( C , D ) p - TIE2 in tumor vessels from control and AMG386 - treated mice ; representative confocal images ; arrowheads point to CD31 + / p - TIE2 - endothelial cells ( scale bars : 50μm ) ( C ) , and quantification ( D ) of p - TIE - 2 / CD31 + in control ( n = 3 ) and AMG386 treated ( n = 3 ) tumors ( 1 , 200 CD31 + cells / group ) . P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 . ( E , F ) p - EphrinB ( E ) in representative tumor vessels from control ( n = 3 ) and AMG386 - treated ( n = 3 ) tumors ; confocal images ( E ) , ( scale bar : 50μm ) and quantification of p - EphrinB / CD31 + ( F ) ; P values from two - tailed Student ' s t - test ; * * * P < 0 . 001 .

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(C,D) p-TIE2 in tumor vessels from control and AMG386-treated mice; representative confocal images; arrowheads point to CD31+/p-TIE2- endothelial cells (scale bars: 50μm) (C), and quantification (D) of p-TIE-2/CD31+ in control (n=3) and AMG386 treated (n=3) tumors (1,200 CD31+ cells/group). P values from two-tailed Student's t-test; ***P<0.001. (E,F) p-EphrinB (E) in representative tumor vessels from control (n=3) and AMG386-treated (n=3) tumors; confocal images (E), (scale bar: 50μm) and quantification of p-EphrinB/CD31+ (F); P values from two-tailed Student's t-test; ***P<0.001.


( G ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 ( red ) identifies the endothelium ; DAPI ( blue ) identifies the nuclei ; representative tumor confocal images ( scale bar : 50μm ) of 3 mice / group ; arrow heads point to FITC - dextran extravasation ; asterisks to extravasated erythrocytes .

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(G) Vessel perfusion visualized by FITC-dextran (green); CD31 (red) identifies the endothelium; DAPI (blue) identifies the nuclei; representative tumor confocal images (scale bar:50μm) of 3 mice/group; arrow heads point to FITC-dextran extravasation; asterisks to extravasated erythrocytes.


( H ) Quantification of cleaved caspase - 3 in the tumor vasculature ( CD31 + cells ) ; n = 3 tumors / group ; number of CD31 + cells counted : control ( n = 311 ) ; SHP099 ( n = 425 ) ; AMG386 ( n = 451 ) ; combined SHP099 + AMG386 ( n = 484 ) . Error bars : ± S . D . ; P values by analysis of variance with Dunnett ' s multiple comparison test ; * * P < 0 . 01 , * * * P < 0 . 001 .

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(H) Quantification of cleaved caspase-3 in the tumor vasculature (CD31+ cells); n=3 tumors/group; number of CD31+ cells counted: control (n=311); SHP099 (n=425); AMG386 (n=451); combined SHP099+AMG386 (n=484). Error bars: ± S.D.; P values by analysis of variance with Dunnett's multiple comparison test; **P<0.01, ***P<0.001.


( A - D ) Nuclear p - STAT3 ( A , B ) and p - ERK1 / 2 ( C , D ) in tumor CD31 + endothelial cells of mice treated for 14 days with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) immunostaining ( A , C ) and quantification of staining intensity / CD31 + cell ( B , D ) ; horizontal line : mean intensity / group ; n = 3 tumors / group ; number of CD31 + cells counted in B : control ( n = 180 ) , SHP099 ( n = 308 ) , AMG386 ( n = 300 ) , combined SHP099 + AMG386 ( n = 322 ) ; number of CD31 + cells counted in D : control ( n = 1 , 733 ) , SHP099 ( n = 821 ) , AMG386 ( n = 1 , 399 ) , combined SHP099 + AMG386 ( n = 783 ) . Scale bars : A , 50μm ; C , 10μm .

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(A-D) Nuclear p-STAT3 (A,B) and p-ERK1/2 (C,D) in tumor CD31+ endothelial cells of mice treated for 14 days with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) immunostaining (A,C) and quantification of staining intensity/CD31+ cell (B, D); horizontal line: mean intensity/group; n=3 tumors/group; number of CD31+ cells counted in B: control (n=180), SHP099 (n=308), AMG386 (n=300), combined SHP099+AMG386 (n=322); number of CD31+ cells counted in D: control (n=1,733), SHP099 (n=821), AMG386 (n=1,399), combined SHP099+AMG386 (n=783). Scale bars: A, 50μm; C, 10μm.


FOXO1 detection in tumor CD31 + endothelial cells after treatment with SHP099 ( 200 mg / kg ) , AMG386 ( 5 . 6mg / kg ) or the combination of SHP099 ( 200 mg / kg ) + AMG386 ( 5 . 6mg / kg ) . Representative ( 3 tumors / group ) confocal

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FOXO1 detection in tumor CD31+ endothelial cells after treatment with SHP099 (200 mg/kg), AMG386 (5.6mg/kg) or the combination of SHP099 (200 mg/kg) + AMG386 (5.6mg/kg). Representative (3 tumors/group) confocal images


( C ) Vessel perfusion visualized by FITC - dextran ( green ) ; CD31 identifies endothelial cells ( red ) ; DAPI ( blue ) identifies the nuclei ; representative ( of 3 mice / group ) confocal images ( scale bar : 50μm ) ; arrow heads point to FITC - dextran extravasation .

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(C) Vessel perfusion visualized by FITC-dextran (green); CD31 identifies endothelial cells (red); DAPI (blue) identifies the nuclei; representative (of 3 mice/group) confocal images (scale bar: 50μm); arrow heads point to FITC-dextran extravasation.


( H ) Representative images depicting Ki67 + cells in control and treated MC38 tumors ; representative images ( scale bar : 50μm ) of 4 tumors / group .

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(H) Representative images depicting Ki67+ cells in control and treated MC38 tumors; representative images (scale bar: 50μm) of 4 tumors/group.


( A ) WT and CITK - / - embryonic ( E14 . 5 ) mousecerebral cortex was stained for γ - tubulin ( green ) and DNA ( gray ) . The ventricular plane is marked by a red dashed line , and the spindle axis of apical progenitors is indicated by a white dashed line . The angle between these two lines represents the mitotic angle . Scale bars = 5 μm . ( B ) Quantification of vertical divisions of apical progenitors in WT and CITK - / - mice . n = 3 per each genotype .

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(A) WT and CITK -/- embryonic (E14.5) mousecerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype.


( C ) Pregnant CITK + / - females , crossed with CITK + / - males , were injected with BrdU at E13 . 5 ; 24 hours later ( E14 . 5 ) WT and CITK - / - embryonic mouse cerebral cortex were fixed in 4 % PFA , cryosectioned and stained for Ki67 ( red ) , BrdU ( green ) , Tbr2 ( blue ) and DAPI ( gray ) . The different cortical regions are indicated : CP = cortical plate ; IZ = intermediate zone ; SVZ = subventricular zone ; VZ = ventricular zone . Scale bars = 10 μm . White arrows in the inset indicate two Ki67 - cells positive for BrdU and Tbr2 .

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(C) Pregnant CITK +/- females, crossed with CITK +/- males, were injected with BrdU at E13.5; 24 hours later (E14.5) WT and CITK -/- embryonic mouse cerebral cortex were fixed in 4% PFA, cryosectioned and stained for Ki67 (red), BrdU (green), Tbr2 (blue) and DAPI (gray). The different cortical regions are indicated: CP=cortical plate; IZ= intermediate zone; SVZ= subventricular zone; VZ= ventricular zone. Scale bars = 10 μm. White arrows in the inset indicate two Ki67- cells positive for BrdU and Tbr2.


( D ) Quantification of cell cycle exit ( ratio of cells BrdU + / Ki67 − ) in the proliferative regions ( VZ / SVZ ) and in the neuronal layers ( IZ and CP ) of sections prepared as in panel C ( n = 4 per each genotype ) .

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(D) Quantification of cell cycle exit (ratio of cells BrdU+/ Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel C (n=4 per each genotype).


( E ) Sections prepared as in C were stained for TUBB3 ( TuJ ) to reveal post - mitotic neurons and with DAPI . Red arrowheads indicate apoptotic neurons ( defined by pyknotic nuclei ) . Quantification of neuronal distribution in VZ - SVZ versus total neurons population is showed on the right . Scale bars = 10 μm .

