( d ) ELISA of IL - 1β secretion by LPS - primed Map1lc3b - / - mice and Becn1 + / - BMDMs incubated for 1 h with z - YVAD - fmk ( z - YVAD ; 10 μM ) or vehicle ( dimethylsulfoxide ( DMSO ) ) , followed by stimulation for 1 h with ATP . * P 0 . 05 ( Student ' s t - test ) . Data are representative of three or four experiments ( mean and s . d . in b - d ) .

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(d) ELISA of IL-1β secretion by LPS-primed Map1lc3b-/- mice and Becn1+/- BMDMs incubated for 1 h with z-YVAD-fmk (z-YVAD; 10 μM) or vehicle (dimethylsulfoxide (DMSO)), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three or four experiments (mean and s.d. in b-d).


( a , b ) Transmission electron microscopy of morphological changes in mitochondria ( arrows ) in Map1lc3b − / − BMDMs ( a ) or Becn1 + / − BMDMs ( b ) left untreated or incubated for 6 h with LPS ( 10 ng / ml ) and then stimulated for 30 min with ATP ( 1 mM ) . Outlined areas ( middle ) enlarged at right . Scale bars , 500 nm .

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(a,b) Transmission electron microscopy of morphological changes in mitochondria (arrows) in Map1lc3b−/− BMDMs (a) or Becn1+/− BMDMs (b) left untreated or incubated for 6 h with LPS (10 ng/ml) and then stimulated for 30 min with ATP (1 mM). Outlined areas (middle) enlarged at right. Scale bars, 500 nm.


( c ) Flow cytometry of Map1lc3b − / − and Becn1 + / − macrophages left unstained or labeled with MitoSOX .

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(c) Flow cytometry of Map1lc3b−/− and Becn1+/− macrophages left unstained or labeled with MitoSOX.


( d ) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP ; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment . Data are representative of three experiments .

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(d) Flow cytometry of macrophages left untreated or stimulated with LPS and ATP; cells were stained for 15 min with MitoTracker Deep Red and MitoTracker Green before ATP treatment. Data are representative of three experiments.


( a ) ELISA of cytokines in supernatants ( left ) and immunoblot analysis of caspase - 1 and IL - 1β in lysates ( right ) of J774A . 1 macrophages exposed to EtBr ( 0 , 10 , 100 or 500 ng / ml ; wedges ) and left unstimulated or stimulated with LPS alone or with LPS and ATP .

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(a) ELISA of cytokines in supernatants (left) and immunoblot analysis of caspase-1 and IL-1β in lysates (right) of J774A.1 macrophages exposed to EtBr (0, 10, 100 or 500 ng/ml; wedges) and left unstimulated or stimulated with LPS alone or with LPS and ATP.


( b ) Immunoblot analysis of caspase - 1 and ASC in lysates of J774A . 1 or ρ0 J774A . 1 macrophages ( treated with 100 ng / ml of EtBr ) incubated with LPS , followed by stimulation for 0 , 15 or 30 min with ATP .

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(b) Immunoblot analysis of caspase-1 and ASC in lysates of J774A.1 or ρ0 J774A.1 macrophages (treated with 100 ng/ml of EtBr) incubated with LPS, followed by stimulation for 0, 15 or 30 min with ATP.


( c ) Cytokine secretion by J774A . 1 or ρ0 J774A . 1 macrophages incubated with various combinations of LPS and ATP ( below graph ) .

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(c) Cytokine secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with various combinations of LPS and ATP (below graph).


( d ) Cytokine secretion ( right ) and immunoblot analysis of caspase - 1 and IL - 1β in lysates ( left ) of LPS - primed macrophages incubated for 1 h with rotenone ( + ; 5 μM ) or DMSO ( − ) before ATP treatment .

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(d) Cytokine secretion (right) and immunoblot analysis of caspase-1 and IL-1β in lysates (left) of LPS-primed macrophages incubated for 1 h with rotenone (+; 5 μM) or DMSO (−) before ATP treatment.


( e ) ELISA of IL - 1β secretion by J774A . 1 or ρ0 J774A . 1 macrophages incubated with LPS and rotenone ( or DMSO ) , followed by ATP .

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(e) ELISA of IL-1β secretion by J774A.1 or ρ0 J774A.1 macrophages incubated with LPS and rotenone (or DMSO), followed by ATP.


( f ) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito - TEMPO and then stimulated with various combinations of LPS and ATP .

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(f) Cytokine secretion by peritoneal macrophages preincubated for 1 h with Mito-TEMPO and then stimulated with various combinations of LPS and ATP.


