNEL-positive myocytenuclei; red, Troponin I-stained cardiomyocytes; blue, DAPI-stained nuclei. Scale bar, 50 μm.


C Quantitative of apoptosis . * p < 0 . 0001 , n = 8 mice per group .

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C Quantitative analysis of apoptosis. * p < 0.0001, n = 8 mice per group.


D SerumLDH in sham - operated mice or those subjected to ischemia a 4 - hour reperfusion . Data from 3 separate experiments was analyzed by one - way of variance . * p < 0 . 0001 , n = 13 mice per group .

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D SerumLDH levels in sham-operated mice or those subjected to ischemia and a 4-hour reperfusion. Data from 3 separate experiments was analyzed by one-way analysis of variance. * p < 0.0001, n = 13 mice per group.


E Echocardiography of left ventricular dimensions cardiac function at 1 week post - I / R in mice . * * p < 0 . 0001 , n = 15 mice in sham - operated group n = 10 mice in I / R group .

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E Echocardiography analysis of left ventricular dimensions and cardiac function at 1 week post-I/R in mice. ** p < 0.0001, n = 15 mice in the sham-operated group and n = 10 mice in the I/R group.


A Immunoblotting for CUEDC2 level . 4 after tail - vein injection of rAAV9 - CUEDC2 , indicated mice tissues were harvested subjected to immunoblotting to detect CUEDC2 level . Ctl : heart of rAAV9 - GFP injected mice .

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A Immunoblotting for CUEDC2 protein level. 4 weeks after tail-vein injection of rAAV9-CUEDC2, indicated mice tissues were harvested and subjected to immunoblotting to detect CUEDC2 protein level. Ctl: heart of rAAV9-GFP injected mice.


B were injected by rAAV9 - CUEDC2 in a different amount of viral genomes ( VG ) . Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction . Tubulin level was examined for normalization purposes .

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B Animals were injected by rAAV9-CUEDC2 in a different amount of viral genomes (VG). Heart tissues were then analyzed by immunoblotting to detect CUEDC2 transduction. Tubulin protein level was examined for normalization purposes.


C ratios of AAR to LV , IRI to AAR IRI to LV are shown . n = 10 mice in WT + rAAV - GFP group Cuedc2 - / - + rAAV - GFP group ; n = 12 mice in Cuedc2 - / - + rAAV - CUEDC2 ( 1 × 1010 VG ) group Cuedc2 - / - + rAAV - CUEDC2 ( 1 × 1011 VG ) group . * p = 0 . 0055 , * * p = 0 . 0146 , * * * p = 0 . 0200 , * * * * p = 0 . 0377 .

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C The ratios of AAR to LV, IRI to AAR and IRI to LV are shown. n = 10 mice in WT + rAAV-GFP group and Cuedc2-/- + rAAV-GFP group; n = 12 mice in Cuedc2-/- + rAAV-CUEDC2 (1×1010 VG) group and Cuedc2-/- + rAAV-CUEDC2 (1×1011 VG) group. * p = 0.0055, ** p = 0.0146, *** p = 0.0200, **** p = 0.0377.


D Effect of rAAV9 - CUEDC2 treatment on echocardiography indices of left ventricle function recovery post - I / R measured by fractional shortening ( FS ) ejection fraction ( EF ) . WT + rAAV - GFP ( n = 10 ) ; Cuedc2 - / - + rAAV - GFP ( n = 10 ) ; Cuedc2 - / - + rAAV - CUEDC2 ( Low dose ) ( n = 12 ) ; Cuedc2 - / - + rAAV - CUEDC2 ( High dose ) ( n = 12 ) . * * p < 0 . 0001 .

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D Effect of rAAV9-CUEDC2 treatment on echocardiography indices of left ventricle function recovery post-I/R measured by fractional shortening (FS) and ejection fraction (EF). WT+ rAAV-GFP (n = 10); Cuedc2-/- + rAAV-GFP (n = 10); Cuedc2-/- + rAAV-CUEDC2 (Low dose) (n = 12); Cuedc2-/- + rAAV-CUEDC2 (High dose) (n = 12). ** p < 0.0001.


A HPLC detection of a superoxide probe oxidized dihydroethidium ( DHE ) product in young ( 8 - week - old ) old mouse ( 20 - month - old ) heart tissue , 2 - hydroxyethidium ( EOH ) , a specific product for superoxide anion radical . * p = 0 . 0087 , n = 5 in each group .

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A HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in young (8-week-old) and old mouse (20-month-old) heart tissue, 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. * p = 0.0087, n = 5 in each group.


B HPLC detection of ethidium ( E ) , oxidized by other reactive oxygen species such as H2O2 ( mainly ) ONOO . * p = 0 . 0021 , n = 5 in each group .

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B HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. * p = 0.0021, n = 5 in each group.


C Comparison of 8 - hydroxy - 2 ' - deoxyguanosine ( 8 - OHdG ) in young old mice . * p = 0 . 0164 , n = 6 mice per group .

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C Comparison of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in young and old mice. * p = 0.0164, n = 6 mice per group.


D Comparison of heart / ( HW / BW ) in WT Cuedc2 - / - mice . * * p = 0 . 036 , n = 15 in young WT Cuedc2 - / - mice , n = 10 in old WT mice n = 12 in old Cuedc2 - / - mice .

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D Comparison of heart weight / body weight (HW/BW) in WT and Cuedc2-/- mice. ** p = 0.036, n = 15 in young WT and Cuedc2-/- mice, n = 10 in old WT mice and n = 12 in old Cuedc2-/- mice.


E Comparison of left ventricle function in WT Cuedc2 - / - mice by transthoracic echocardiography . * p = 0 . 0384 , * * p = 0 . 0389 . n = 15 in young WT Cuedc2 - / - mice , n = 10 in old WT mice n = 12 in old Cuedc2 - / - mice .

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E Comparison of the left ventricle function in WT and Cuedc2-/- mice by transthoracic echocardiography. *p = 0.0384, **p = 0.0389. n = 15 in young WT and Cuedc2-/- mice, n = 10 in old WT mice and n = 12 in old Cuedc2-/- mice.


F Left ventriclecardiomyocytes apoptosis was tested by TUNEL . * p = 0 . 0017 , n = 5 in each group .

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F Left ventriclecardiomyocytes apoptosis was tested by TUNEL staining. *p = 0.0017, n = 5 in each group.


G Left ventricle myocardial fibrosis was tested by Masson in WT Cuedc2 - / - mice . represent results were shown in ( G ) quantitative of myocardial fibrosis was shown in ( H ) . * p = 0 . 0177 , n = 5 in each group .

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G Left ventricle myocardial fibrosis was tested by Masson staining in WT and Cuedc2-/- mice. The represent results were shown in (G) and the quantitative analysis of myocardial fibrosis was shown in (H). *p = 0.0177, n = 5 in each group.


Volcano plot representation of AFG3L2E408Q - FLAG interaction partners . Mitochondria isolated from Flp - In HEK293T - REx expressing AFG3L2E408Q - FLAG were subjected to immunoprecipitation . Co - purifying were identified by quantitative mass spectrometry ( MS ) ( n = 5 independent experiments ) followed by an unpaired two - sided t - permutation - FDR estimation to correct for multiple testing ( FDR < 0 . 05 . Significantly enriched MitoCarta 3 . 0 are colored in red , gene names indicate previously verified AFG3L2 interactors TMBIM5 ( GHITM , MICS1 ) .

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Volcano plot representation of AFG3L2E408Q-FLAG interaction partners. Mitochondria isolated from Flp-In HEK293T-REx cells expressing AFG3L2E408Q-FLAG were subjected to immunoprecipitation. Co-purifying proteins were identified by quantitative mass spectrometry (MS) (n=5 independent experiments) followed by an unpaired two-sided t-test and permutation-based FDR estimation to correct for multiple testing (FDR<0.05. Significantly enriched MitoCarta 3.0 proteins are colored in red, gene names indicate previously verified AFG3L2 interactors and TMBIM5 (GHITM, MICS1).