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(E) Sections prepared as in C were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ-SVZ versus total neurons population is showed on the right. Scale bars = 10 μm.


( F ) Quantification of the percentage of Tbr2 - positive cells in the BrdU - positive population of the same sections analyzed for panel D ( n = 4 per each genotype ) .

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(F) Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel D (n=4 per each genotype).


( G ) Neuroblasts of wild type and dck alleles immunostained for α - tubulin and Miranda . Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis , in dck alleles the spindle show a more oblique orientation respect to Mira crescent . Scale bar = 5 μm . ( H ) Distribution of spindle angle amplitude ( ° ) in neuroblasts of wild type and ck alleles ( n = 58 ) .

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(G) Neuroblasts of wild type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle show a more oblique orientation respect to Mira crescent. Scale bar = 5 μm.(H) Distribution of spindle angle amplitude (°) in neuroblasts of wild type and ck alleles (n=58).


( A ) Selected frames from time - lapse imaging experiments ( see Movies EV1 and EV2 ) showing two dividing cells transfected with either control ( CTRL ) or CITK - specific siRNAs , respectively . Note one of the two daughter cells in the lower panel ( red arrow ) going out of focus as a consequence of oblique division . ( B ) Graphical representation of vertical and oblique divisions . The cleavage plane is indicated by a black dashed line . ( C ) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis ( oblique division ) in CITK siRNA treated HeLa cells compared to control ( n > 50 cells , 3 independent experiments ) .

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(A) Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division.(B) Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line.(C) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK siRNA treated HeLa cells compared to control (n > 50 cells, 3 independent experiments).


( D ) Control or CITK - depleted cells were immunostained for γ - tubulin ( red ) and DNA ( blue ) and imaged in z ( 0 . 3 µm - thick sections ) . Upper panel : maximum intensity projections of confocal z - stacks are shown . Lower panel : cross - section ( XZ ) through the two poles of the same cell . ( E ) Distribution of spindle angles ( ° ) in control and in CITK - depleted cells . The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip . ( n ≥ 150 cells , 6 independent experiments ) .

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(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).


( F ) Quantification of spindle angles average in control cells and in cells depleted of CITK , ASPM or co - depleted of the two proteins ( n ≥ 150 cells , in at least 3 independent experiments ) .

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(F) Quantification of spindle angles average in control cells and in cells depleted of CITK, ASPM or co-depleted of the two proteins (n ≥ 150 cells, in at least 3 independent experiments).


( A ) HeLa cells pre - extracted for 1 min with 0 . 5 % Triton X - 100 in PHEM buffer and immunostained for CITK ( green ) , γ - tubulin ( red ) and DNA ( blue ) . Scale bars ,

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(A) HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red) and DNA (blue). Scale bars, 10μm


( B ) HeLa cells expressing CITK - GFP ( green ) from a BAC transgene treated as in ( A ) and immunostained for γ - tubulin ( red ) and DNA ( blue ) . Scale bars ,

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(B) HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Scale bars, 5μm


( C ) Western blot of centrosome - containing fractions from HeLa cells , showing that CITK co - purifies with γ ‑ tubulin .

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(C) Western blot of centrosome-containing fractions from HeLa cells, showing that CITK co-purifies with γ‑tubulin.


( D ) HeLa cells pre - extracted 1 min with 0 . 5 % Triton X - 100 in PHEM buffer and immunostained for CITK ( green ) , ASPM ( red ) and DNA ( blue ) .

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(D) HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red) and DNA (blue).


( E ) HeLa cells expressing CITK - GFP treated as in ( A ) and immunostained for GFP ( green ) , ASPM ( red ) and DNA ( blue ) .

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(E) HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red) and DNA (blue).


( F ) Close physical proximity between CITK and ASPM , revealed by PLA on ASPM - GFP - expressing HeLa cells , incubated with GFP and CITK antibodies and developed with PLA - specific secondary reagents .

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(F) Close physical proximity between CITK and ASPM, revealed by PLA on ASPM-GFP-expressing HeLa cells, incubated with GFP and CITK antibodies and developed with PLA-specific secondary reagents.


( G ) Control or CITK - depleted cells immunostained for ASPM ( green ) , γ - tubulin ( red ) and DNA ( blue ) .

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(G) Control or CITK-depleted cells immunostained for ASPM (green), γ-tubulin (red) and DNA (blue).


( H ) Control or ASPM - depleted cells treated as in ( A ) and immunostained for CITK ( green ) , γ - tubulin ( red ) and DNA ( blue ) . ( I ) Quantification of CITK positive centrosomes and of the ratio of CITKspindle pole intensity versus total cell mean intensity , in control and ASPM - depleted cells . ( n ≥ 75 cells , 4 independent experiments ) .

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(H) Control or ASPM-depleted cells treated as in (A) and immunostained for CITK (green), γ-tubulin (red) and DNA (blue).(I) Quantification of CITK positive centrosomes and of the ratio of CITKspindle pole intensity versus total cell mean intensity, in control and ASPM-depleted cells. (n ≥ 75 cells, 4 independent experiments).


( A ) Control , CITK or ASPM - depleted HeLa cells immunostained for α - tubulin ( red ) and DNA ( blue ) . Maximum intensity projections of confocal z - stacks are shown . ( B ) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL , CITK or ASPM siRNA ( n > 130 cells in 3 independent experiments ) .

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(A) Control, CITK or ASPM-depleted HeLa cells immunostained for α-tubulin (red) and DNA (blue). Maximum intensity projections of confocal z-stacks are shown.(B) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL, CITK or ASPM siRNA (n > 130 cells in 3 independent experiments).


( C ) Quantification of the rescue of astral MT number after CITK overexpression in ASPM - depleted HeLa cells ( n > 50 cells in 3 independent experiments ) . ( D ) Quantification of spindle orientation rescue after overexpression of CITK , CITN ( lacking the kinase domain ) or CKD ( Citron kinase dead ) mutants in ASPM - depleted HeLa cells ( n > 50 cells in 3 independent experiments ) .

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(C) Quantification of the rescue of astral MT number after CITK overexpression in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).(D) Quantification of spindle orientation rescue after overexpression of CITK, CITN (lacking the kinase domain) or CKD (Citron kinase dead) mutants in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).


( E ) Synchronized HeLa transfected with control , ASPM - or CITK - specific siRNAs treated with DMSO or with 250 pM Paclitaxel ( taxol ) 30 min before immunostaining for α - Tubulin . Spindle angles were measured as above ( n > 100 cells in 6 independent experiments ) .

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(E) Synchronized HeLa transfected with control, ASPM- or CITK-specific siRNAs treated with DMSO or with 250 pM Paclitaxel (taxol) 30 min before immunostaining for α-Tubulin. Spindle angles were measured as above (n > 100 cells in 6 independent experiments).


( A ) Exemplar image of EB3 - tdTomatoHeLa cell used for kymograph analyses . To assess astral MT number and dynamics a line ( blue ) was added to images from the pole to the cortex .

(A) Exemplar image of EB3-tdTomatoHeLa cell used for kymograph analyses. To assess astral MT number and dynamics a line (blue) was added to images from the pole to the cortex.


( B ) Kymographs of pole ( bottom ) - to - cortex ( top ) line scans of control and CITK - depleted EB3 - tdTomatoHeLa cells . The maximum projection of a 20 - pixel - wide line ( exemplified above ) was plotted at 500 ms time intervals from left to right . MT growth results in diagonal lines .

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(B) Kymographs of pole (bottom)-to-cortex (top) line scans of control and CITK-depleted EB3-tdTomatoHeLa cells. The maximum projection of a 20-pixel-wide line (exemplified above) was plotted at 500 ms time intervals from left to right. MT growth results in diagonal lines.


( C ) Quantification of astral MT dynamics . MT stability , length , growth speed ( n > 250 MT , 30 cells ) and nucleation frequency were calculated ( n > 30 cells , 3 independent experiments ) .

(C) Quantification of astral MT dynamics. MT stability, length, growth speed (n > 250 MT, 30 cells) and nucleation frequency were calculated (n > 30 cells, 3 independent experiments).