( g , h ) ELISA of IL - 1β secretion ( left ) and immunoblot analysis for caspase - 1 in lysates ( right ) of Map1lc3b + / + and Map1lc3b − / − ( g ) or Becn1 + / + and Becn1 + / − ( h ) peritoneal macrophages incubated for 1 h with Mito - TEMPO ( 500 μM ) , followed by LPS and ATP . * P 0 . 05 ( Student ' s t - test ) . Data represent three experiments ( mean and s . d . in a , c , d - h ) .

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(g,h) ELISA of IL-1β secretion (left) and immunoblot analysis for caspase-1 in lysates (right) of Map1lc3b+/+ and Map1lc3b−/− (g) or Becn1+/+ and Becn1+/− (h) peritoneal macrophages incubated for 1 h with Mito-TEMPO (500 μM), followed by LPS and ATP. *P 0.05 (Student's t-test). Data represent three experiments (mean and s.d. in a,c,d-h).


( a ) ELISA of cytokine secretion by LPS - primed Map1lc3b + / + and Map1lc3b − / − BMDMs incubated with cyclosporine A or DMSO , followed by stimulation for 1 h with ATP .

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(a) ELISA of cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− BMDMs incubated with cyclosporine A or DMSO, followed by stimulation for 1 h with ATP.


( b ) ELISA of cytokine secretion by LPS - primed Becn1 + / + or Becn1 + / − BMDMs incubated with cyclosporine A ( 10 μM ) or DMSO , followed by ATP stimulation .

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(b) ELISA of cytokine secretion by LPS-primed Becn1+/+ or Becn1+/− BMDMs incubated with cyclosporine A (10 μM) or DMSO, followed by ATP stimulation.


( c ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed BMDMs incubated for 1 h with rotenone ( 5 μM ) or vehicle ( DMSO ) before ATP stimulation .

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(c) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDMs incubated for 1 h with rotenone (5 μM) or vehicle (DMSO) before ATP stimulation.


( d ) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito - TEMPO ( 500 μM ) and then incubated with LPS , followed by ATP stimulation .

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(d) Quantitative PCR analysis of cytosolic mtDNA in macrophages preincubated for 1 h with Mito-TEMPO (500 μM) and then incubated with LPS, followed by ATP stimulation.


( e ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed BMDM incubated with cyclosporine A ( 10 μM ) , plus either rotenone ( 5 μM ) or DMSO , followed by ATP stimulation .

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(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed BMDM incubated with cyclosporine A (10 μM), plus either rotenone (5 μM) or DMSO, followed by ATP stimulation.


( f ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed Map1lc3b − / − and Becn1 + / − BMDMs stimulated for 30 min with ATP . The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells , set as 1 . * P 0 . 05 ( Student ' s t - test ) . Data are representative of three experiments ( mean and s . d . ) .

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(f) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed Map1lc3b−/− and Becn1+/− BMDMs stimulated for 30 min with ATP. The amount of cytosolic mtDNA is presented relative to the amount in untreated control cells, set as 1. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).


( a ) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I , lactate dehydrogenase LDH ( Control ) or heat - inactivated DNase I ( HI DNase I ) and then stimulated with LPS , followed by stimulation for 1 h with ATP .

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(a) ELISA of cytokines in supernatants of BMDMs incubated for 4 h after delivery of 3 μg DNase I, lactate dehydrogenase LDH (Control) or heat-inactivated DNase I (HI DNase I) and then stimulated with LPS, followed by stimulation for 1 h with ATP.


( b ) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS , followed by stimulation for 15 min with ATP .

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(b) Quantitative PCR analysis of cytosolic mtDNA in BMDMs transfected as in a and then stimulated with LPS, followed by stimulation for 15 min with ATP.


( c ) ELISA of IL - 1β secretion by Aim2 + / + and Aim2 − / − BMDMs primed for 4 h with LPS ( 200 ng / ml ) , then transfected for 6 h with 1 μg mtDNA ( through the use of liposomes as the vehicle ) , followed by stimulation for 1 h with ATP . * P 0 . 05 , versus Aim2 − / − cells treated with LPS and ATP ( Student ' s t - test ) .

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(c) ELISA of IL-1β secretion by Aim2+/+ and Aim2−/− BMDMs primed for 4 h with LPS (200 ng/ml), then transfected for 6 h with 1 μg mtDNA (through the use of liposomes as the vehicle), followed by stimulation for 1 h with ATP. *P 0.05, versus Aim2−/− cells treated with LPS and ATP (Student's t-test).


