Hypoxia mimetic DMOG induced HIF - 1 / 2α , CA9 & GPRC5A protein expression . Dual HIF - 1 / 2α depletion reduced GPRC5A induction by DMOG .

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Hypoxia mimetic DMOG induced HIF-1/2α, CA9 & GPRC5A protein expression. Dual HIF-1/2α depletion reduced GPRC5A induction by DMOG.


qRT - PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


qRT - PCR demonstrating that HIF - 1 / 2α depletion decreased GPRC5A induction during hypoxia . GPRC5A was normalised to HPRT ( error bars ± SD ) .

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qRT-PCR demonstrating that HIF-1/2α depletion decreased GPRC5A induction during hypoxia. GPRC5A was normalised to HPRT (error bars ± SD).


ChIP / PCR analyses identify HIF - 1α a binding to the GPRC5A promoter region containing a putative optimal HRE ( error bars ± SD ) .

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ChIP/PCR analyses identify HIF-1α a binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD).


Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced CA9 expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of CA9 in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced CA9 expression with siRNA confirms antibody specificity (scale bars: 200um).


Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC . Reduced ; GPRC5A expression with siRNA confirms antibody specificity ( scale bars : 200um ) .

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Expression of ; GPRC5A in formalin fixed paraffin embedded hypoxic SW620 cells by IHC. Reduced ; GPRC5A expression with siRNA confirms antibody specificity (scale bars: 200um).


IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia ( strangulated colon ) . GPRC5A is co - expressed with CA9 in the colonic epithelial cells ( scale bars : 50um ) .

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IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co-expressed with CA9 in the colonic epithelial cells (scale bars: 50um).


Quantitative RT - PCR analysis of mouse intestinal tissue . Gene expression was normalised to housekeeping gene Tbp . Raw data from three independent experiments ( n = 3 mice ) are shown ( error bars ± SEM ) .

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Quantitative RT-PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp. Raw data from three independent experiments (n=3 mice) are shown (error bars ± SEM).


Tg [ fli1 : eGFP ; vhl - / - ] & Tg [ fli1 : eGFP ] zebrafish embryos ( 5 days post - fertilisation ) demonstrate excessive angiogenesis & increased expression of HIF target genes ( scale bars : 100um ) .

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Tg[fli1:eGFP; vhl-/-] & Tg[fli1:eGFP] zebrafish embryos (5 days post-fertilisation) demonstrate excessive angiogenesis & increased expression of HIF target genes (scale bars: 100um).


gprc5ba was induced in vhl mutant zebrafish embryos & fli1 : eGFP zebrafish embryos exposed to 5 % O2 ( vs normoxia ) for 24 hours ( RT - PCR ) .

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gprc5ba was induced in vhl mutant zebrafish embryos & fli1:eGFP zebrafish embryos exposed to 5% O2 (vs normoxia) for 24 hours (RT-PCR).


Kaplan - Meier curve following analysis of transcriptomics dataset GSE24551 . Event - free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA .

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Kaplan-Meier curve following analysis of transcriptomics dataset GSE24551. Event-free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA.


GPRC5A depletion markedly increases caspase - 3 activation / PARP cleavage during hypoxia .

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GPRC5A depletion markedly increases caspase-3 activation/PARP cleavage during hypoxia.


Three independent siRNA sequences targeting GPRC5A induce caspase - 3 activation / PARP cleavage during hypoxia .

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Three independent siRNA sequences targeting GPRC5A induce caspase-3 activation/PARP cleavage during hypoxia.


GPRC5A depletion reduces hypoxic cell growth / survival . Crystal violet cell assays show reduced cell growth / survival in GPRC5A depleted cells during hypoxia [ n = 3 ] .

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GPRC5A depletion reduces hypoxic cell growth/survival. Crystal violet cell assays show reduced cell growth/survival in GPRC5A depleted cells during hypoxia [n=3].


Expression of an siRNA - resistant GPRC5A cDNA rescues hypoxic GPRC5A - depleted cells from apoptosis . Upper : doxycycline - induced expression GPRC5Asi1R rescues increases caspase - 3 / PARP cleavage induced by GPRC5A depletion in hypoxia . Lower : generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations .

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Expression of an siRNA-resistant GPRC5A cDNA rescues hypoxic GPRC5A-depleted cells from apoptosis. Upper: doxycycline-induced expression GPRC5Asi1R rescues increases caspase-3/PARP cleavage induced by GPRC5A depletion in hypoxia. Lower: generation of an siRNA1 resistant GPRC5A cDNA by synonymous mutations.