( D ) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0 , 1 , and 2 min after nocodazole washout . Control and CITK - depleted cells were immunostained for α - tubulin ( red ) and DNA ( blue ) . ( E ) Percentage of cells showing detectable MT nucleation ( aster size > 1 µm ) 0 , 1 , and 2 min after nocodazole washout ( n > 100 cells , 4 independent experiments ) . ( F ) Quantification of aster size 2 min after nocodazole washout ( n > 100 cells , 4 independent experiments ) .

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(D) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0, 1, and 2 min after nocodazole washout. Control and CITK-depleted cells were immunostained for α-tubulin (red) and DNA (blue).(E) Percentage of cells showing detectable MT nucleation (aster size > 1 µm) 0, 1, and 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).(F) Quantification of aster size 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).


( A ) The activities ( mUnits / g of protein ) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆ 4 - CYB cells and normalized by the percentage of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 4 - 6 biological replicates ) . Unpaired Student ' s t - test * * p = 0 . 0100 ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 .

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(A) The activities (mUnits/g of protein) of the MRC enzymes were determined by spectrophotometric kinetic measurements in WT and ∆4-CYB cells and normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=4-6 biological replicates). Unpaired Student's t-test **p=0.0100; ***p=0.0002; ****p<0.0001.


( B ) Complex I in gel activity assays ( IGA ) after Blue - native gel electrophoresis ( BNGE ) of WT and ∆ 4 - CYB samples solubilized either with 1 . 6 mg DDM / mg protein or 4 mg digitonin / mg protein . The gels were incubated in the reaction mixture for 1 . 5 hours ( lighter signals in DDM gels ) or were left to continue the reaction for 24 hours to obtain darker signals ( DDM and Digitonin gels ) .

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(B) Complex I in gel activity assays (IGA) after Blue-native gel electrophoresis (BNGE) of WT and ∆4-CYB samples solubilized either with 1.6 mg DDM/mg protein or 4 mg digitonin/mg protein. The gels were incubated in the reaction mixture for 1.5 hours (lighter signals in DDM gels) or were left to continue the reaction for 24 hours to obtain darker signals (DDM and Digitonin gels).


( C ) BNGE , Western blot and immunodetection , with anti - NDUFS1 ( cI ) , anti - ATP5A ( cV ) and anti - SDHB ( cII ) antibodies , of samples from the WT cybrids and from ∆ 4 - CYB clones E ( # 17 . 3E ) and B ( # 17 . 3B ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) BNGE, Western blot and immunodetection, with anti-NDUFS1 (cI), anti-ATP5A (cV) and anti-SDHB (cII) antibodies, of samples from the WT cybrids and from ∆4-CYB clones E (#17.3E) and B (#17.3B).


( A ) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light ( H / L ) and ( L / H ) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆ 4 - CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Scatter plot generated from the peptide content analyzed by mass spectrometry in each of the 64 slices excised from BNGE and after quantifying the heavy to light (H/L) and (L/H) ratios in both reciprocal labeling experiments performed with mitochondria isolated from WT and ∆4-CYB cells The logarithmic ratios were calculated using MaxQuant and the statistical significance of the differences for the enrichment or depletion of the proteins were determined with Perseus


( B ) Labeling of the thirteen mtDNA - encoded MRC structural subunits . Cells were incubated with [ 35S ] - L - Met for one hour in the presence of emetine 100 μg / ml to inhibit cytoplasmic translation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Labeling of the thirteen mtDNA-encoded MRC structural subunits. Cells were incubated with [35S]-L-Met for one hour in the presence of emetine 100 μg/ml to inhibit cytoplasmic translation.


( C ) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 ; * * * p = 0 . 0007 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Immunodetection of complex III structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; ***p=0.0007.


( D ) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 ; * * p = 0 . 0024 ( NDUFS3 ) ; * * p = 0 . 0061 ( NDUFB8 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunodetection of complex I structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001; **p=0.0024 (NDUFS3); **p=0.0061 (NDUFB8).


( E ) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0011 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunodetection of complex IV structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0011.


( F ) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Immunodetection of complex II structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). 2-way ANOVA with Sidak's multiple comparisons test ****p<0.0001.


( G ) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS - PAGE , from three independent replicates of WT and ∆ 4 - CYB cells . The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β - Actin . The mean of the three control ( WT ) samples was set to 1 . 0 and all the measurements were referenced to that value . The values plotted in the graphs are the mean ± SD ( n = 3 ) . There were no differences in the steady - state levels of the tested subunits ( 2 - way ANOVA with Sidak ' s multiple comparisons test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunodetection of complex V structural subunits on Western blots of total cell lysates separated by SDS-PAGE, from three independent replicates of WT and ∆4-CYB cells. The graph shows the densitometric quantification of the signals corresponding to each subunit normalized to that of the β-Actin. The mean of the three control (WT) samples was set to 1.0 and all the measurements were referenced to that value. The values plotted in the graphs are the mean ± SD (n=3). There were no differences in the steady-state levels of the tested subunits (2-way ANOVA with Sidak's multiple comparisons test).


( A ) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane , setting the maximum to 1 . 0 , vs . the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve . The relative amounts of the proteins between the two cell lines was determined by calculating the H / L ratios of peptides that were present in both WT ( blue traces ) and ∆ 4 - CYB samples ( red traces ) . The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Complexome profiles of cIII2 structural subunits generated by analyzing the peptide content in each of the 64 slices in which the gel lanes were excised The graphs plot the relative peptide peak intensities along the lane, setting the maximum to 1.0, vs. the molecular mass calculated using the individual complexes and supercomplexes as the standards to generate a calibration curve. The relative amounts of the proteins between the two cell lines was determined by calculating the H/L ratios of peptides that were present in both WT (blue traces) and ∆4-CYB samples (red traces). The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Second - dimension BNGE of digitonin - solubilized samples from WT and ∆ 4 - CYB cells , western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies . The immunodetection patterns were equivalent to the complexome profiles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Second-dimension BNGE of digitonin-solubilized samples from WT and ∆4-CYB cells, western blot and immunodetection of the indicated cIII2 structural subunits with specific antibodies. The immunodetection patterns were equivalent to the complexome profiles.


( C ) Quantification of the total peak area under the curves ( AUC ) defined by the peptide intensity peaks for the indicated cIII2 subunits . The x - axis values were the slice number ( 1 - 64 ) and the y - axis values were the relative peptide intensity . The graph shows the mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0083 ( UQCRC2 ) ; * * p = 0 . 0033 ( UQCRFS1 ) ; * p = 0 . 0224 ; n . s . = non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the total peak area under the curves (AUC) defined by the peptide intensity peaks for the indicated cIII2 subunits. The x-axis values were the slice number (1-64) and the y-axis values were the relative peptide intensity. The graph shows the mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0083 (UQCRC2); **p=0.0033 (UQCRFS1); *p=0.0224; n.s.=non-significant.


( D ) Complexome profiles of two cIII2 assembly factors ( BCS1L and LYRM7 or MZM1L ) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Complexome profiles of two cIII2 assembly factors (BCS1L and LYRM7 or MZM1L) generated The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( A ) SDS - PAGE , Western blot an immunodetection , with the indicated specific antibodies , of ∆ 4 - CYB and WT cells expressing HA - tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert ( Empty ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SDS-PAGE, Western blot an immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing HA-tagged versions of UQCRQ and UQCR10 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).


( B ) BNGE , Western blot and immunodetection , with an anti - HA tag antibody , of samples from the same cell lines as in ( A ) solubilized either with digitonin or DDM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the same cell lines as in (A) solubilized either with digitonin or DDM.


( C ) BNGE , Western blot and immunodetection , with the monoclonal ( M ) anti - UQCRQ antibody ( Abcam ab110255 ) , of non - transduced ∆ 4 - CYB and WT cells . The mitoplast samples were solubilized with

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) BNGE, Western blot and immunodetection, with the monoclonal (M) anti-UQCRQ antibody (Abcam ab110255), of non-transduced ∆4-CYB and WT cells. The mitoplast samples were solubilized with DDM


( D ) Scatter plot generated from the analysis of the logarithmic heavy ( H ) to light ( L ) ratios in the x - axis and the reverse in the y - axis , in the two reciprocal labeling SILAC experiments ( 1 and 2 ) and anti - HA immunopurification of ∆ 4 - CYB and WT cells expressing UQCR10HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments (1 and 2) and anti-HA immunopurification of ∆4-CYB and WT cells expressing UQCR10HA.