Representative immunoblot of HeLa wildtype ( WT ) transfected with scrambled siRNA ( control ) or siRNA targeting TMBIM5 treated with indicated drugs for 16 h . Control ( 0 . 1 % DMSO ) , actinomycin D ( 1 µM ) , venetoclax ( 1 µM ) , A - 1155463 ( 1 µM ) , staurosporine ( 1 µM ) , thapsigargin ( 2 µM ) . n = 3 independent experiments .

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Representative immunoblot of HeLa wildtype (WT) cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5 and treated with the indicated drugs for 16 h. Control (0.1% DMSO), actinomycin D (1 µM), venetoclax (1 µM), A-1155463 (1 µM), staurosporine (1 µM), thapsigargin (2 µM). n=3 independent experiments.


Representative immunoblot of HeLa WT TMBIM5 - / - transfected with scrambled siRNA ( control ) or siRNA targeting MCU for 72 h . Where indicated samples were treated with staurosporine ( STS ; 1 µM ) for 16 h . ( n = 3 independent experiments ) .

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Representative immunoblot of HeLa WT and TMBIM5-/- cells transfected with scrambled siRNA (control) or siRNA targeting MCU for 72 h. Where indicated samples were treated with staurosporine (STS; 1 µM) for 16 h. (n=3 independent experiments).


Viability of WT TMBIM5 - / - ( depleted of MCU when indicated ) after incubation with staurosporine ( STS ; 0 . 1 µM ) for 16 h . viability was assessed cytofluorimetrically monitoring NucView488 RedDot2 expressed as of . n = 3 independent experiments ; two - t - ; mean ± SD . A p - value of < 0 . 05 was considered statistically significant .

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Viability of WT and TMBIM5-/- cells (depleted of MCU when indicated) after incubation with staurosporine (STS; 0.1 µM) for 16 h. Cell viability was assessed cytofluorimetrically monitoring NucView488 and RedDot2 staining and expressed as percentage of all cells. n=3 independent experiments; two-tailed t-test; mean ±SD. A p-value of <0.05 was considered statistically significant.


WT TMBIM5 - / - HeLa were transfected with indicated scrambled siRNA ( control ) siRNA targeting MCU for 48 h incubated with staurosporine ( 0 . 1 µM ) for 16 h in presence of Z - VAD - FMK ( 50 µM ) epoxomicin ( 1 µM ) to prevent apoptosis . were subjected to immunofluorescence with antibodies directed against cytochrome c ( green ) TOMM20 ( magenta ) . * with cytochrome c . ( n = 3 independent experiments ; number of > 150 each experiment ) .

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WT and TMBIM5-/- HeLa cells were transfected with the indicated scrambled siRNA (control) and siRNA targeting MCU for 48 h and incubated with staurosporine (0.1 µM) for 16 h in the presence of Z-VAD-FMK (50 µM) and epoxomicin (1 µM) to prevent apoptosis. Cells were subjected to immunofluorescence analysis with antibodies directed against cytochrome c (green) and TOMM20 (magenta). *Cells with cytosolic cytochrome c. (n=3 independent experiments; number of cells > 150 each experiment).


Calcium retention capacity ( CRC ) was assessed in isolated mitochondria of HEK293T WT transfected with indicated siRNA . experiment was performed in sucrose - medium containing respiratory substrates - impermeable Ca2 + sensor Ca2 + Green - 5N . Ca2 + Green - 5N was monitored following repeated addition of Ca2 + pulses ( 10 µM ) . Data show representative traces ( 2 independent experiments run in triplicate each condition ) .

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Calcium retention capacity (CRC) was assessed in isolated mitochondria of HEK293T WT cells transfected with the indicated siRNA. The experiment was performed in sucrose-based medium containing respiratory substrates and the membrane-impermeable Ca2+ sensor Ca2+ Green-5N. Ca2+ Green-5N fluorescence was monitored following repeated addition of Ca2+ pulses (10 µM). Data show representative traces (2 independent experiments run in triplicate each condition).


( F ) F / F0 quantifications , where F0 is average of first ten seconds F is peak measured after thapsigargin ( 2 µM ) administration ( left panel ) . right shows mtRED - GECO traces of HeLa transfected with scrambled siRNA ( control ) siRNA targeting TMBIM5 . Each measurement was performed in at least 10 from 6 different preparations . Mean ± SD ; two - t - . A p - value of < 0 . 05 was considered statistically significant .

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Fluorescence (F) F/F0 ratio quantifications, where F0 is the average of the first ten seconds and F is the fluorescence peak measured after thapsigargin (2 µM) administration (left panel). The right shows mtRED-GECO ratio traces of HeLa cells transfected with scrambled siRNA (control) and siRNA targeting TMBIM5. Each measurement was performed in at least 10 cells from 6 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Matrix pH was measured with Sypher3smito in wildtye ( WT ) HeLa transfected with scrambled siRNA ( control ) or siRNA targeting TMBIM5 for 72 h . probe was calibrated in KRB buffer by stepwise addition / removal of permeant weak acid . Each measurement was performed in at least 80 from 7 different preparations . Mean ± SD ; two - t - . A p - value of < 0 . 05 was considered statistically significant .

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Matrix pH was measured with Sypher3smito in wildtye (WT) HeLa cells transfected with scrambled siRNA (control) or siRNA targeting TMBIM5 for 72 h. The probe was calibrated in KRB buffer by the stepwise addition/removal of permeant weak acid. Each measurement was performed in at least 80 cells from 7 different preparations. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Mitochondrial potential was monitored with TMRM in WT TMBIM5 - / - HeLa . was calculated as to value upon CCCP addition ( 15 µM ) . Values are expressed as mean ± SD relative to control . Each measurement was performed in triplicate from 4 different preparations .

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Mitochondrial membrane potential was monitored with TMRM in WT and TMBIM5-/- HeLa cells. Fluorescent intensity was calculated as ratio to the value upon CCCP addition (15 µM). Values are expressed as mean ±SD relative to control. Each measurement was performed in triplicate from 4 different preparations.


Matrix pH was measured with Sypher3smito in WT HeLa transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h . of absorbance at 488 nm 405 nm is shown in arbitrary units ( a . u . ) . Each measurement was performed in at least 20 from 3 different preparations . Representative immunoblot of WT HeLa transfected with TMBIM5 or TMBIM5D294K D325K for 24 h ( right panel ) . Mean ± SD ; two - t - . A p - value of < 0 . 05 was considered statistically significant .

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Matrix pH was measured with Sypher3smito in WT HeLa cells transiently expressing TMBIM5 or TMBIM5D294K D325K for 24 h. The ratio of absorbance at 488 nm and 405 nm is shown in arbitrary units (a.u.). Each measurement was performed in at least 20 cells from 3 different preparations. Representative immunoblot of WT HeLa cells transfected with TMBIM5 or TMBIM5D294K D325K for 24 h (right panel). Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Liposome . Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura - 2 . CaCl2 was added later at of 100 µM . Rmax was obtained at end of experiment after addition of ionomycin ( 10 µM ) . Data are expressed as mean of R / Rmax , where R is peak after CaCl2 addition . Where indicated GdCl3 ( 100 µM ) was added 10 min before experiment . n = 3 independent experiments , shown as black , grey dots . Mean ± SD ; two - t - . A p - value of < 0 . 05 was considered statistically significant . Liposome ( as in E ) . Slope was calculated in 100 s after 100 µM CaCl2 addition as expressed as mean ± SD . n = 3 independent experiments , shown as black , grey dots . Two - t - . A p - value of < 0 . 05 was considered statistically significant .