Caspase inhibitor QVD prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

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Caspase inhibitor QVD prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe / dead cell apoptosis assay and flow

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GPRC5A depletion in hypoxia induces apoptosis as determined by the violet ratiometric membrane asymmetry probe/dead cell apoptosis assay and flow cytometry


Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells .

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Hypoxia induced YAP stabilisation via Ser397 dephosphorylated was abrogated in GPRC5A depleted cells.


Hypoxia - induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells .

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Hypoxia-induced nuclear localisation of YAP was attenuated in GPRC5A depleted cells.


Hypoxia activated TEAD activity ( 8x GTIIC - luc reporter ) but this was reduced by GPRC5A depletion . A representative triplicate experiment is shown ( n = 3 ) .

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Hypoxia activated TEAD activity (8x GTIIC-luc reporter) but this was reduced by GPRC5A depletion. A representative triplicate experiment is shown (n=3).


Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion .

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Hypoxia reduced LATS activity and expression but this was prevented by GPRC5A depletion.


Constitutively active RhoA ( G14V ) expression restored YAP stabilisation ( Ser397 dephosphorylation ) by hypoxia in GPRC5A depleted cells .

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Constitutively active RhoA (G14V) expression restored YAP stabilisation (Ser397 dephosphorylation) by hypoxia in GPRC5A depleted cells.


The YAP / TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay ( n = 3 independent experiments ) .

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The YAP/TEAD inhibitor verteporfin selectively inhibited cancer cell survival in hypoxia by crystal violet assay (n=3 independent experiments).


YAP knockdown was sufficient to induce caspase - 3 activation / PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion .

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YAP knockdown was sufficient to induce caspase-3 activation/PARP cleavage in hypoxia and was not further enhanced by GPRC5A depletion.


Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival . GPRC5A depleted cells were rescued by expression of an siRNA - resistant GPRC5A cDNA ( GPRC5Asi1R ) but this was prevented by co - depletion of YAP ( n = 3 independent experiments ) .

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Crystal violet assays show that YAP was required downstream of GPRC5A to promote cell survival. GPRC5A depleted cells were rescued by expression of an siRNA-resistant GPRC5A cDNA (GPRC5Asi1R) but this was prevented by co-depletion of YAP (n=3 independent experiments).


Expression of an siRNA - resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion . GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia - induced YAP stabilisation ( Ser397 dephosphorylation ) ; these phenotypes were reversed by YAP depletion .

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Expression of an siRNA-resistant GPRC5A rescued the critical phenotypes of GPRC5A depletion. GPRC5Asi1R expression prevented PARP cleavage in hypoxia as well as restoring hypoxia-induced YAP stabilisation (Ser397 dephosphorylation); these phenotypes were reversed by YAP depletion.


GPRC5A depletion attenuated hypoxia - induced BCL - XL expression .

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GPRC5A depletion attenuated hypoxia-induced BCL-XL expression.


Constitutively active YAP ( S127A ) expression induced BCL - XL expression and prevents caspase - 3 activation / PARP cleavage by GPRC5A depletion in hypoxia .

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Constitutively active YAP (S127A) expression induced BCL-XL expression and prevents caspase-3 activation/PARP cleavage by GPRC5A depletion in hypoxia.


GPRC5Asi1R expression restored BCL - XL expression and prevents the appearance of cleaved caspase - 3 induced by GPRC5A depletion in hypoxia .

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GPRC5Asi1R expression restored BCL-XL expression and prevents the appearance of cleaved caspase-3 induced by GPRC5A depletion in hypoxia.


A CUEDC2protein levels in mouse tissues . Adult ( 12 - week - old ) mouse was sacrifices and total protein was extracted from the liver , kidney , lung , spleen , heart , stomach , intestine , prostate , testis , brain , and thymus .

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A CUEDC2protein levels in mouse tissues. Adult (12-week-old) mouse was sacrifices and total protein was extracted from the liver, kidney, lung, spleen, heart, stomach, intestine, prostate, testis, brain, and thymus.


B ( top ) In vivo cardiac ischemia - reperfusion model protocol ( 30 - minute ischemia followed by indicated times of reperfusion ) . ( bottom ) Mice were subjected to reversible ischemia in vivo for 30 minutes and reperfused for different time period . Protein was extracted from the area at risk of heart at the indicated time points and subjected to immunoblot analysis of CUEDC2 and CUEDC1 . Representative results were shown from 2 mice at each time point , and experiments were repeated for three times .