( E ) Complexome profiles for the proteins found specifically enriched in ∆ 4 - CYB UQCR10HA , according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Complexome profiles for the proteins found specifically enriched in ∆4-CYB UQCR10HA, according to the SILAC immunopurification experiments The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( F ) SDS - PAGE , Western blot and immunodetection , with the indicated specific antibodies , of ∆ 4 - CYB and WT cells expressing an HA - tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert ( Empty ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) SDS-PAGE, Western blot and immunodetection, with the indicated specific antibodies, of ∆4-CYB and WT cells expressing an HA-tagged version of CYC1 and of cells transduced with the lentiviral expression vector without any cDNA insert (Empty).


( G ) BNGE , Western blot and immunodetection , with an anti - HA tag antibody , of samples from the cell lines solubilized either with digitonin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) BNGE, Western blot and immunodetection, with an anti-HA tag antibody, of samples from the cell lines solubilized either with digitonin.


( H ) Scatter plot generated from the analysis of the logarithmic heavy ( H ) to light ( L ) ratios in the x - axis and the reverse in the y - axis , in the two reciprocal labeling SILAC experiments of anti - HA immunopurification of ∆ 4 - CYB and WT cells expressing CYC1HA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Scatter plot generated from the analysis of the logarithmic heavy (H) to light (L) ratios in the x-axis and the reverse in the y-axis, in the two reciprocal labeling SILAC experiments of anti-HA immunopurification of ∆4-CYB and WT cells expressing CYC1HA.


( A ) Complexome profiles of the different cI structural modules . The graphs were generated but in this case , the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Complexome profiles of the different cI structural modules. The graphs were generated but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of the cI assembly factor NDUFAF2 in WT ( blue ) and ∆ 4 - CYB ( red ) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Complexome profiles of the cI assembly factor NDUFAF2 in WT (blue) and ∆4-CYB (red) mitochondria The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) Quantification of the total area under the curve in the profiles corresponding to each cI module ( top graph ) . The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules . The plotted values are the mean mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * * p = 0 . 0006 ; * * p = 0 . 0012 ( N - module ) ; * * p = 0 . 0016 ( ND1 - module ) ; * * p = 0 . 0032 ( ND2 - module ) ; * * p = 0 . 0057 ( ND4 - module ) ; * * p = 0 . 0020 ( ND5 - module ) ; * p = 0 . 0140 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of the total area under the curve in the profiles corresponding to each cI module (top graph). The area of the NDUFA9 subunit was calculated separately as its profile did not correspond to any of the other structural subunit modules. The plotted values are the mean mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test ***p=0.0006; **p=0.0012 (N-module); **p=0.0016 (ND1-module); **p=0.0032 (ND2-module); **p=0.0057 (ND4-module); **p=0.0020 (ND5-module); *p=0.0140.


( D ) Quantification of the area of the N - module peak corresponding to the molecular mass of the fully assembled cI : 1 , 172 kDa in ∆ 4 - CYB samples and the respirasome ( cI + cIII2 + cIV ) SCs in # 4 . 1 WT samples ( 2 , 110 kDa ) . The plotted values are the mean mean ± SD ( n = 2 ) . Unpaired Student ' s t - test * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of the area of the N-module peak corresponding to the molecular mass of the fully assembled cI: 1,172 kDa in ∆4-CYB samples and the respirasome (cI+cIII2+cIV) SCs in #4.1 WT samples (2,110 kDa). The plotted values are the mean mean ± SD (n=2). Unpaired Student's t-test ****p<0.0001.


( A ) SDS - PAGE resolving the radioactively labeled mitochondrial translation products after a 2 - hour pulse ( P ) . The 35S - Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times ( 2 , 5 and 24 hours ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) SDS-PAGE resolving the radioactively labeled mitochondrial translation products after a 2-hour pulse (P). The 35S-Met and the cycloheximide were removed from the medium and cells were collected at the indicated chase times (2, 5 and 24 hours).


( B ) First dimension ( 1D ) BNGE analysis of the same cells prepared with digitonin .

(B) First dimension (1D) BNGE analysis of the same cells prepared with digitonin.


( C ) Denaturing second dimension ( 2D ) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Denaturing second dimension (2D) BNGE analysis of the same samples allowed following the incorporation of the individual labeled subunits inside their corresponding complex and supercomplex species.


( D ) Complex I IGA ( cI - IGA ) analysis of digitonin - solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days ( 0h ) . After removing the drug and restoring synthesis of the mtDNA - encoded subunits , the cells were collected at the indicated times to follow the appearance of cI reactivity with time . The gels were incubated in the reaction mixture for 24 hours . SS = steady state . The asterisk indicates the presence of a high molecular weight cI - containing band of unknown

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Complex I IGA (cI-IGA) analysis of digitonin-solubilized samples after inhibiting mitochondrial translation with doxycycline for 6 days (0h). After removing the drug and restoring synthesis of the mtDNA-encoded subunits, the cells were collected at the indicated times to follow the appearance of cI reactivity with time. The gels were incubated in the reaction mixture for 24 hours. SS = steady state. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature


( E ) 2D BNGE , Western blot and immunodetection analysis of WT and ∆ 4 - CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear - encoded subunits , belonging to different structural modules . The blots shown were either exposed for 16 sec . ( low exposures ) or 160 sec . ( high exposures ) in order to visualize the qualitative signals in the ∆ 4 - CYB samples .

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

(E) 2D BNGE, Western blot and immunodetection analysis of WT and ∆4-CYB mitochondria from cells collected at the same times after doxycycline treatment to follow the incorporation kinetics of the indicated cI nuclear-encoded subunits, belonging to different structural modules. The blots shown were either exposed for 16 sec. (low exposures) or 160 sec. (high exposures) in order to visualize the qualitative signals in the ∆4-CYB samples.


( A ) SDS - PAGE , Western blot and immunodetection of AOXHA expression in whole - cell lysates from WT and ∆ 4 - CYB cells transduced with the AOXHA / pWPXLd - ires - HygroR lentiviral vector . The transduction and selection controls were the same cell lines transfected with an empty pWPXLd - ires - HygroR vector ( EV ) .

(A) SDS-PAGE, Western blot and immunodetection of AOXHA expression in whole-cell lysates from WT and ∆4-CYB cells transduced with the AOXHA/pWPXLd-ires-HygroR lentiviral vector. The transduction and selection controls were the same cell lines transfected with an empty pWPXLd-ires-HygroR vector (EV).


( B ) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls . Cell growth was monitored every six hours after substituting the medium in two replicate 24 - well plates , one plate with medium without uridine ( Uridine - ) , and the second plate with medium supplemented with 50 µg / ml uridine ( Uridine + ) . The graphs show the average confluence ± SD at each time point ( n = 6 wells per cell line ) .

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

(B) Growth curves of the AOXHA expressing cell lines and their corresponding EV controls. Cell growth was monitored every six hours after substituting the medium in two replicate 24-well plates, one plate with medium without uridine (Uridine-), and the second plate with medium supplemented with 50 µg/ml uridine (Uridine+). The graphs show the average confluence ± SD at each time point (n=6 wells per cell line).


( C ) Immunodetection of cI structural subunits and NDUFAF2 in the same

(C) Immunodetection of cI structural subunits and NDUFAF2 in the same samples


( D ) 1D BNGE , Western blot and immunodetection analyses of digitonin - solubilized mitochondria from WT and ∆ 4 - CYB cybrids expressing AOXHA and their corresponding EV controls .

(D) 1D BNGE, Western blot and immunodetection analyses of digitonin-solubilized mitochondria from WT and ∆4-CYB cybrids expressing AOXHA and their corresponding EV controls.


