( 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 B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD 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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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

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(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 .

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(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 .

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(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 ) .

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(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 B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE 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 .

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(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 B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 .

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(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 .

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(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 .

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(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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY B-SUBCELLULAR O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE 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 .

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(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

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(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 .

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(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 ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O O 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 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 B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY 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

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(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 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O

(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

O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O

(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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O O 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-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY 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 .

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(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 B-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD 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 ) .

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(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 ) .

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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 .

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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 B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O B-ORGANISM O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O B-ORGANISM O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O B-SUBCELLULAR O B-GENEPROD O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-ORGANISM O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

B-GENEPROD B-EXP_ASSAY O B-ORGANISM O O O O B-ORGANISM O 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-GENEPROD O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-CELL_TYPE 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 .

B-GENEPROD B-EXP_ASSAY O B-ORGANISM O 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-GENEPROD O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR B-CELL_TYPE 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 B-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE B-ORGANISM O O B-ORGANISM O O O O O O O O O O O O O O O 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-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 B-EXP_ASSAY O B-ORGANISM O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 .

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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 .

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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 .

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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 .

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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 .

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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 B-GENEPROD O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O 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 .

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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.


D . Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase - 12 and cleaved PARP levels in tunicamycin - treated Nox4 KO mice compared to WT .

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D. Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase-12 and cleaved PARP levels in tunicamycin-treated Nox4 KO mice compared to WT.


E . Survival curves showed that a very high proportion of Nox4 KO mice died after AKI . Guanabenz ( Gbz ) treatment dramatically improved survival in tunicamycin - treated KO mice . Number of animals as indicated . Levels of significance by Kaplan Meier analysis are reported to the right .

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E. Survival curves showed that a very high proportion of Nox4 KO mice died after AKI. Guanabenz (Gbz) treatment dramatically improved survival in tunicamycin-treated KO mice. Number of animals as indicated. Levels of significance by Kaplan Meier analysis are reported to the right.


H - I . Fibulin 7 immunostaining in 2 - month - vs . 2 - year - old tail section ( H ) and its intensity quantification per basal epidermal stem cell / basement membrane ( normalized to 2 - month - old ) ( I ) . Dotted box areas were enlarged in the lower panels . White arrows indicate fibulin 7 basement membrane staining .

O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O O 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-GENEPROD I-GENEPROD B-TISSUE I-TISSUE B-EXP_ASSAY O

H-I. Fibulin 7 immunostaining in 2-month- vs. 2-year-old tail section (H) and its intensity quantification per basal epidermal stem cell/basement membrane (normalized to 2-month-old) (I). Dotted box areas were enlarged in the lower panels. White arrows indicate fibulin 7 basement membrane staining.


D - E . Ki67 immunostaining in 1 - week - chase mice ( D ) and its quantitation ( E ) . White arrows show Ki67 and tdTomato double positive cells , which were counted and summarized in ( E ) . Scale bar : 20 μm . N = 5 WT / het and 3 KO mice .

O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O 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-ORGANISM O

D-E. Ki67 immunostaining in 1-week-chase mice (D) and its quantitation (E). White arrows show Ki67 and tdTomato double positive cells, which were counted and summarized in (E). Scale bar: 20 μm. N=5 WT/het and 3 KO mice.


F . Representative pictures from tail wounds of 1 - year - old Fbln7 WT and KO mice . G . Measurements of wound area over time in Fbln7 WT ( N = 5 ) and KO ( N = 7 ) mice .

O O O O O B-TISSUE B-DISEASE O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-DISEASE O O O O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM O

F. Representative pictures from tail wounds of 1-year-old Fbln7 WT and KO mice. G. Measurements of wound area over time in Fbln7 WT (N=5) and KO (N=7) mice.


H - J . Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs . KO mice ( H ) and quantitation of the re - epithelialization length ( I ) and thickness at the healing front ( J ) . Scale bar : 200 μm . N = 3 WT and 5 KO mice .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O B-TISSUE O O B-DISEASE B-GENEPROD O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O 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-ORGANISM O

H-J. Hematoxylin and eosin staining from tail sections of wounded Fbln7 WT vs. KO mice (H) and quantitation of the re-epithelialization length (I) and thickness at the healing front (J). Scale bar: 200 μm. N=3 WT and 5 KO mice.