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B (top) In vivo cardiac ischemia-reperfusion model protocol (30-minute ischemia followed by indicated times of reperfusion). (bottom) Mice were subjected to reversible ischemia in vivo for 30 minutes and reperfused for different time period. Protein was extracted from the area at risk of heart at the indicated time points and subjected to immunoblot analysis of CUEDC2 and CUEDC1. Representative results were shown from 2 mice at each time point, and experiments were repeated for three times.


C Isolated primary ratneonatal cardiomyocytes were treated to hypoxia with serum free - medium for 6h and reoxygenated accompanying adding serum back for different time period in vitro and the protein level of CUEDC2 was tested at different time points during reoxygenation . Each experiment was repeated for three times .

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C Isolated primary ratneonatal cardiomyocytes were treated to hypoxia with serum free-medium for 6h and reoxygenated accompanying adding serum back for different time period in vitro and the protein level of CUEDC2 was tested at different time points during reoxygenation. Each experiment was repeated for three times.


D CUEDC2 protein level was plotted using the immunohistochemical scores as described in methods . ( left ) Plot of CUEDC2 scores in each BZ and DZ ( n = 9 patients ) . ( right ) Representative images from immunohistochemistry staining for CUEDC2 in tissues from patients who suffered acute myocardial infarctions . The boxed areas in the left images are magnified in the middle and right images . DZ , distant zone ; BZ , border zone . Scale bars , 250 μm ( left ) , 50 μm ( middle and right ) . * p = 0 . 001 .

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D CUEDC2 protein level was plotted using the immunohistochemical scores as described in methods. (left) Plot of CUEDC2 scores in each BZ and DZ (n = 9 patients). (right) Representative images from immunohistochemistry staining for CUEDC2 in tissues from patients who suffered acute myocardial infarctions. The boxed areas in the left images are magnified in the middle and right images. DZ, distant zone; BZ, border zone. Scale bars, 250 μm (left), 50 μm (middle and right). * p = 0.001.


A Primary mousecardiomyocytes were treated with H2O2 ( 1 mM ) for 3 hours and cardiomyocyteviability was determined by flow cytometry . Representative results of flow cytometry analysis .

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A Primary mousecardiomyocytes were treated with H2O2 (1 mM) for 3 hours and cardiomyocyteviability was determined by flow cytometry. Representative results of flow cytometry analysis.


B Quantitative analysis of PI - positive cells treated with H2O2 was shown ( 42 . 9 ± 3 . 1 % in wild - type group vs . 10 . 1 ± 4 . 4 % in Cuedc2 - / - group , * p = 0 . 0004 , n = 5 wells per group and repeated for three times ) .

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B Quantitative analysis of PI-positive cells treated with H2O2 was shown (42.9 ± 3.1% in wild-type group vs. 10.1 ± 4.4% in Cuedc2-/- group, * p = 0.0004, n = 5 wells per group and repeated for three times).


C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes . Cardiomyocytes were then stained with 5 μM CellROX ® Deep Red Reagent and analyzed by flow cytometry . Representative results of flow cytometry analysis are shown , and all experiments were repeated for three times . D ROS levels were quantitated and summarized . * p = 0 . 0108 , n = 3 wells per group and repeated for three times .

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C Mouse neonatal cardiomyocytes were subjected to hypoxia for 6 hours and then reoxygenation for 30 minutes. Cardiomyocytes were then stained with 5 μM CellROX® Deep Red Reagent and analyzed by flow cytometry. Representative results of flow cytometry analysis are shown, and all experiments were repeated for three times.D ROS levels were quantitated and summarized. * p = 0.0108, n = 3 wells per group and repeated for three times.


E HPLC detection of a superoxide probe oxidized dihydroethidium ( DHE ) product in sham and I / R injury heart tissue ( 30 - min ischemia followed by 30 - min reperfusion ) , 2 - hydroxyethidium ( EOH ) , a specific product for superoxide anion radical . * p < 0 . 0001 , n = 5 in each group . F In the same samples of ( E ) , HPLC detection of ethidium ( E ) , oxidized by other reactive oxygen species such as H2O2 ( mainly ) and ONOO . * p = 0 . 0001 , n = 5 in each group .