( E ) Complex I in gel activity assays ( IGA ) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours . The asterisk indicates the presence of a high molecular weight cI - containing band of unknown

(E) Complex I in gel activity assays (IGA) after BNGE The gels were incubated in the IGA reaction mixture for 5 hours. The asterisk indicates the presence of a high molecular weight cI-containing band of unknown nature


( F ) Spectrophotometric kinetic measurements of cI activity in WT and ∆ 4 - CYB AOXHA and EV samples , normalized by the percentage of citrate synthase ( CS ) activity . Results are expressed as mean ± SD ( n = 5 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * p = 0 . 0077 ( WT AOXHA vs . ∆ 4 - CYB AOXHA ) ; * * p = 0 . 0044 ( ∆ 4 - CYB EV vs . ∆ 4 - CYB AOXHA ) ; * * * p = 0 . 0003 ; * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Spectrophotometric kinetic measurements of cI activity in WT and ∆4-CYB AOXHA and EV samples, normalized by the percentage of citrate synthase (CS) activity. Results are expressed as mean ± SD (n=5 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test **p=0.0077 (WT AOXHA vs. ∆4-CYB AOXHA); **p=0.0044 (∆4-CYB EV vs. ∆4-CYB AOXHA); ***p=0.0003; ****p<0.0001.


( G ) High resolution respirometry analyses performed in intact cells in an Oroboros instrument . ROUTINE : cellular basal oxygen consumption rate ( in pmol O2 / sec ) per million cells in DMEM medium . LEAK is the non - phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin . ETS : Maximal respiration rate in the presence of the uncoupler CCCP . AOX : oxygen consumption in the presence of antimycin A , inhibiting cIII2 activity but not that of AOX . ROTENONE : Oxygen consumption in the presence of the cI inhibitor rotenone . In all cases this was equal to the background . Results are expressed as mean ± SD ( n = 2 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * * * p < 0 . 0001 .

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

(G) High resolution respirometry analyses performed in intact cells in an Oroboros instrument. ROUTINE: cellular basal oxygen consumption rate (in pmol O2/sec) per million cells in DMEM medium. LEAK is the non-phosphorylating respiration in the presence of the ATP synthase inhibitor oligomycin. ETS: Maximal respiration rate in the presence of the uncoupler CCCP. AOX: oxygen consumption in the presence of antimycin A, inhibiting cIII2 activity but not that of AOX. ROTENONE: Oxygen consumption in the presence of the cI inhibitor rotenone. In all cases this was equal to the background. Results are expressed as mean ± SD (n=2 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( H ) Respirometry analyses , in WT EV controls and AOXHA - expressing cells untreated or treated with 2 . 5 µM antimycin A for 7 days ( + AA ) . Results are expressed as mean ± SD ( n = 4 biological replicates ) . 2 - way ANOVA with Tukey ' s multiple comparisons test * * * * p < 0 . 0001 .

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

(H) Respirometry analyses , in WT EV controls and AOXHA- expressing cells untreated or treated with 2.5 µM antimycin A for 7 days (+AA). Results are expressed as mean ± SD (n=4 biological replicates). 2-way ANOVA with Tukey's multiple comparisons test ****p<0.0001.


( I ) 1D BNGE followed by cI - IGA ( right ) or Western blot and immunodetection of cIII2 subunit CYC1 ( left ) in digitonin - solubilized samples from WT EV controls and AOXHA - expressing cells untreated ( - ) or treated ( + ) with 2 . 5 µM antimycin A for 7 days .

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

(I) 1D BNGE followed by cI-IGA (right) or Western blot and immunodetection of cIII2 subunit CYC1 (left) in digitonin-solubilized samples from WT EV controls and AOXHA- expressing cells untreated (-) or treated (+) with 2.5 µM antimycin A for 7 days.


( A ) Complexome profiles of the two cV structural and assembly modules . The graphs were generated as in Figure 3 but in this case , the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis . The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Complexome profiles of the two cV structural and assembly modules. The graphs were generated as in Figure 3 but in this case, the peptide intensity values for the individual subunits belonging to each module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( B ) Complexome profiles of the three detected cII subunits . The represented values are the mean ± SEM of the two reciprocal labeling experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Complexome profiles of the three detected cII subunits. The represented values are the mean ± SEM of the two reciprocal labeling experiments.


( C ) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated ( NDUFA4 ) . , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis . The represented values are the mean ± SEM of the two reciprocal labeling experiments . The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module . The plotted values are mean ± SD ( n = 2 ) . 2 - way ANOVA with Sidak ' s multiple comparisons test * * p = 0 . 0088 ( Early ) ; * * p = 0 . 0063 ( MT - CO2 ) ; * * p = 0 . 0040 ( NDUFA4 ) ; * p = 0 . 0264 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Complexome profiles of the different cIV assembly modules and of the last subunit to be incorporated (NDUFA4). , the peptide intensity values for the individual subunits belonging to each assembly module were averaged to simplify the analysis. The represented values are the mean ± SEM of the two reciprocal labeling experiments. The bar graph represents the quantification of the total area under the curve in the profiles corresponding to each cIV module. The plotted values are mean ± SD (n=2). 2-way ANOVA with Sidak's multiple comparisons test **p=0.0088 (Early); **p=0.0063 (MT-CO2); **p=0.0040 (NDUFA4); *p=0.0264.


( D ) 1D BNGE , Western blot and immunodetection of two cIV subunits ( MT - CO1 and COX7B ) in samples from WT and ∆ 4 - CYB cells solubilized with DDM and digitonin ( Dig ) .

(D) 1D BNGE, Western blot and immunodetection of two cIV subunits (MT-CO1 and COX7B) in samples from WT and ∆4-CYB cells solubilized with DDM and digitonin (Dig).


D ) Percentage of zone 1 - 3 uninfected hepatocytes day 5 post plating ( + / - s . d . ) of 4 different donors ( blue dots ) . For each donor , > 500 cells were characterized for the final percentages . Kruskal Wallis test ( p = 0 . 0002 ) followed by Dunn ' s multiple comparisons test where Z1 / hGK + + / hGS - versus Z2 / hGK + / hGS - has a p value of 0 . 0140 ( the other comparisons of Z1 / hGK + + / hGS - vs Z3 / hGK - / hGS + and Z2 / hGK + / hGS - vs Z3 / hGK - / hGS + were non - significant ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Percentage of zone 1-3 uninfected hepatocytes day 5 post plating (+/- s.d.) of 4 different donors (blue dots). For each donor, >500 cells were characterized for the final percentages. Kruskal Wallis test (p = 0.0002) followed by Dunn's multiple comparisons test where Z1/hGK++/hGS- versus Z2/hGK+/hGS- has a p value of 0.0140 (the other comparisons of Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ were non-significant).


C : Expression of typical liver stage protein markers : Circumsporozoite protein ( CSP ) top left ; Exported Protein 2 ( EXP2 ) top right ; Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) bottom left ; and Merozoite surface protein 1 ( MSP1 ) bottom right . Scale bar is 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C: Expression of typical liver stage protein markers: Circumsporozoite protein (CSP) top left; Exported Protein 2 (EXP2) top right; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bottom left; and Merozoite surface protein 1 (MSP1) bottom right. Scale bar is 25 µm.