F - G . K31 and K14 immunostaining in tail sections of 2 - month - vs . 2 - year - old C57BL / 6J WT mice ( F ) and the quantification ( G ) . Dotted box areas are enlarged in the lower panels . Scale bar : 50 μm . Quantification of K14 intensity in the suprabasal area was normalized to basal area per cell . N = 4 ( 2 - 3 - month - old ) and N = 5 ( 2 - year - old ) C57BL / 6J mice ( G ) .

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O B-ORGANISM O O O O 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-GENEPROD O O O B-TISSUE O O O O B-TISSUE O O O O O O 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-ORGANISM O O O O

F-G. K31 and K14 immunostaining in tail sections of 2-month- vs. 2-year-old C57BL/6J WT mice (F) and the quantification (G). Dotted box areas are enlarged in the lower panels. Scale bar: 50 μm. Quantification of K14 intensity in the suprabasal area was normalized to basal area per cell. N=4 (2-3-month-old) and N=5 (2-year-old) C57BL/6J mice (G).


H - J . K31 and K14 immunostaining in tail sections of 1 - year - old Fbln7 WT vs . KO compared with 2 - month - old Fbln7 KO mice ( H ) and the quantitation ( I , J ) . Scale bar : 50 μm . N = 5 for both WT and KO of 2 - 3 - month - old mice ( I ) . N = 5 and N = 7 for 1 - year - old WT vs . KO , respectively ( J ) . Data information ( F - J )

O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O 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-ORGANISM O O O O O O O O O O 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-J. K31 and K14 immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO compared with 2-month-old Fbln7 KO mice (H) and the quantitation (I, J). Scale bar: 50 μm. N=5 for both WT and KO of 2-3-month-old mice (I). N=5 and N=7 for 1-year-old WT vs. KO, respectively (J). Data information (F-J)


A . Collagen IV immunostaining in tail sections of 1 - year - old Fbln7 WT vs . KO . Dotted box regions are enlarged .

O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-TISSUE O O O O O O O B-GENEPROD O O O O O O O O O O O

A. Collagen IV immunostaining in tail sections of 1-year-old Fbln7 WT vs. KO. Dotted box regions are enlarged.


B . Quantification of Collagen IV basement membrane intensity per cell in Fbln7 KO mice normalized to WT mice . N = 4 WT and N = 6 KO mice . Graphs show mean ± S . D . Mann - Whitney test . * * ; P < 0 . 01 , ns ; not significant .

O O O O B-GENEPROD I-GENEPROD B-TISSUE I-TISSUE O O O O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O 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. Quantification of Collagen IV basement membrane intensity per cell in Fbln7 KO mice normalized to WT mice. N=4 WT and N=6 KO mice. Graphs show mean ± S.D. Mann-Whitney test. **; P<0.01, ns; not significant.


A Representative 3D tile scan images of young adrenal gland , ovary , pituitary gland , testis , pancreas and thyroid gland stained with the antibodies indicated in the panel . Scale bars are 200 µm . The white and dashed lines represent the outlines of each organ .

O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE B-EXP_ASSAY O O 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 Representative 3D tile scan images of young adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland stained with the antibodies indicated in the panel. Scale bars are 200 µm. The white and dashed lines represent the outlines of each organ.


B Representative 3D images at single - cell resolution level of adrenal gland , ovary , pituitary gland , testis , pancreas and thyroid gland . Insets ( * ) indicate higher magnification images of regions with single - cell resolution and surface rendering for all markers . Scale bars for tile scan 3D images are 200 µm , for single - cell resolution insets are 5 µm . The dashed lines represent the outlines of each organ .

O O O B-EXP_ASSAY O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY 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 Representative 3D images at single-cell resolution level of adrenal gland, ovary, pituitary gland, testis, pancreas and thyroid gland. Insets (*) indicate higher magnification images of regions with single-cell resolution and surface rendering for all markers. Scale bars for tile scan 3D images are 200 µm, for single-cell resolution insets are 5 µm. The dashed lines represent the outlines of each organ.