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Liposome assay. Recombinant TMBIM5 was reconstituted in liposomes containing 100 µM Fura-2. CaCl2 was added later at the concentration of 100 µM. Rmax was obtained at the end of the experiment after the addition of ionomycin (10 µM). Data are expressed as mean of R/Rmax, where R is the peak after CaCl2 addition. Where indicated GdCl3 (100 µM) was added 10 min before the experiment. n=3 independent experiments, shown as black, white and grey dots. Mean ±SD; two-tailed t-test. A p-value of <0.05 was considered statistically significant. Liposome assay (as in E). Slope was calculated in the 100 s after 100 µM CaCl2 addition as expressed as mean ±SD. n=3 independent experiments, shown as black, white and grey dots. Two-tailed t-test. A p-value of <0.05 was considered statistically significant.


Alterations in mitochondrial proteome in WT TMBIM5 - / - HeLa upon depletion of AFG3L2 . WT TMBIM5 - / - HeLa were transfected with scrambled siRNA ( control ) or siRNA targeting AFG3L2 for 48 h . , which were downregulated in absence of TMBIM5 whose steady state was altered upon depletion of AFG3L2 , were identified using a two - way ANOVA . An interaction p - value of 0 . 01 was used as a cutoff . Log2 - transformed LFQ intensities of MitoCarta 3 . 0 were Z - Score normalized visualized in a hierarchical cluster ( Euclidean , complete method ) . dendrogram is omitted . accumulating upon depletion of AFG3L2 ( cluster 1 , C1 ) whose steady state are decreased upon depletion of AFG3L2 ( cluster 2 , C2 ) are shown .

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Alterations in mitochondrial proteome in WT and TMBIM5-/- HeLa cells upon depletion of AFG3L2. WT and TMBIM5-/- HeLa cells were transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Proteins, which were downregulated in the absence of TMBIM5 and whose steady state levels was altered upon depletion of AFG3L2, were identified using a two-way ANOVA. An interaction p-value of 0.01 was used as a cutoff. Log2-transformed LFQ intensities of MitoCarta 3.0 proteins were Z-Score normalized and visualized in a hierarchical cluster analysis (Euclidean distance, complete method). The dendrogram is omitted. Proteins accumulating upon depletion of AFG3L2 (cluster 1, C1) and proteins whose steady state levels are decreased upon depletion of AFG3L2 (cluster 2, C2) are shown.


Representative immunoblot of TMBIM5 - / - HeLa transfected with scrambled siRNA ( control ) or siRNA targeting AFG3L2 for 48 h . Samples were treated with synthesis inhibitor cycloheximide ( CHX ; 10 μg / ml ) collected at indicated time points ( n = 3 independent experiments ) .

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Representative immunoblot of TMBIM5-/- HeLa cells transfected with scrambled siRNA (control) or siRNA targeting AFG3L2 for 48 h. Samples were treated with the protein synthesis inhibitor cycloheximide (CHX; 10 μg/ml) and collected at the indicated time points (n=3 independent experiments).


Volcano plot of mitochondrial ( MitoCarta 3 . 0 ) changes in WT HeLa treated with oligomycin ( 10 μM for 16 h ) when compared to untreated . Before MitoCarta 3 . 0 - filtering , significantly altered were identified using a two - sided t - followed by permutation - FDR estimation to 0 . 05 are colored in blue ( n = 5 independent experiments ) .

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Volcano plot of mitochondrial protein (MitoCarta 3.0) changes in WT HeLa cells treated with oligomycin (10 μM for 16 h) when compared to untreated cells. Before MitoCarta 3.0-filtering, significantly altered proteins were identified using a two-sided t-test followed by permutation-based FDR estimation to 0.05 and are colored in blue (n=5 independent experiments).


Representative immunoblot of WT HeLa treated with indicated drugs for 16 h . Antimycin A ( AA ; 10 μM ) , piericidin A ( 10 μM ) oligomycin ( 10 μM ) .

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Representative immunoblot of WT HeLa cells treated with the indicated drugs for 16 h. Antimycin A (AA; 10 μM), piericidin A (10 μM) and oligomycin (10 μM).


Representative immunoblot of WT HeLa transfected with scrambled siRNA ( control ) siRNA targeting AFG3L2 for 48 h , treated when indicated with oligomycin ( 10 μM ) for 16 h . of TMBIM5 is shown on right panel . TMBIM5 in untreated WT was set to 1 ( n = 3 independent experiments ; mean ± SD ) .

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Representative immunoblot of WT HeLa cells transfected with scrambled siRNA (control) and siRNA targeting AFG3L2 for 48 h, treated when indicated with oligomycin (10 μM) for 16 h. Quantification of TMBIM5 levels is shown on the right panel. TMBIM5 levels in untreated WT cells was set to 1 (n=3 independent experiments; mean ±SD).


TMBIM5 abundance determined by mass spectrometry in WT HeLa treated with oligomycin ( 10 µM ; 16 h ) depleted of AFG3L2 when indicated . n = 5 independent experiments . P - value was calculated using a two - sided unpaired t - . central band of each box is median value , box defines 25th ( lower ) 75th ( higher ) quantile . whiskers represent minimum maximum value in data excluding outliers ( greater than 1 . 5 * inter quantile range to median - no outliers found here ) .

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TMBIM5 abundance determined by mass spectrometry in WT HeLa cells treated with oligomycin (10 µM; 16 h) and depleted of AFG3L2 when indicated. n=5 independent experiments. P-value was calculated using a two-sided unpaired t-test. The central band of each box is the median value, and the box defines the 25th (lower) and 75th (higher) quantile. The whiskers represent the minimum and maximum value in the data excluding outliers (greater than 1.5 * inter quantile range distance to median - no outliers found here).


Zoom in to selected clusters of hierarchical clustering of Z - Score normalized log2 LFQ intensities . that were significantly ( FDR < 0 . 05 ) accumulated between HeLa WT TMBIM5 - / - after oligomycin treatment ( 10 µM ) for 16 h were clustered using Euclidean complete method on features . figure shows identified cluster C4 C5 with gene names MitoCarta 3 . 0 localization annotations . row dendrogram is omitted .

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Zoom in to selected clusters of hierarchical clustering of Z-Score normalized log2 LFQ intensities. Proteins that were significantly (FDR<0.05) accumulated between HeLa WT and TMBIM5-/- cells after oligomycin treatment (10 µM) for 16 h were clustered using Euclidean distance and the complete method on protein features. The figure shows identified cluster C4 and C5 with gene names and MitoCarta 3.0 localization annotations. The row dendrogram is omitted.


A . AICD was induced in hPBT as described in Materials methods . Apoptotic were detected at indicated times after AICD induction by flow cytometry as AnnexinV / PI double positive between AICD Ctrl values obtained are shown . Data represent mean ± SE of six independent experiments .

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A. AICD was induced in hPBT cells as described in Materials and methods. Apoptotic cells were detected at indicated times after AICD induction by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown. Data represent mean ± SE of six independent experiments.


B . AICD was induced in Jurkat as described in Materials methods . Apoptotic were detected as in ( A ) . Data represent mean ± SE of five independent experiments .

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B. AICD was induced in Jurkat cells as described in Materials and methods. Apoptotic cells were detected as in (A). Data represent mean ± SE of five independent experiments.


C . hPBT were transfected with mtYFP after 24h AICD was induced . Representative reconstructions of confocal z - stacks of mtYFP 30 ' after AICD induction is shown . Scale bar , 5μm .

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C. hPBT cells were transfected with mtYFP and after 24h AICD was induced. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 30' after AICD induction is shown. Scale bar, 5μm.


D . Morphometric of mitochondrial shape of treated as in ( B ) . Data represent mean ± SE of five independent experiments ( n = 30 per condition in each experiment ) .