D ) Percentage schizonts ( from total number of schizonts in a well + / - s . d . ) located in the different hepatocyte sub - populations by NF54 , NF135 , NF175 on day 3 p . i . . Each dot represents a biological replicate . For each parasite line , at least 100 schizonts were characterized from 2x 96 wells for the final percentage . The values from three biological replicates were used in a RM ANOVA test followed by a Tukey ' s multiple comparison test . Percentage of schizonts in Z1 / hGK + + / hGS - hepatocytes is not different between the three parasite strains . Percentage of schizonts in Z2 / hGK + / hGS - for NF54 is significantly different from NF135 ( p = 0 . 0015 ) and NF175 ( p = 0 . 0015 ) . Percentage of schizonts in Z3 / hGK - / hGS + hepatocytes for NF54 is significantly different from NF135 ( p = 0 . 0002 ) and NF175 ( p = 0 . 0 . 0002 ) . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Percentage schizonts (from total number of schizonts in a well +/- s.d.) located in the different hepatocyte sub-populations by NF54, NF135, NF175 on day 3 p.i.. Each dot represents a biological replicate. For each parasite line, at least 100 schizonts were characterized from 2x 96 wells for the final percentage. The values from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Percentage of schizonts in Z1/hGK++/hGS- hepatocytes is not different between the three parasite strains. Percentage of schizonts in Z2/hGK+/hGS- for NF54 is significantly different from NF135 (p = 0.0015) and NF175 (p = 0.0015). Percentage of schizonts in Z3/hGK-/hGS+ hepatocytes for NF54 is significantly different from NF135 (p = 0.0002) and NF175 (p = 0.0.0002). Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


F ) Size of schizonts in Z1 / hGK + + / hGS - , Z2 / hGK + / hGS - and Z3 / hGK - / hGS + hepatocytes on day 3 post invasion for the different Pf strains . For each parasite line , at least 100 schizonts were measured from each biological replicate . The median values + / - s . d . ( as the sizes within the population is not normally distributed ) from three biological replicates were used in a RM ANOVA test followed by a Tukey ' s multiple comparison test . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F) Size of schizonts in Z1/hGK++/hGS-, Z2/hGK+/hGS- and Z3/hGK-/hGS+ hepatocytes on day 3 post invasion for the different Pf strains. For each parasite line, at least 100 schizonts were measured from each biological replicate. The median values +/- s.d. (as the sizes within the population is not normally distributed) from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


hGS interaction with NF135 ( A ) , NF175 ( B ) schizonts on days 3 ( top ) , 5 ( middle ) and 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) and phalloidin ( grey ) . The U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hGS interaction with NF135 (A), NF175 (B) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


hGS interaction with NF54 ( C ) schizonts on days 3 ( top ) , 5 ( middle ) and 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) and phalloidin ( grey ) . The U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

hGS interaction with NF54 (C) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


D ) Median sizes ( + / - s . d . ) of NF135 ( green ) , NF175 ( purple ) and NF54 ( blue ) schizonts divided based on the presence ( solid line ) or absence ( dashed line ) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates . A two - way RM ANOVA was performed on the median followed by a Tukey ' s multiple comparisons test . The significant differences between hGS + and hGS - population within a strain is showed with asterisks ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . See Appendix Fig S1 for the raw measurements .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Median sizes (+/- s.d.) of NF135 (green), NF175 (purple) and NF54 (blue) schizonts divided based on the presence (solid line) or absence (dashed line) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates. A two-way RM ANOVA was performed on the median followed by a Tukey's multiple comparisons test. The significant differences between hGS+ and hGS- population within a strain is showed with asterisks (* p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001). See Appendix Fig S1 for the raw measurements.


A ) The total hGS signal within intracellular NF135 and B ) NF175 schizonts on day 3 and 5 . Each graph represents a biological replicate where each dot represents an individual schizont .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) The total hGS signal within intracellular NF135 and B) NF175 schizonts on day 3 and 5. Each graph represents a biological replicate where each dot represents an individual schizont.


A ) Normalized percentage + / - s . d . of intracellular schizont size of NF54 ( blue ) and NF135 ( green ) after treatment of GS inhibitors at different concentrations of AIP . Median size of the schizonts were normalized to that of H2O - treated NF135 ( which is set at 100 % ) . AIP was added one day post invasion and kept on for 48 hours . Each dot represents a biological replicate where ≥ 100 schizonts were measured on day 5 p . i . . A two - way ANOVA was performed followed by a Tukey ' s multiple comparison test with the * * * * < 0 . 0001 to compare conditions within the same parasite strain . A two - way ANOVA was performed followed by a Sidak ' s multiple comparisons test with * * * * < 0 . 0001 to compare conditions between parasite strains ) .

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

A) Normalized percentage +/- s.d. of intracellular schizont size of NF54 (blue) and NF135 (green) after treatment of GS inhibitors at different concentrations of AIP. Median size of the schizonts were normalized to that of H2O-treated NF135 (which is set at 100%). AIP was added one day post invasion and kept on for 48 hours. Each dot represents a biological replicate where ≥100 schizonts were measured on day 5 p.i.. A two-way ANOVA was performed followed by a Tukey's multiple comparison test with the **** < 0.0001 to compare conditions within the same parasite strain. A two-way ANOVA was performed followed by a Sidak's multiple comparisons test with **** <0.0001 to compare conditions between parasite strains).


B ) Size difference between the GS positive ( closed circle ) and negative ( open circle ) NF135 schizonts after treatment of AIP . Each dot represents the median of a biological replicate where ≥ 100 schizonts were measured . Three biological replicates were performed with the median from each plotted + / - s . d . . A two - way ANOVA was performed followed by a Dunnett ' s multiple comparison test with * * * = 0 . 0002 and * * * * = 0 . 0001 .

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

B) Size difference between the GS positive (closed circle) and negative (open circle) NF135 schizonts after treatment of AIP. Each dot represents the median of a biological replicate where ≥100 schizonts were measured. Three biological replicates were performed with the median from each plotted +/- s.d.. A two-way ANOVA was performed followed by a Dunnett's multiple comparison test with *** = 0.0002 and **** = 0.0001.


A . Tunicamycin ( Tn , 2 µg / ml ) increased Nox4 protein levels in H9c2 cells . Tubulin was used as a loading control . n = 4 - 6 / group . * , significant compared to baseline .

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

A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.


B . Thapsigargin ( Tp , 1 µg / ml ) increased Nox4 protein levels in H9c2 cells . n = 4 - 6 / group . * , significant compared to baseline .

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

B. Thapsigargin (Tp, 1 µg/ml) increased Nox4 protein levels in H9c2 cells. n=4-6/group. *, significant compared to baseline.


C . Effect of Nox4 on the unfolded protein response . Nox4 was depleted in H9c2 cells by shRNA - mediated knockdown ( Ad . shNox4 ) or cells were treated with a control adenovirus ( Ad . Ctl ) . In cells with Nox4 knockdown , tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94 , Grp78 and calreticulin than in conrol cells . Nuclear protein levels of ATF4 were substantially lower in Nox4 - depleted cells than control cells but the levels of cleaved ATF6 ( ATF6c ) were similar . Histone was used as a loading control . The relative mRNA levels of Xbp1 - s ( a readout of IRE1 signaling ) were unaltered after Nox4 knockdown . Mean data are shown in Appendix Fig S1E . Similar results were obtained with an independent siRNA approach ( Appendix Fig S2A ) . D . Effect of adenoviral - mediated overexpression of Nox4 ( Ad . Nox4 ) or a control β - galactosidase protein ( Ad . β - Gal ) on tunicamycin responses of H9c2 cells . Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin - induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels . Mean data are shown in Appendix Fig S2B .

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

C. Effect of Nox4 on the unfolded protein response. Nox4 was depleted in H9c2 cells by shRNA-mediated knockdown (Ad.shNox4) or cells were treated with a control adenovirus (Ad.Ctl). In cells with Nox4 knockdown, tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94, Grp78 and calreticulin than in conrol cells. Nuclear protein levels of ATF4 were substantially lower in Nox4-depleted cells than control cells but the levels of cleaved ATF6 (ATF6c) were similar. Histone was used as a loading control. The relative mRNA levels of Xbp1-s (a readout of IRE1 signaling) were unaltered after Nox4 knockdown. Mean data are shown in Appendix Fig S1E. Similar results were obtained with an independent siRNA approach (Appendix Fig S2A).D. Effect of adenoviral-mediated overexpression of Nox4 (Ad.Nox4) or a control β-galactosidase protein (Ad.β-Gal) on tunicamycin responses of H9c2 cells. Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin-induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels. Mean data are shown in Appendix Fig S2B.


E , F . Effect of Nox4 knockdown or overexpression , respectively , on the tunicamycin - induced changes in mRNA levels of ATF4 target genes . n = 4 / group . Psat1 , phosphoserine aminotransferase ; Phgdh , 3 - phosphoglycerate dehydrogenase ; Asns , asparagine synthetase ; Slc6a9 , glycine transporter 1 .

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

E,F. Effect of Nox4 knockdown or overexpression, respectively, on the tunicamycin-induced changes in mRNA levels of ATF4 target genes. n=4/group. Psat1, phosphoserine aminotransferase; Phgdh, 3-phosphoglycerate dehydrogenase; Asns, asparagine synthetase; Slc6a9, glycine transporter 1.


G . Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin - treated H9c2 cells . Scrambled siRNAs were used as a control ( Ctl ) . Representative immunoblots shown to the top ( captions at bottom of bar graphs refer also to the immunoblots ) ; tubulin was used as a loading control . n = 4 / group . * , significant compared to baseline ; # , significant comparing siATF4 versus corresponding siCtl .

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

G. Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin-treated H9c2 cells. Scrambled siRNAs were used as a control (Ctl). Representative immunoblots shown to the top (captions at bottom of bar graphs refer also to the immunoblots); tubulin was used as a loading control. n=4/group. *, significant compared to baseline; #, significant comparing siATF4 versus corresponding siCtl.


H . Effect of ATF4 overexpression on Nox4 mRNA and protein levels . n = 3 / group .

H. Effect of ATF4 overexpression on Nox4 mRNA and protein levels. n=3/group.


A . The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin - induced eIF2a phosphorylation in H9c2 cells , with no change in phospho - Thr980 - PERK ( PERK - P ) levels . GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels . B . Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin - induced eIF2a phosphorylation , with minimal change in phospho - PERK levels . C , D . Mean levels of phosphorylated eIF2a relative to total eIF2a protein in tunicamycin - treated cells after Nox4 knockdown or overexpression , respectively . n = 3 / group . * , significant compared to baseline ; # , significant comparing Nox4 knockdown ( Ad . shNox4 ) or overexpression ( Ad . Nox4 ) versus corresponding controls ( Ad . Ctl or Ad . β - Gal , respectively ) .

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A. The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin-induced eIF2a phosphorylation in H9c2 cells, with no change in phospho-Thr980-PERK (PERK-P) levels. GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels.B. Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin-induced eIF2a phosphorylation, with minimal change in phospho-PERK levels.C,D. Mean levels of phosphorylated eIF2a relative to total eIF2a protein in tunicamycin-treated cells after Nox4 knockdown or overexpression, respectively. n=3/group. *, significant compared to baseline; #, significant comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).


E , F . Effect of Nox4 knockdown or overexpression , respectively , on okadaic acid - resistant Ser / Thr phosphatase activity in membrane fractions of tunicamycin - treated H9c2 cells . n = 4 / group . * p < 0 . 05 , * * p < 0 . 01 cf . baseline ; # , p < 0 . 05 , # # , p < 0 . 01 comparing Nox4 knockdown ( Ad . shNox4 ) or overexpression ( Ad . Nox4 ) versus corresponding controls ( Ad . Ctl or Ad . β - Gal , respectively ) .

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E,F. Effect of Nox4 knockdown or overexpression, respectively, on okadaic acid-resistant Ser/Thr phosphatase activity in membrane fractions of tunicamycin-treated H9c2 cells. n=4/group. *p<0.05, **p<0.01 cf. baseline; #, p<0.05, ##, p<0.01 comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).


G , H . Nox4 knockdown or overexpression , respectively , had no effect on the phosphorylation of glycogen synthase at Ser641 ( GS - P ) or Histone H3 at Ser57 ( H3 - P ) in H9c2 cells .

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G,H. Nox4 knockdown or overexpression, respectively, had no effect on the phosphorylation of glycogen synthase at Ser641 (GS-P) or Histone H3 at Ser57 (H3-P) in H9c2 cells.


I . Nox4 - / - MEF cells ( KO ) showed blunted tunicamycin - induced increases in levels of phospho - eIF2a , ATF4 and ER chaperones as compared to wild - type ( WT ) MEFs , a response that was rescued by re - introduction of Nox4 ( KO + Nox4 ) . The latter had no effect on GS - P or H3 - P levels .

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I. Nox4-/- MEF cells (KO) showed blunted tunicamycin-induced increases in levels of phospho-eIF2a, ATF4 and ER chaperones as compared to wild-type (WT) MEFs, a response that was rescued by re-introduction of Nox4 (KO+Nox4). The latter had no effect on GS-P or H3-P levels.


A . Sub - cellular localization of Nox4 . Tunicamycin ( Tn 2 µg / ml , 6h ) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy ( scale bar , 10 µm ) . 3D SIM images ( scale bars , 2 µm ) showed localisation of Nox4 ( green ) to the ER , which was labeled with an anti - KDEL antibody ( red ) . At higher magnification ( right ) , yellow dots denote co - localization of Nox4 and KDEL signals . Cell nuclei were stained with DAPI ( blue ) . 1 Z slice from 3D stack is shown .

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A. Sub-cellular localization of Nox4. Tunicamycin (Tn 2 µg/ml, 6h) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy (scale bar, 10 µm).3D SIM images (scale bars, 2 µm) showed localisation of Nox4 (green) to the ER, which was labeled with an anti-KDEL antibody (red). At higher magnification (right), yellow dots denote co-localization of Nox4 and KDEL signals. Cell nuclei were stained with DAPI (blue). 1 Z slice from 3D stack is shown.


B . Progressive enrichment of GADD34 , PP1 , eIF2α and Nox4 in membrane fractions of tunicamycin - treated H9c2 cells .

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B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells.


C . After sucrose gradient fractionation of lysates of tunicamycin - treated H9c2 cells , GADD34 , PP1 and Nox4 co - eluted in fractions 12 and 13 ( F12 , F13 ) .

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C. After sucrose gradient fractionation of lysates of tunicamycin-treated H9c2 cells, GADD34, PP1 and Nox4 co-eluted in fractions 12 and 13 (F12, F13).


D . Immunoprecipitation ( IP ) of pooled fractions 12 / 13 with an anti - GADD34 antibody revealed the presence of both PP1 and Nox4 .

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D. Immunoprecipitation (IP) of pooled fractions 12/13 with an anti-GADD34 antibody revealed the presence of both PP1 and Nox4.


E . The association of Nox4 with GADD34 was validated in HEK293 cells co - transfected with Nox4 and either Flag - tagged or non - tagged GADD34 , followed by IP with an anti - Flag antibody .

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E. The association of Nox4 with GADD34 was validated in HEK293 cells co-transfected with Nox4 and either Flag-tagged or non-tagged GADD34, followed by IP with an anti-Flag antibody.


F . Co - transfection of HEK293 cells with GADD34 - Flag and different myc - tagged Nox4 constructs , followed by IP with an anti - myc antibody . GADD34 binds to full - length Nox4 ( FL ) and the Nox4 transmembrane domain ( TD ) but not the C - terminal domain ( CD ) .

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F. Co-transfection of HEK293 cells with GADD34-Flag and different myc-tagged Nox4 constructs, followed by IP with an anti-myc antibody. GADD34 binds to full-length Nox4 (FL) and the Nox4 transmembrane domain (TD) but not the C-terminal domain (CD).


G . Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto , respectively , in tunicamycin - treated MEF cells . Redox - insensitive mutant probes were used as negative controls and to exclude pH changes . Extracellular H2O2 ( 200 nM ) was added as a positive control . The pseudocolor scale is shown along the left vertical edge of each image . KO = Nox4 - / - . Scale bars , 2 µm .

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G. Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto, respectively, in tunicamycin-treated MEF cells. Redox-insensitive mutant probes were used as negative controls and to exclude pH changes. Extracellular H2O2 (200 nM) was added as a positive control. The pseudocolor scale is shown along the left vertical edge of each image. KO = Nox4-/-. Scale bars, 2 µm.


H . Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity ( bar graph ) and decreased phospho - eIF2 levels ( immunoblots ) . Co - transfection of full - length Nox4 reduced PP1 activity and increased phospho - eIF2 levels ( captions at bottom refer both to the bar graphs and immunoblots ) . These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain ( TD ) were transfected . Nox4 did not affect phosphorylation of glycogen synthase ( GS - P ) or histone H3 ( H3 - P ) . All experiments were n = 3 / group . Values below the immunoblots are mean ± SEM levels for phospho - eIF2 / total - eIF2 . * , p < 0 . 05 comparing third and fourth lanes ; Levels of significance for comparisons of PP1 activities are shown above the bar columns . See also Fig EV1 and Appendix Fig S4 .