C Exemplar image illustrates the cell - cell interactome analysis with the distance estimates of CD68 + macrophages to blood vessels ( Endoglin ) in cortex , medulla and the transition zone ( TZ ) of an aged adrenal gland . The colour code indicates the distance which is very close in violet and very far in red . The scale bar is 50 µm . The white and dashed lines represent the outline of the transition zone .

O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE O B-TISSUE I-TISSUE O B-GENEPROD O O B-TISSUE O B-TISSUE O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O 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 Exemplar image illustrates the cell-cell interactome analysis with the distance estimates of CD68+ macrophages to blood vessels (Endoglin) in cortex, medulla and the transition zone (TZ) of an aged adrenal gland. The colour code indicates the distance which is very close in violet and very far in red. The scale bar is 50 µm. The white and dashed lines represent the outline of the transition zone.


A Young and aged adrenal gland immunolabelled with HSPG2 , NG2 and Podocalyxin . Higher magnification insets show cortex ( Co ) and medulla ( Me ) . Combined box and whiskers , and scatter plot with quantification of vessel density in young and aged adrenal glands . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O B-TISSUE O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O

A Young and aged adrenal gland immunolabelled with HSPG2, NG2 and Podocalyxin. Higher magnification insets show cortex (Co) and medulla (Me). Combined box and whiskers, and scatter plot with quantification of vessel density in young and aged adrenal glands. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


B Young and aged ovary immunolabelled with Sca - 1 , PDGFRβ and Emcn . Higher magnification insets show cortex and medulla . Combo plot with quantification of vessel density in young and aged ovary . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Co : Cortex ; Me : Medulla Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE O B-TISSUE O O O O O O B-TISSUE B-EXP_ASSAY O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O

B Young and aged ovary immunolabelled with Sca-1, PDGFRβ and Emcn. Higher magnification insets show cortex and medulla. Combo plot with quantification of vessel density in young and aged ovary. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Co: Cortex; Me: Medulla Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


C Young ( 10 - weeks - old ) and aged pituitary gland immunolabelled with ESM - 1 , Collagen IV and Emcn . Insets with higher magnifications of pars distalis and pars nervosa regions . Combo plot shows quantification of vessel density . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Pd : Pars Distalis ; Pn : Pars Nervosa . Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O O O O O O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O B-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-EXP_ASSAY O O O O O O O O

C Young (10-weeks-old) and aged pituitary gland immunolabelled with ESM-1, Collagen IV and Emcn. Insets with higher magnifications of pars distalis and pars nervosa regions. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Pd: Pars Distalis; Pn: Pars Nervosa. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


D Young and aged testis immunolabelled with Tie - 2 and HSPG2 . Higher magnifications of cortex and medulla . Combo plot shows quantification of vessel density . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-TISSUE O B-TISSUE O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O

D Young and aged testis immunolabelled with Tie-2 and HSPG2. Higher magnifications of cortex and medulla. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


E Young and aged pancreas immunolabelled with SM22α and Endoglin . Insets representative of higher magnifications of regions in young and aged pancreas . Combo plot shows quantification of vessel density . Data information : The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-TISSUE O O O O O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O

E Young and aged pancreas immunolabelled with SM22α and Endoglin. Insets representative of higher magnifications of regions in young and aged pancreas. Combo plot shows quantification of vessel density. Data information: The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


F Young and aged thyroid gland immunolabelled with CXCR4 , Laminin and Emcn . Combo plot shows quantification of vessel density in young and aged thyroid glands . Insets : periphery and centre of the thyroid gland . The white and dashed lines in each panel represent the outlines of each organ . ( n = 5 ) , P - values and two - tailed unpaired t - tests . ns : not significant ; * * : P < 0 . 01 ; * * * : P < 0 . 001 . Nuclei : TO - PRO - 3 . For the Combo plots , the boxes represent mean ± s . d . , line in the box is the median , and the lower and upper lines show the minimum and the maximum of the values . The line on the right side of these combined plots represents the sample distribution . ; Pe : Periphery Ct : Center Scale bars for tile scan 3D images 200 µm , for insets 50 µm .