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D. Morphometric analysis of mitochondrial shape of cells treated as in (B). Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


E . Jurkat were transfected with mtYFP , 24h after AICD induction were fixed . Representative reconstructions of confocal z - stacks of mtYFP 24h after AICD induction are shown . Scale bar , 5μm . F . Morphometric of mitochondrial shape in treated as in ( E ) is shown . Data represent mean ± SE of five independent experiments ( n = 30 per condition in each experiment ) .

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E. Jurkat cells were transfected with mtYFP, 24h after AICD induction cells were fixed. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.F. Morphometric analysis of mitochondrial shape in cells treated as in (E) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


G . Representative electron of hPBT treated as indicated . 30min after AICD induction were fixed processed for electron microscopy . Scale bar , 2μm . H . 24h after AICD induction Jurkat were fixed processed for electron microscopy . Representative electron of treated as indicated are shown . In EM arrowheads point to autophagic vesicles ( late autophagosomes or most likely autophagolysosomes , Aφ ) ; black arrows point to mitochondria ( mito ) with remodelled cristae , specifically touching a representative disorganized crista . Scale bar , 2μm .

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G. Representative electron micrographs of hPBT cells treated as indicated. 30min after AICD induction cells were fixed and processed for electron microscopy. Scale bar, 2μm.H. 24h after AICD induction Jurkat cells were fixed and processed for electron microscopy. Representative electron micrographs of cells treated as indicated are shown. In EM images white arrowheads point to autophagic vesicles (late autophagosomes or most likely autophagolysosomes, Aφ); black arrows point to mitochondria (mito) with remodelled cristae, specifically touching a representative disorganized crista. Scale bar, 2μm.


I . Representative confocal microscopy of hPBT transfected with mtYFP , fixed 30 ' after AICD induction immunostained with an anti - cytochrome c antibody ( red ) . J . Cytochrome c localization index was calculated from 30 randomly selected treated as in ( I ) . Data represent mean ± SE of three independent experiments . K . Representative confocal of Jurkat transfected with mtYFP , fixed at 32h after AICD induction immunostained with an anti - cytochrome c antibody . L . Cytochrome c localization index was calculated from 30 randomly selected per condition . Data represent mean ± SE of five independent experiments .

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I. Representative confocal microscopy images of hPBT cells transfected with mtYFP, fixed 30' after AICD induction and immunostained with an anti-cytochrome c antibody (red).J. Cytochrome c localization index was calculated from 30 randomly selected cells treated as in (I). Data represent mean ± SE of three independent experiments.K. Representative confocal images of Jurkat cells transfected with mtYFP, fixed at 32h after AICD induction and immunostained with an anti-cytochrome c antibody.L. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of five independent experiments.


A . samples ( 20 μg ) from Jurkat treated as indicated were separated by SDS - PAGEand immunoblotted with indicated antibodies . WB are representative of at least three independent experiments .

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A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGEand immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.


B . Jurkat were transfected with pE - GFP indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as of GFP - positive , annexin - V - PE - negative . Data represent mean ± SE of five independent experiments .

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B. Jurkat cells were transfected with pE-GFP and the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of five independent experiments.


C . Jurkat were transfected as indicated . Representative reconstructions of confocal z - stacks of mtYFP 24h after AICD induction are shown . Scale bar , 5μm . D . Morphometric of mitochondrial shape of transfected as in ( C ) is shown . Data represent mean ± SE of five independent experiments ( n = 30 per condition in each experiment ) .

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C. Jurkat cells were transfected as indicated. Representative reconstructions of confocal z-stacks of the mtYFP fluorescence 24h after AICD induction are shown. Scale bar, 5μm.D. Morphometric analysis of mitochondrial shape of cells transfected as in (C) is shown. Data represent mean ± SE of five independent experiments (n = 30 cells per condition in each experiment).


E . AICD was induced in Jurkat over expressing pE - GFP indicated plasmids sorted for GFP positive . 24h after AICD induction were fixed processed for electron microscopy . Representative electron of randomly selected mitochondria from treated as indicated are shown . arrow points to tight cristae structure , black arrow points to remodelled cristae to a swollen cristae junction . Scale bar , 0 . 2μm . F . Morphometric of cristae width in transfected as in ( E ) .

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E. AICD was induced in Jurkat cells over expressing pE-GFP and the indicated plasmids and sorted for GFP positive fluorescence. 24h after AICD induction cells were fixed and processed for electron microscopy. Representative electron micrographs of randomly selected mitochondria from cells treated as indicated are shown. White arrow points to tight cristae structure, black arrow points to remodelled cristae and to a swollen cristae junction. Scale bar, 0.2μm.F. Morphometric analysis of cristae width in cells transfected as in (E).


G . Representative confocal microscopy of transfected as indicated , fixed 32h after AICD induction immunostained with an anti - cytochrome c antibody ( red ) . H . Cytochrome c localization index was calculated from 30 randomly selected ( per condition ) transfected as in ( G ) . Data represent mean ± SE of five independent experiments .

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G. Representative confocal microscopy images of cells transfected as indicated, fixed 32h after AICD induction and immunostained with an anti-cytochrome c antibody (red).H. Cytochrome c localization index was calculated from 30 randomly selected cells (per condition) transfected as in (G). Data represent mean ± SE of five independent experiments.


A . Representative confocal microscopy of transfected GFP - LC3 Parkin - Cherry , treated as indicated , fixed 24h after AICD induction immunostained with anti - TOM20 antibody are shown . Scale bar , 5μm .

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A. Representative confocal microscopy images of cells transfected GFP-LC3 and Parkin-Cherry, treated as indicated, fixed 24h after AICD induction and immunostained with anti-TOM20 antibody are shown. Scale bar, 5μm.


B . Statistical of Parkin localization in subjected to AICD or to 10μM CCCP for indicated times is shown . Data represent mean ± SE of three independent experiments ( n = 30 per condition in each experiment ) .

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B. Statistical analysis of Parkin localization in cells subjected to AICD or to 10μM CCCP for the indicated times is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).


C . were treated as in ( A ) . Where indicated , lysosomal proteases inhibitors E64D PepA were added . LC3 - Parkin - mitochondria co - localization was calculated from 30 randomly selected per condition . Data represent mean ± SE of three independent experiments . Scale bar ,

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C. Cells were treated as in (A). Where indicated, lysosomal proteases inhibitors E64D and PepA were added. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected cells per condition. Data represent mean ± SE of three independent experiments. Scale bar, 5μm


D . hPBT were transfected with LC3 - cherry treated as indicated . 30min after AICD induction were fixed stained with TUNEL DAPI . Representative reconstructions of confocal z - stacks of LC3 - Cherry single stacks for DAPI are shown . Scale bar , 5μm . E . Statistical of autophagy in treated as in ( D ) is shown . Data represent mean ± SE of three independent experiments ( n = 20 per condition in each experiment ) .

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D. hPBT cells were transfected with LC3-cherry and treated as indicated. 30min after AICD induction cells were fixed and stained with TUNEL and DAPI. Representative reconstructions of confocal z-stacks of the LC3-Cherry fluorescence and single stacks for DAPI are shown. Scale bar, 5μm.E. Statistical analysis of the autophagy in cells treated as in (D) is shown. Data represent mean ± SE of three independent experiments (n = 20 cells per condition in each experiment).


F . Jurkat were transfected with GFP - LC3 treated as indicated . Representative reconstructions of confocal z - stacks of GFP - LC3 16h after AICD induction are shown . Scale bar , 5μm . G . Statistical of autophagic flux in treated as in ( F ) is shown . Data represent mean ± SE of three independent experiments ( n = 30 per condition in each experiment ) .

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F. Jurkat cells were transfected with GFP-LC3 and treated as indicated. Representative reconstructions of confocal z-stacks of the GFP-LC3 fluorescence 16h after AICD induction are shown. Scale bar, 5μm.G. Statistical analysis of the autophagic flux in cells treated as in (F) is shown. Data represent mean ± SE of three independent experiments (n = 30 cells per condition in each experiment).