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E HPLC detection of a superoxide probe oxidized dihydroethidium (DHE) product in sham and I/R injury heart tissue (30-min ischemia followed by 30-min reperfusion), 2-hydroxyethidium (EOH), a specific product for superoxide anion radical. *p < 0.0001, n = 5 in each group.F In the same samples of (E), HPLC detection of ethidium (E), oxidized by other reactive oxygen species such as H2O2 (mainly) and ONOO. *p = 0.0001, n = 5 in each group.


A Protein was extracted from the area at risk of left ventricle from wild - type or Cuedc2 - / - mice with 30 - minute ischemia followed by 30 - minute reperfusion and subjected to immunoblot analysis . Representative results from 2 mice are shown , and each experiment was repeated for three times .

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A Protein was extracted from the area at risk of left ventricle from wild-type or Cuedc2-/- mice with 30-minute ischemia followed by 30-minute reperfusion and subjected to immunoblot analysis. Representative results from 2 mice are shown, and each experiment was repeated for three times.


B Neonatal mousecardiomyocytes were subjected to hypoxia for 6 hours , followed by the indicated times of reoxygenation , before protein was extracted and subjected to immunoblot analysis .

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B Neonatal mousecardiomyocytes were subjected to hypoxia for 6 hours, followed by the indicated times of reoxygenation, before protein was extracted and subjected to immunoblot analysis.


C Luciferase assay of WT or Cuedc2 - / - MEFs transiently transfected with an NF - κB - responsive luciferase reporter and then , 1 d later , treated for 6 h with TNF ( 10 ng / ml ; TNF ) or for H / R . * p = 0 . 0032 , n = 3 per group , * * p = 0 . 013 , n = 3 per group .

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C Luciferase assay of WT or Cuedc2-/- MEFs transiently transfected with an NF-κB-responsive luciferase reporter and then, 1 d later, treated for 6 h with TNF (10 ng/ml; TNF) or for H/R. * p = 0.0032, n = 3 per group, ** p = 0.013, n = 3 per group.


D Wild - type and Cuedc2 - / - mice were treated with a sham or I / R operation in vivo . Three hours after the onset of reperfusion , the mRNA levels of tumor necrosis factor α ( TNFα ) , interleukin 6 ( IL - 6 ) and interleukin 23 ( IL - 23 ) were tested . * p = 0 . 0005 , * * p = 0 . 0001 , n = 3 per group .

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D Wild-type and Cuedc2-/- mice were treated with a sham or I/R operation in vivo. Three hours after the onset of reperfusion, the mRNA levels of tumor necrosis factor α (TNFα), interleukin 6 (IL-6) and interleukin 23 (IL-23) were tested. * p = 0.0005, ** p = 0.0001, n = 3 per group.


A Protein was extracted from the left ventricle of wild - type or Cuedc2 - / - adult mice ( 12 week - old ) and subjected to immunoblot analysis ( 3 mice per group ) .

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A Protein was extracted from the left ventricle of wild-type or Cuedc2-/- adult mice (12 week-old) and subjected to immunoblot analysis (3 mice per group).


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 .

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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 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 .

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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 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 .

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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 Primary neonatal mousecardiomyocytes were transfected with lentivirus carrying control or GPX1 shRNA , and the 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 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 .

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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 - / - cells treated with 20μM cycloheximide ( CHX ) for the indicated times were subjected to immunoblotting ( left ) . Relative GPX1 levels 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 - / - 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were transfected with increasing amounts of Flag - FBXW7 expression vectors and 48 hours after transfection , cell 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 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 .

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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 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 .

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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 , 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 .

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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 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 .

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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 analysis 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 ) 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 .

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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 ) and 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 and 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 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 .

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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 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 .

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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 staining . * 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 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 .

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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 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 ) .

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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 ) 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 .

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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 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 ) .

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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 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 .

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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 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 ) .

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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 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 ) .

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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).


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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 cells treated with the indicated drugs for 16 h . Antimycin A ( AA ; 10 μM ) , piericidin A ( 10 μM ) and 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 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 ) .

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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 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 ) .

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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 . 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 .

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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 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 .

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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 cells as described in Materials and methods . Apoptotic cells 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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 . 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 ,

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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 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 ) .

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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 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 ) .

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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 . 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 .

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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 . 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 .

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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 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 .

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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 . 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 .

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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 . 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 .

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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 cells were transfected with the 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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 / + 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 .

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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 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 .

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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 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 .

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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 cells 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.