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H. Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity (bar graph) and decreased phospho-eIF2 levels (immunoblots). Co-transfection of full-length Nox4 reduced PP1 activity and increased phospho-eIF2 levels (captions at bottom refer both to the bar graphs and immunoblots). These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain (TD) were transfected. Nox4 did not affect phosphorylation of glycogen synthase (GS-P) or histone H3 (H3-P). All experiments were n=3/group. Values below the immunoblots are mean ± SEM levels for phospho-eIF2/total-eIF2. *, p<0.05 comparing third and fourth lanes; Levels of significance for comparisons of PP1 activities are shown above the bar columns. See also Fig EV1 and Appendix Fig S4.


A . Recombinant PP1 was inhibited by H2O2 ( 0 . 2 mM ) and activity was not restored by glutathione ( GSH ) , cysteine ( Cys ) or dithiothreitol ( DTT ) . A Cys127Ser / Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild - type PP1 . Values above bars denote level of significance for the inhibitory effect of H2O2 .

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A. Recombinant PP1 was inhibited by H2O2 (0.2 mM) and activity was not restored by glutathione (GSH), cysteine (Cys) or dithiothreitol (DTT). A Cys127Ser/Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild-type PP1. Values above bars denote level of significance for the inhibitory effect of H2O2.


B . Ascorbate ( Asc ) dose - dependently restored PP1 activity . # , significant effect of Asc compared to H2O2 alone .

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B. Ascorbate (Asc) dose-dependently restored PP1 activity. #, significant effect of Asc compared to H2O2 alone.


C . EPR spectra of PP1 incubated with ascorbate ( 1 mM ) alone ( a ) or PP1 exposed to H2O2 followed by catalase treatment , then incubation with ascorbate ( b ) . Panel b shows a typical spectrum for the ascorbyl radical ( hyperfine splitting constant , aH = 1 . 8G ) , similar to the positive control obtained by exposing ascorbate to H2O2 ( c ) . Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1 , prior to ascorbate addition .

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C. EPR spectra of PP1 incubated with ascorbate (1 mM) alone (a) or PP1 exposed to H2O2 followed by catalase treatment, then incubation with ascorbate (b). Panel b shows a typical spectrum for the ascorbyl radical (hyperfine splitting constant, aH = 1.8G), similar to the positive control obtained by exposing ascorbate to H2O2 (c). Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1, prior to ascorbate addition.


D . Cartoon representation of the active site of H2O2 - treated PP1 as in Fig EV2D . 2mFo - DFc electron density map at the 2 . 2 Å resolution is shown in yellow at the 1 . 1 level . H2O2 - treatment causes an overall shrinkage of the PP1 coordination sphere by 0 . 12 Å compared to ascorbate - treated crystals consistent with the oxidation of the dinuclear center . This increases the energy barrier for the catalytic steps involving - OH - attack on the phosphorous center of the bridging phosphate and rupture of the P - O scissile bond with the assistance of H125 ( black arrows ) . Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit . See also Appendix Table S1 .

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D. Cartoon representation of the active site of H2O2-treated PP1 as in Fig EV2D. 2mFo-DFc electron density map at the 2.2 Å resolution is shown in yellow at the 1.1 level. H2O2-treatment causes an overall shrinkage of the PP1 coordination sphere by 0.12 Å compared to ascorbate-treated crystals consistent with the oxidation of the dinuclear center. This increases the energy barrier for the catalytic steps involving -OH- attack on the phosphorous center of the bridging phosphate and rupture of the P-O scissile bond with the assistance of H125 (black arrows). Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit. See also Appendix Table S1.


E . Effect of Nox4 on GADD34 / PP1 - mediated eIF2 dephosphorylation in transfected HEK293 cells . Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1 , and resulted in even higher phospho - eIF2α levels in cells transfected with N124D or D64N PP1 variants .

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E. Effect of Nox4 on GADD34/PP1-mediated eIF2 dephosphorylation in transfected HEK293 cells. Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1, and resulted in even higher phospho-eIF2α levels in cells transfected with N124D or D64N PP1 variants.


F . Effect of ascorbate ( Asc , 0 . 5 mM ) on phosphatase inhibition in tunicamycin - treated H9c2 cells with overexpression or knockdown of Nox4 ( Ad . Nox4 and Ad . shNox4 , respectively ) . In control cells , ascorbate enhanced tunicamycin - stimulated increases in phosphatase activity . Phosphatase activity was lower in Nox4 - overexpressing than control cells but was normalized by ascorbate to the same level as in control cells . In Nox4 knockdown cells , tunicamycin - induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect .

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F. Effect of ascorbate (Asc, 0.5 mM) on phosphatase inhibition in tunicamycin-treated H9c2 cells with overexpression or knockdown of Nox4 (Ad.Nox4 and Ad.shNox4, respectively). In control cells, ascorbate enhanced tunicamycin-stimulated increases in phosphatase activity. Phosphatase activity was lower in Nox4-overexpressing than control cells but was normalized by ascorbate to the same level as in control cells. In Nox4 knockdown cells, tunicamycin-induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect.


A . H9c2 cells treated with tunicamycin ( 2 µg / ml , 12h ) showed significantly lower survival when endogenous Nox4 was silenced ( siNox4 ) as compared to cells treated with a scrambled siRNA ( siCtl ) . Cell survival was restored by treatment with either guanabenz ( Gbz , 5 µM ) or salubrinal ( Sal , 50 µM ) but was unaffected by clonidine ( Cld , 5 µM ) . n = 3 / group .

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A. H9c2 cells treated with tunicamycin (2 µg/ml, 12h) showed significantly lower survival when endogenous Nox4 was silenced (siNox4) as compared to cells treated with a scrambled siRNA (siCtl). Cell survival was restored by treatment with either guanabenz (Gbz, 5 µM) or salubrinal (Sal, 50 µM) but was unaffected by clonidine (Cld, 5 µM). n=3/group.


B . Nox4 - depleted cells had lower levels of phospho - eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz ( Gbz ) .

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B. Nox4-depleted cells had lower levels of phospho-eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz (Gbz).


D . Hearts from Nox4 knockout ( KO ) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion ( I / R ) . Infarct size assessed by triphenyltetrazolium chloride ( TTC ) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz ( Gbz ) . In the representative heart sections shown at the top , white denotes infarct area and red the viable area . Scale bars , 1 mm . Numbers of hearts are indicated within the bars .

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D. Hearts from Nox4 knockout (KO) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion (I/R). Infarct size assessed by triphenyltetrazolium chloride (TTC) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz (Gbz). In the representative heart sections shown at the top, white denotes infarct area and red the viable area. Scale bars, 1 mm. Numbers of hearts are indicated within the bars.


E . Immunoblotting of heart homogenates after I / R showed lower levels of phospho - eIF2a , ATF4 and ER chaperones , and higher levels of cleaved caspase - 12 , in Nox4 KO compared to WT . Tubulin was used as a loading control . Treatment with guanabenz ( Gbz ) reversed these changes ( blots shown to the right ) .

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E. Immunoblotting of heart homogenates after I/R showed lower levels of phospho-eIF2a, ATF4 and ER chaperones, and higher levels of cleaved caspase-12, in Nox4 KO compared to WT. Tubulin was used as a loading control. Treatment with guanabenz (Gbz) reversed these changes (blots shown to the right).


A . Plasma urea levels were elevated to a greater extent in tunicamycin - treated Nox4 KO mice than WT . Co - treatment with guanabenz ( Gbz ) reduced urea levels in both groups . Numbers of animals are indicated within bars .

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A. Plasma urea levels were elevated to a greater extent in tunicamycin-treated Nox4 KO mice than WT. Co-treatment with guanabenz (Gbz) reduced urea levels in both groups. Numbers of animals are indicated within bars.


B . 48 hours after systemic tunicamycin treatment , kidneys of Nox4 KO mice showed a marked surface pallor ( bottom right ) .

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B. 48 hours after systemic tunicamycin treatment, kidneys of Nox4 KO mice showed a marked surface pallor (bottom right).


C . TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin - treated Nox4 KO mice . n = 4 / group .

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C. TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin-treated Nox4 KO mice. n=4/group.