H . samples ( 20 μg ) from Jurkat treated as indicated were separated by SDS - PAGE immunoblotted with indicated antibodies . Where indicated , lysosomal proteases inhibitors E64D PepA were added . WB are representative of at least three independent experiments .

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H. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Where indicated, lysosomal proteases inhibitors E64D and PepA were added. WB are representative of at least three independent experiments.


A - B . samples ( 20 μg ) from Jurkat treated as indicated were separated by SDS - PAGE immunoblotted with indicated antibodies . Wb are representative of at least four independent experiments . of phosphorylation status of AMPK were quantified analyzed as shown . In these panels , Time states for " time , hours , after AICD treatment " indicates at which time upon AICD induction samples have been collected .

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A-B. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. Wb are representative of at least four independent experiments. Levels of proteins and phosphorylation status of AMPK were quantified and analyzed as shown. In these panels, Time states for "time, hours, after AICD treatment" and indicates at which time upon AICD induction samples have been collected.


C . Jurkat were transfected with indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as of GFP - positive , annexin - V - PE - negative . Data represent mean ± SE of three independent experiments .

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C. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.


D . samples from Jurkat over expressing empty vector or AMPKS173A - S485A were immunoblotted for LC3 LC3 - II / Actin was quantified analyzed as shown . Data represent mean ± SE of three independent experiments .

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D. Protein samples from Jurkat cells over expressing empty vector or AMPKS173A-S485A were immunoblotted for LC3 and LC3-II/Actin ratio was quantified and analyzed as shown. Data represent mean ± SE of three independent experiments.


E - F . samples ( 20 μg ) from Jurkat treated as indicated were separated by SDS - PAGE immunoblotted with indicated antibodies . WB are representative of at least three independent experiments . of phosphorylation status of LC3 were quantified analyzed as shown .

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E-F. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments. Levels of proteins and phosphorylation status of LC3 were quantified and analyzed as shown.


G . Jurkat were transfected with indicated plasmids . 32h after AICD induction , viability was determined cytofluorimetrically as described above .

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G. Jurkat cells were transfected with the indicated plasmids. 32h after AICD induction, viability was determined cytofluorimetrically as described above.


H - I . where treated as in ( A ) where indicated 10μM H89 was added . Quantitative of P - AMPK Ser485 - 491 phosphorylation ( H ) of LC3 - II / LC3 - I ( I ) during AICD are shown .

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H-I. Cells where treated as in (A) and where indicated 10μM H89 was added. Quantitative analysis of P-AMPK Ser485-491 phosphorylation (H) and of LC3-II/LC3-I ratio (I) during AICD are shown.


J - K . AICD was induced in Jurkat ( J ) in hPBT ( K ) in presence of 10μM H89 . At indicated time points ( hours ) after AICD induction , apoptotic were detected by flow cytometry as AnnexinV / PI double positive between AICD Ctrl values obtained are shown .

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J-K. AICD was induced in Jurkat cells (J) and in hPBT cells (K) in presence of 10μM H89. At indicated time points (hours) after AICD induction, apoptotic cells were detected by flow cytometry as AnnexinV/PI double positive cells and the ratio between AICD and Ctrl values obtained are shown.


A . Apoptotic were detected at indicated time points after AICD induction in hPBT by flow cytometry as AnnexinV / PI double positive . Where indicated hPBT were pre - incubated with 100nM rapamycin for 24h before AICD induction . Data represent mean ± SE of six independent experiments .

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A. Apoptotic cells were detected at indicated time points after AICD induction in hPBT cells by flow cytometry as AnnexinV/PI double positive cells. Where indicated hPBT cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Data represent mean ± SE of six independent experiments.


B . AICD was induced in Jurkat where indicated 100nM rapamycin was added at time 0h of AICD induction . Apoptotic was carried out as in ( A ) , except that were analysed 32h after AICD induction . Data represent mean ± SE of six independent experiments .

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B. AICD was induced in Jurkat cells and where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of six independent experiments.


C . Morphometric of mitochondrial shape 24h after AICD induction . Jurkat were transfected with mtYFP 24h before AICD induction , where indicated , 100nM rapamycin was added . Data represent mean ± SE of four independent experiments ( n = 30 per condition in each experiment ) .

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C. Morphometric analysis of mitochondrial shape 24h after AICD induction. Jurkat cells were transfected with mtYFP 24h before AICD induction and, where indicated, 100nM rapamycin was added. Data represent mean ± SE of four independent experiments (n = 30 cells per condition in each experiment).


D . Representative electron of subjected to AICD untreated ( upper panel ) or treated with 100nM rapamycin ( lower panel ) . 24h after AICD induction were fixed processed for electron microscopy , Scale bar , 2μm .

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D. Representative electron micrographs of cells subjected to AICD untreated (upper panel) or treated with 100nM rapamycin (lower panel). 24h after AICD induction cells were fixed and processed for electron microscopy, Scale bar, 2μm.


E . Jurkat were transfected with mtYFP treated as indicated . 32h after AICD induction were fixed immunostained with an anti - cytochrome c antibody . Cytochrome c localization index was calculated from 30 randomly selected per condition . Data represent mean ± SE of four independent experiments .

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E. Jurkat cells were transfected with mtYFP and treated as indicated. 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index was calculated from 30 randomly selected images per condition. Data represent mean ± SE of four independent experiments.


F . Jurkat were transfected with DRP1S637A - GFP , 24h after transfection AICD was induced . 32h after AICD induction viability was determined cytofluorimetrically as of GFP - positive , annexin - V - PE - negative . Data represent mean ± SE of three independent experiments .

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F. Jurkat cells were transfected with DRP1S637A-GFP, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage of GFP-positive, annexin-V-PE-negative cells. Data represent mean ± SE of three independent experiments.


G - H . Jurkat were co - transfected with mt - RFP indicated plasmids , 32h after AICD induction were fixed immunostained with an anti - cytochrome c antibody . Cytochrome c localization index ( G ) mitochondrial ( H ) were calculated from 30 randomly selected . Data represent mean ± SE of three independent experiments .

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G-H. Jurkat cells were co-transfected with mt-RFP and the indicated plasmids, 32h after AICD induction cells were fixed and immunostained with an anti-cytochrome c antibody. Cytochrome c localization index (G) and mitochondrial morphology (H) were calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments.


I . Jurkat were transfected with DRP1K38A , 24h after transfection AICD was induced . 32h after AICD induction viability was determined cytofluorimetrically as AnnexinV / PI double negative . Data represent mean ± SE of three independent experiments .

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I. Jurkat cells were transfected with DRP1K38A, 24h after transfection AICD was induced. 32h after AICD induction viability was determined cytofluorimetrically as the percentage AnnexinV/PI double negative cells. Data represent mean ± SE of three independent experiments.


A . samples ( 20 μg ) from Jurkat treated as indicated were separated by SDS - PAGE immunoblotted with indicated antibodies . WB are representative of at least three independent experiments . B - C . Quantitative of between of mitochondrial markers Actin during AICD is shown . Data represent mean ± SE of three independent experiments .

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A. Protein samples (20 μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of at least three independent experiments.B-C. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and Actin during AICD is shown. Data represent mean ± SE of three independent experiments.


D . Jurkat were co - transfected with GFP - LC3 Parkin - Cherry , 32h after AICD induction were fixed immunostained with an anti - TOM20 antibody . Representative for each condition are presented . Scale bar , 5μm . E . LC3 - Parkin - mitochondria co - localization was calculated from 30 randomly selected . Data represent mean ± SE of three independent experiments . Data referring to Ctrl AICD - stimulated without rapamycin correspond to ones presented in Fig . 3C .