O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O O O O O B-TISSUE B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O

F Young and aged thyroid gland immunolabelled with CXCR4, Laminin and Emcn. Combo plot shows quantification of vessel density in young and aged thyroid glands. Insets: periphery and centre of the thyroid gland. The white and dashed lines in each panel represent the outlines of each organ. (n=5), P-values and two-tailed unpaired t-tests. ns: not significant; **: P < 0.01; ***: P < 0.001. Nuclei: TO-PRO-3. For the Combo plots, the boxes represent mean ± s.d., line in the box is the median, and the lower and upper lines show the minimum and the maximum of the values. The line on the right side of these combined plots represents the sample distribution. ; Pe: Periphery Ct: Center Scale bars for tile scan 3D images 200 µm, for insets 50 µm.


A Whole - endocrine gland imaging of young and aged adrenal glands immunolabelled with α - SMA and Emcn acquired on a light - sheet microscope . Asterisks indicate higher magnifications of regions in adrenal glands from young and aged mice . Combined box and whiskers , and scatter plots with quantifications of vessel density and numbers of arteries .

O O O B-TISSUE I-TISSUE B-EXP_ASSAY O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE I-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O

A Whole-endocrine gland imaging of young and aged adrenal glands immunolabelled with α-SMA and Emcn acquired on a light-sheet microscope. Asterisks indicate higher magnifications of regions in adrenal glands from young and aged mice. Combined box and whiskers, and scatter plots with quantifications of vessel density and numbers of arteries.


B Whole - endocrine gland light - sheet imaging - based 3D images of young and aged ovaries stained with α - SMA and Emcn . Asterisks represent higher magnifications of regions in ovaries from young and aged mice . Combo plots show quantifications of vessel density and numbers of arteries in young and aged ovary .

O O O B-TISSUE I-TISSUE B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O O O O B-TISSUE O

B Whole-endocrine gland light-sheet imaging-based 3D images of young and aged ovaries stained with α-SMA and Emcn. Asterisks represent higher magnifications of regions in ovaries from young and aged mice. Combo plots show quantifications of vessel density and numbers of arteries in young and aged ovary.


C 3D images of whole cleared young and aged testes stained with α - SMA and Emcn . Asterisks indicate higher magnifications of indicated regions . Combo plots show quantifications of vessel density and numbers of arteries .

O O B-EXP_ASSAY O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O

C 3D images of whole cleared young and aged testes stained with α-SMA and Emcn. Asterisks indicate higher magnifications of indicated regions. Combo plots show quantifications of vessel density and numbers of arteries.


D Light - sheet imaging of whole cleared young and aged pancreases immunolabelled with α - SMA and Emcn . Asterisks indicate higher magnifications of regions . Combo plots with quantifications of vessel density and numbers of arteries in young and aged pancreas .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O O O O B-TISSUE O

D Light-sheet imaging of whole cleared young and aged pancreases immunolabelled with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots with quantifications of vessel density and numbers of arteries in young and aged pancreas.


E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α - SMA and Emcn . Asterisks indicate higher magnifications of regions . Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands .

O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-TISSUE I-TISSUE B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-TISSUE O O O O B-TISSUE I-TISSUE O

E 3D images acquired on a light sheet microscope of whole cleared young and aged thyroid glands stained with α-SMA and Emcn. Asterisks indicate higher magnifications of regions. Combo plots show quantifications of vessel density and numbers of arteries in young and aged thyroid glands.


F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands . The colour code based on immunolabelling intensities indicates very high expression in red , high expression in yellow , medium in light green , low in dark green and no expression in grey .

O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O O O O B-EXP_ASSAY O O O

F Heatmap of 3D spatial proteomic data showing qualitative analysis of differences in cell type markers and matrix expressions between young and aged murine endocrine glands. The colour code based on immunolabelling intensities indicates very high expression in red, high expression in yellow, medium in light green, low in dark green and no expression in grey.