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D. Jurkat cells were co-transfected with GFP-LC3 and Parkin-Cherry, 32h after AICD induction cells were fixed and immunostained with an anti-TOM20 antibody. Representative images for each condition are presented. Scale bar, 5μm.E. LC3-Parkin-mitochondria co-localization was calculated from 30 randomly selected images. Data represent mean ± SE of three independent experiments. Data referring to Ctrl and AICD-stimulated cells without rapamycin correspond to the ones presented in Fig.3C.


A . Splenocytes were isolated from Ambragt / + wt C57 / BL6mice activated as indicated in materials methods . At day 7 after activation , AICD was induced with 0 . 1μg / ml of plate - bound anti - mCD3 ( Clone 17A2 ) . Apoptotic were detected 12h after AICD induction by flow cytometry as AnnexinV / PI double positive .

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A. Splenocytes were isolated from Ambragt/+ and wt C57/BL6mice and activated as indicated in materials and methods. At day 7 after activation, AICD was induced with 0.1μg/ml of plate-bound anti-mCD3 (Clone 17A2). Apoptotic cells were detected 12h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.


B . Splenocytes were isolated from ATG7 fl / fl wt C57 / BL6mice . Activation of splenocytes AICD were performed as previously described . At day 5 after activation , were infected with retroviral constructs expressing CRE recombinase . Where indicated , were pre - incubated with 100nM rapamycin for 24h before AICD induction . Apoptotic were detected 2h after AICD induction by flow cytometry as AnnexinV / PI double positive .

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B. Splenocytes were isolated from ATG7 fl/fl and wt C57/BL6mice. Activation of splenocytes and AICD were performed as previously described. At day 5 after activation, cells were infected with retroviral constructs expressing CRE recombinase. Where indicated, cells were pre-incubated with 100nM rapamycin for 24h before AICD induction. Apoptotic cells were detected 2h after AICD induction by flow cytometry as AnnexinV/PI double positive cells.


C . Jurkat were transfected with scramble or ATG7 siRNA 24h before AICD induction . AICD was induced as described in materials methods . Where indicated 100nM rapamycin was added at time 0h of AICD induction . Apoptotic was carried out as in ( A ) , except that were analysed 32h after AICD induction . Data represent mean ± SE of five independent experiments .

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C. Jurkat cells were transfected with scramble or ATG7 siRNA 24h before AICD induction. AICD was induced as described in materials and methods. Where indicated 100nM rapamycin was added at time 0h of AICD induction. Apoptotic cell death analysis was carried out as in (A), except that cells were analysed 32h after AICD induction. Data represent mean ± SE of five independent experiments.


D . Mitochondrial depolarization upon AICD induction was analysed in treated as indicated . Data represent mean ± SE of five independent experiments .

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D. Mitochondrial depolarization upon AICD induction was analysed in cells treated as indicated. Data represent mean ± SE of five independent experiments.


E . samples ( 20μg ) from Jurkat treated as indicated were separated by SDS - PAGE immunoblotted with indicated antibodies . WB are representative of three independent experiments . F . Quantitative of between of mitochondrial markers GRP75 during AICD is shown . Data represent mean ± SE of three independent experiments .

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E. Protein samples (20μg) from Jurkat cells treated as indicated were separated by SDS-PAGE and immunoblotted with the indicated antibodies. WB are representative of three independent experiments.F. Quantitative analysis of the ratio between the protein levels of mitochondrial markers and GRP75 during AICD is shown. Data represent mean ± SE of three independent experiments.


A Concentrations of amylase , lipase , CRP , ANGPTL4 in sera of ( n = 90 ) diagnosed with pancreatitis . box plots show a typical display consisting of a median value depicted by in center of box ; an interquartile range ( IQR ; 25th to 75th percentile ) depicted by box ; maximum ( Q3 + 1 . 5 * IQR ) minimum ( Q1 - 1 . 5 * IQR ) values depicted by whisker . Statistical significance of Mann - Whitney U - is indicated ( * * P < 0 . 01 * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . ANGPTL4 CRP from pancreatitis were determined by sandwich ELISA . correlation between ANGPTL4 CRP is depicted with Pearson correlation coefficient ( R ) . Each dot represents of ANGPTL4 CRP from an ( n = 90 ) .

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A Concentrations of amylase, lipase, CRP, and ANGPTL4 in the sera of patients (n = 90) diagnosed with pancreatitis. The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (** P < 0.01 and *** P < 0.001). Exact P values are shown in Appendix Table S2. ANGPTL4 and CRP concentration from pancreatitis patients were determined by sandwich ELISA. The correlation between ANGPTL4 and CRP is depicted with Pearson correlation coefficient (R). Each dot represents the levels of ANGPTL4 and CRP from an individual patient (n = 90).


B Pancreatitis adjacent normal regions ( n = 6 ) were stained by immunofluorescence for amylase ANGPTL4 . Scale bar represents 30

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B Pancreatitis and adjacent normal regions (n = 6) were stained by immunofluorescence for amylase and ANGPTL4. Scale bar represents 30 µm


C H & E , ANGPTL4 amylase expression by immunofluorescence after induction of mild ( AP ) severe acute pancreatitis ( SAP ) for 2 h ( n = 15 , each group ) . Scale bar represents 50 µm .

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C H&E staining, ANGPTL4 and amylase expression by immunofluorescence analysis after the induction of mild (AP) and severe acute pancreatitis (SAP) for 2 h (n = 15, each group). Scale bar represents 50 µm.


D Amylase , lipase , ANGPTL4 were assessed in serum from AP SAP models ANGPTL4 expression were determined in blood pancreas ( n = 15 , each group ) .

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D Amylase, lipase, and ANGPTL4 were assessed in the serum from AP and SAP animal models and ANGPTL4 protein expression were determined in the blood and pancreas (n = 15, each group).


A AP was induced by 5 injections ( IP ) of cerulein at a dosage of 50 μg / kg . SAP was induced by AP one injection of lipopolysaccharide ( LPS , IP ) at a dosage of 20 mg / kg . AP + ANGPTL4 ( 2 mg / kg ) was induced by AP one injection of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . ANGPTL4 ( 4 mg / kg ) was induced by two injections of ANGPTL4 ( IP ) at a dosage of 2 mg / kg . Mice were sacrificed 6 h after pancreatitis induction . ( H & E immunofluorescent for IL - 1β ( red ) amylase ( green ) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues ( n = 10 , each group ) . Scale bar represents 50

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A AP was induced by 5 injections (IP) of cerulein at a dosage of 50 μg/kg. SAP was induced by AP and one injection of lipopolysaccharide (LPS, IP) at a dosage of 20 mg/kg. AP+ANGPTL4 (2 mg/kg) was induced by AP and one injection of ANGPTL4 (IP) at a dosage of 2 mg/kg. ANGPTL4 (4 mg/kg) was induced by two injections of ANGPTL4 (IP) at a dosage of 2 mg/kg. Mice were sacrificed 6 h after pancreatitis induction. (H&E and immunofluorescent staining for IL-1β (red) and amylase (green) was performed along with immunohistochemistry for ANGPTL4 in pancreas tissues (n = 10, each group). Scale bar represents 50 µm


B Concentrations of inflammatory cytokines TGF - β , IL - 1β , IFN - γ as well as ANGPTL4 in sera ( n = 10 , each group ) .

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B Concentrations of the inflammatory cytokines TGF-β, IL-1β, and IFN-γ as well as ANGPTL4 in the sera (n = 10, each group).


C TUNEL for apoptosis vacuoles formation ( ) . Red arrow , apoptotic ; dotted , vacuoles region .

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C TUNEL staining for apoptosis and vacuoles formation (TEM). Red arrow, apoptotic cells; white dotted line, vacuoles region.


D T . E . M . ANGPTL4 immunogold labelling were performed in pancreas tissues from AP , SAP ANGPTL4 - treated models ( n = 5 , each group ) .

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D T.E.M. ANGPTL4 immunogold labelling were performed in pancreas tissues from the AP, SAP and ANGPTL4-treated models (n = 5, each group).


A AP SAP were induced in wild type ( WT ) ANGPTL4 - / - mice ( n = 15 , each group ) , as described in material methods . H & E , TUNEL for apoptosis , immunofluorescence for ANGPTL4 ( red ) amylase ( green ) were performed in pancreatic tissues . ANGPTL4 IL - 6 expressions were also determined in pancreatic tissues from ANGPTL4 WT - / - mice by Western blotting . Scale bar represents 100 µm .

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A AP and SAP were induced in wild type (WT) and ANGPTL4 -/- mice (n = 15, each group), as described in the material and methods. H&E staining, TUNEL staining for apoptosis, and immunofluorescence staining for ANGPTL4 (red) and amylase (green) were performed in the pancreatic tissues. ANGPTL4 and IL-6 protein expressions were also determined in pancreatic tissues from ANGPTL4 WT and -/- mice by Western blotting. Scale bar represents 100 µm.


B of ANGPTL4 , amylase , lipase , MPO in serum ( n = 15 , each group ) .

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B Levels of ANGPTL4, amylase, lipase, and MPO in serum (n = 15, each group).


C Immunofluorescence of TNF - α IL - 6 , of inflammatory cytokines TGF - β , IFN - γ , IL - 1β in sera from ANGPTL4 WT - / - mice ( n = 10 , each group ) . Scale bar represents 150

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C Immunofluorescence staining of TNF-α and IL-6, and the levels of the inflammatory cytokines TGF-β, IFN-γ, and IL-1β in the sera from ANGPTL4 WT and -/- mice (n = 10, each group). Scale bar represents 150 µm


A For macrophage depletion models , mice ( six ) were injected with GdCl3 ( 15 mg / kg ) . After induction of macrophage depletion in mice , pancreatitis was induced by cerulein , LPS , ANGPTL4 for 24 h ( n = 10 , each group ) . H & E of pancreatic tissue , of amylase lipase in serum . Scale bar represents 100

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A For macrophage depletion models, mice (six weeks) were injected with GdCl3 (15 mg/kg). After the induction of macrophage depletion in the mice, pancreatitis was induced by cerulein, LPS, and ANGPTL4 for 24 h (n = 10, each group). H&E staining of pancreatic tissue, and levels of amylase and lipase in the serum. Scale bar represents 100 µm


B Immunohistochemistry for macrophage marker CD11b ANGPTL4 in pancreas tissues . of ANGPTL4 in serum were measured in AP , SAP , ANGPTL4 ( 4 mg / kg ) groups of macrophage WT or depletion mice h ( n = 10 , each group ) . Scale bar represents 100

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B Immunohistochemistry for the macrophage marker CD11b and ANGPTL4 in pancreas tissues. Levels of ANGPTL4 in the serum were measured in the AP, SAP, and ANGPTL4 (4 mg/kg) groups of macrophage WT or depletion mice h (n = 10, each group). Scale bar represents 100 µm


C Immunofluorescent of F4 / 80 macrophage marker of macrophage - positive in AP SAP groups from ANGPTL4 - / - WT mice ( n = 10 ) . Scale bar represents 150

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C Immunofluorescent staining of the F4/80 macrophage marker and quantification of macrophage-positive cells in the AP and SAP groups from the ANGPTL4 -/-and WT mice (n = 10). Scale bar represents 150 µm


A mRNA of inflammatory cytokines , including TNF - α , IL - 6 , IL - 1β in mouse macrophages treated with LPS ANGPTL4 ( 100 ng / ml ) using qPCR RT - PCR , of TGF - β NO in LPS - ANGPTL4 - treated macrophages ( n = 5 ) . Scale bar represents 100

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A Cell morphology and the mRNA levels of inflammatory cytokines, including and TNF-α, IL-6, and IL-1β in mouse primary macrophages treated with LPS and ANGPTL4 (100 ng/ml) using qPCR and RT-PCR, and levels of TGF-β and NO in the LPS- and ANGPTL4-treated primary macrophages (n = 5). Scale bar represents 100 µm


B Migration assays were performed with mouse acinar macrophages using IBIDI removable 2 - well silicone culture inserts that were placed in a culture μ - Dish transwell at 24 h ( n = 5 ) . Scale bar represents 50

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B Migration assays were performed with mouse primary acinar cells and macrophages using IBIDI removable 2-well silicone culture inserts that were placed in a cell culture μ-Dish and transwell at 24 h (n = 5). Scale bar represents 50 µm


C Mouse acinar macrophages were labelled with dyes ( macrophages , red acinar , green ) . Co - cultured treated with 100 ng / ml of ANGPTL4 or LPS were analyzed by microscopy at 12 48 h . Scale bars represent 80 µm ( red ) 40 µm ( ) .

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C Mouse primary acinar cells and macrophages were labelled with fluorescence dyes (macrophages, red and acinar cells, green). Co-cultured cells treated with 100 ng/ml of ANGPTL4 or LPS were analyzed by microscopy at 12 and 48 h. Scale bars represent 80 µm (red) and 40 µm (white).


A Macrophage were treated with ANGPTL4 ( 100 ng / ml ) for 24 h then cytokine were analyzed by a mouse cytokine profiler ( n = 3 ) . Mean pixel density was quantified using ImageJ software .

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A Macrophage cells were treated with ANGPTL4 (100 ng/ml) for 24 h and then cytokine levels were analyzed by a mouse cytokine profiler assay (n=3). Mean pixel density was quantified using ImageJ software analysis.


B ANGPTL4 C5a expression were measured in bone marrow - derived macrophages from pancreatitis - induced ANGPTL4 - / - WT mice .

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B ANGPTL4 and C5a expression levels were measured in bone marrow-derived macrophages from pancreatitis-induced ANGPTL4-/- and WT mice.


C Phospho - kinase array was performed to find mechanism by which ANGPTL4 increased C5a in human THP1 - derived macrophage ( n = 3 ) . were treated with ANGPTL4 ( 100 ng / ml ) for 6 h . After phospho - kinase array , expression of AMPKα , JNK , AKT , P70S6K , EGFR was confirmed by western blotting .

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C Phospho-kinase array was performed to find the mechanism by which ANGPTL4 increased C5a in human THP1-derived macrophage cells (n=3). Cells were treated with ANGPTL4 (100 ng/ml) for 6 h. After phospho-kinase array analysis, the expression of AMPKα, JNK, AKT, P70S6K, and EGFR was confirmed by western blotting.


D To explore whether ANGPTL4 affects JNK , AMPK , PI3K / AKT signaling , each inhibitor ( SP600125 , compound C , HS - 173 , 1 µM ) was treated to in THP1 - derived macrophages for 2 h after ANGPTL4 treatment ( 100 ng / ml ) for 6 h .

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D To explore whether ANGPTL4 affects JNK, AMPK, and PI3K/AKT signaling, each inhibitor (SP600125, compound C, and HS-173, 1 µM) was treated to in THP1-derived macrophages for 2 h after ANGPTL4 treatment (100 ng/ml) for 6 h.


E C5a , C5aR , TNF - α , IL - 6 , IL - 1β mRNA were identified by qPCR in mouse macrophages or acinar after treatment with ANGPTL4 LPS ( 100 ng / ml ) . In transwell experiment , mouse acinar were cultured in lower well macrophages were incubated in upper transwell bucket treated with ANGPTL4 ( 100 ng / ml ) . Expression of ANGPTL4 , C5aR , C5a was determined in siANGPTL4 - treated THP1 - derived macrophage after LPS activation .

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E C5a, C5aR, TNF-α, IL-6, and IL-1β mRNA levels were identified by qPCR in mouse macrophages or primary acinar cells after treatment with ANGPTL4 and LPS (100 ng/ml). In the transwell experiment, mouse primary acinar cells were cultured in the lower well and macrophages were incubated in the upper transwell bucket treated with ANGPTL4 (100 ng/ml). Expression of ANGPTL4, C5aR, and C5a was determined in siANGPTL4-treated THP1-derived macrophage after LPS activation.


F In transwell experiment , TUNEL positive C5aR expression were identified in acinar after C5aR antagonist ( W - 54011 , 10 nM ) pretreatment for 12 h prior to treatment with ANGPTL4 , LPS , C5a ( 100 ng / ml ) for 24 h to macrophage in transwell . Scale bar represents 20

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F In the transwell experiment, TUNEL positive cells and C5aR expression were identified in primary acinar cells after C5aR antagonist (W-54011, 10 nM) pretreatment for 12 h prior to treatment with ANGPTL4, LPS, and C5a (100 ng/ml) for 24 h to macrophage in transwell. Scale bar represents 20 µm


A Concentrations of C5a in sera from pancreatitis , correlation between ANGPTL4 C5a in sera from pancreatitis ( n = 90 ) . box plots show a typical display consisting of a median value depicted by in center of box ; an interquartile range ( IQR ; 25th to 75th percentile ) depicted by box ; maximum ( Q3 + 1 . 5 * IQR ) minimum ( Q1 - 1 . 5 * IQR ) values depicted by whisker . Statistical significance of Mann - Whitney U - is indicated ( * * * P < 0 . 001 ) . Exact P values are shown in Appendix Table S2 . correlation between C5a ANGPTL4 is depicted with Pearson correlation coefficient ( R ) . Each dot represents of C5a ANGPTL4 from an ( n = 90 ) . immunofluorescent expression of C5a amylase was observed in pancreatitis adjacent normal regions in tissues . Scale bar represents 50

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A Concentrations of C5a in the sera from pancreatitis patients, and correlation between ANGPTL4 and C5a in the sera from pancreatitis patients (n = 90). The box plots show a typical display consisting of a median value depicted by the line in the center of the box; an interquartile range (IQR; 25th to the 75th percentile) depicted by the box; and the maximum (Q3+1.5*IQR) and minimum (Q1-1.5*IQR) values depicted by the whisker. Statistical significance of Mann-Whitney U-tests is indicated (*** P < 0.001). Exact P values are shown in Appendix Table S2. The correlation between C5a and ANGPTL4 is depicted with Pearson correlation coefficient (R). Each dot represents the levels of C5a and ANGPTL4 from an individual patient (n = 90). The immunofluorescent expression of C5a and amylase was observed in pancreatitis and adjacent normal regions in patient tissues. Scale bar represents 50 µm


B Immunofluorescence of C5a in pancreas tissues from ANGPTL4 - / - WT macrophage depletion / WT models with pancreatitis . Scale bar represents 150 µm . of C5a were measured in serum ( n = 7 ) .

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B Immunofluorescence staining of C5a in pancreas tissues from ANGPTL4 -/- and WT animals and macrophage depletion/WT models with pancreatitis. Scale bar represents 150 µm. Levels of C5a were measured in serum (n = 7).


C After macrophages were activated with LPS ( 100 ng / ml ) , were treated with either neutralizing ANGPTL4 or C5a antibody ( 100 ng / ml ) or an isotype control ( IgG ) . ANGPTL4 , C5a , TNF - α , IL - 6 were measured in culture media ( n = 5 ) .

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C After macrophages were activated with LPS (100 ng/ml), the cells were treated with either neutralizing ANGPTL4 or C5a antibody (100 ng/ml) or an isotype control (IgG). ANGPTL4, C5a, TNF-α, and IL-6 levels were measured in culture media (n = 5).


D neutralizing ANGPTL4 antibody ( 10 mg / kg ) was injected twice ( IP ) into AP SAP models . H & E of pancreatic tissues concentrations of ANGPTL4 , C5a , TNF - α , amylase in serum ( n = 10 , each group ) . Scale bar represents 50

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D The neutralizing ANGPTL4 antibody (10 mg/kg) was injected twice (IP) into the AP and SAP models. H&E staining of pancreatic tissues and the concentrations of ANGPTL4, C5a, TNF-α, and amylase in the serum (n = 10, each group). Scale bar represents 50 µm


( A ) . Human neutrophils stained for CD66b after incubation with RBCs infected with GFP + P . falciparum ( arrows ) . Nuclei were stained with DAPI ( blue ) , neutrophils were stained with CD66b ( red ) , GFP expressing ( green ) . Scale bar , 5 µm . upper lower panel show two different .

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(A). Human neutrophils stained for CD66b after incubation with RBCs infected with GFP+ P. falciparum parasites (white arrows). Nuclei were stained with DAPI (blue), neutrophils were stained with CD66b (red), GFP expressing parasites (green). Scale bar, 5 µm. The upper and lower panel show two different cells.


( B ) . Giemsa of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P . falciparum ( black arrows ) .

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(B). Giemsa staining of freshly isolated human neutrophils from a healthy donor incubated with iRBCs harboring late stages P. falciparum parasites (black arrows).


( C ) . Flow cytometry of human neutrophils incubated with opsonized or non - opsonized GFP + - late - stage iRBCs . Free neutrophils were used as control .

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(C). Flow cytometry analysis of human neutrophils incubated with opsonized or non-opsonized GFP+-late-stage iRBCs. Free neutrophils were used as control.


( D ) . of GFP + iRBCs phagocytosed by neutrophils . phagocytosis was analysed by flow cytometry ( FACS ) . Data information :

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(D). Quantification of GFP+ iRBCs phagocytosed by neutrophils. The phagocytosis was analysed by flow cytometry (FACS). Data information:


( A ) . Interaction between neutrophils opsonized ( red ) or non - opsonized ( blue ) GFP + iRBCs by time in a " - chase " experiment . of GFP + - neutrophils following 5 min of co - culture was set as 100 % .

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(A). Interaction between neutrophils and opsonized (red) or non-opsonized (blue) GFP+ iRBCs by time in a "pulse-chase" experiment. The percentage of GFP+-neutrophils following 5 min of co-culture was set as 100%.


( C ) . Parasitemia ( % ) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils .

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(C). Parasitemia (percentage %) of NF54 iRBCs cultures with or without daily addition of freshly isolated neutrophils.


( D ) . Parasitemia ( % ) of cultures of RBC infected by different P . falciparum lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) . were kept in culture for six days in presence ( Neut . ) or absence ( Cont . ) of neutrophils .

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(D). Parasitemia (percentage %) of cultures of RBC infected by different P. falciparum lines (NF54, Dd2, SL, PfEMP1-KO). The parasites were kept in culture for six days in the presence (Neut.) or absence (Cont.) of neutrophils.


( E ) . change in parasitemia following neutrophil challenge of RBCs infected by different lines ( NF54 , Dd2 , SL , PfEMP1 - KO ) .

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(E). Percentage change in parasitemia following neutrophil challenge of RBCs infected by different parasite lines (NF54, Dd2, SL, PfEMP1-KO).


( F ) . Flow cytometric of effect of neutrophil challenge on cycle progression . Neutrophils were stained with an anti - CD11b antibody , while iRBCs were labeled with SYBR green . Gating for iRBCs were performed by gating for SYBR green CD11b negative population , excluding neutrophils that were SYBR green negative , but CD11b positive .

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(F). Flow cytometric analysis of the effect of neutrophil challenge on parasite cell cycle progression. Neutrophils were stained with an anti-CD11b antibody, while iRBCs were labeled with SYBR green. Gating for iRBCs were performed by gating for the SYBR green and CD11b negative population, excluding the neutrophils that were SYBR green negative, but CD11b positive.


( G ) . Short term neutrophil killing of late - stage NF54 - luciferase positive RBCs in presence ( red ) or absence ( blue ) of catalase .

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(G). Short term neutrophil killing of late-stage NF54-luciferase positive RBCs in the presence (red) or absence (blue) of catalase.