( d ) ELISA of cytokine secretion by LPS - primed Aim2 − / − BMDMs transfected for 6 h with mtDNA , poly ( dA : dT ) or mtDNA predigested by DNase I ( + DNase ; far left ) , followed by stimulation for 1 h with ATP . * P 0 . 05 ( Student ' s t - test ) . Data are representative of three experiments ( mean and s . d . ) .

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(d) ELISA of cytokine secretion by LPS-primed Aim2−/− BMDMs transfected for 6 h with mtDNA, poly(dA:dT) or mtDNA predigested by DNase I (+ DNase; far left), followed by stimulation for 1 h with ATP. *P 0.05 (Student's t-test). Data are representative of three experiments (mean and s.d.).


( a ) Cytokine secretion by LPS - primed Map1lc3b + / + and Map1lc3b − / − ( a ) or Becn1 + / + and Becn1 + / − ( b ) peritoneal macrophages incubated for 15 min with glybenclamide ( Glyb ; 100 μM ( a ) or 50 - 100 μM ( b ) ) , Bay 11 - 7082 ( Bay ; 12 μM ) or DMSO ( - ) , followed by ATP .

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(a) Cytokine secretion by LPS-primed Map1lc3b+/+ and Map1lc3b−/− (a) or Becn1+/+ and Becn1+/− (b) peritoneal macrophages incubated for 15 min with glybenclamide (Glyb; 100 μM (a) or 50-100 μM (b)), Bay 11-7082 (Bay; 12 μM) or DMSO (-), followed by ATP.


( c ) Cytokine secretion by LPS - primed wild - type ( WT ) and NALP3 - deficient ( NALP3 - KO ) macrophages incubated with rotenone , followed by stimulation for 1 h with ATP .

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(c) Cytokine secretion by LPS-primed wild-type (WT) and NALP3-deficient (NALP3-KO) macrophages incubated with rotenone, followed by stimulation for 1 h with ATP.


( d ) Quantitative PCR analysis of cytosolic mtDNA in wild - type and NALP3 - deficient BMDMs left unstimulated ( - ) or stimulated with LPS , followed by treatment for 15 or 30 min with ATP ( left ) , and immunoblot analysis of cytosolic cytochrome c and activation of caspase - 1 in the cells at left ( right ) .

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(d) Quantitative PCR analysis of cytosolic mtDNA in wild-type and NALP3-deficient BMDMs left unstimulated (-) or stimulated with LPS, followed by treatment for 15 or 30 min with ATP (left), and immunoblot analysis of cytosolic cytochrome c and activation of caspase-1 in the cells at left (right).


( e ) Quantitative PCR analysis of cytosolic mtDNA in LPS - primed wild - type , NALP3 - deficient or ASC - deficient ( ASC - KO ) BMDMs stimulated for 15 min with ATP . * P 0 . 05 , NALP3 - deficient or ASC - deficient versus wild - type treated with LPS and ATP ( Student ' s t - test ) .

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(e) Quantitative PCR analysis of cytosolic mtDNA in LPS-primed wild-type, NALP3-deficient or ASC-deficient (ASC-KO) BMDMs stimulated for 15 min with ATP. *P 0.05, NALP3-deficient or ASC-deficient versus wild-type treated with LPS and ATP (Student's t-test).


( f ) Flow cytometry of untreated macrophages ( control ) or of LPS - primed macrophages left unstained or stained with MitoSOX , then stimulated for 15 min with ATP .

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(f) Flow cytometry of untreated macrophages (control) or of LPS-primed macrophages left unstained or stained with MitoSOX, then stimulated for 15 min with ATP.


( g ) Flow cytometry of untreated macrophages ( control ) or of macrophages stimulated with LPS and ATP , stained with MitoTracker Deep Red . Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential .

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(g) Flow cytometry of untreated macrophages (control) or of macrophages stimulated with LPS and ATP, stained with MitoTracker Deep Red. Numbers above bracketed lines indicate percent cells with loss of mitochondrial membrane potential.


( h ) Flow cytometry of LPS - primed wild - type , ASC - deficient and NALP3 - deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V . Data represent three experiments ( mean and s . d . in a - e ) .

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(h) Flow cytometry of LPS-primed wild-type, ASC-deficient and NALP3-deficient peritoneal macrophages stimulated for 30 min with ATP and stained with annexin V. Data represent three experiments (mean and s.d. in a-e).


( a ) ELISA of serum IL - 1β and IL - 18 at 24 h after intraperitoneal injection of LPS ( 12 mg per kg body weight ) into male Map1lc3b + / + and Map1lc3b − / − mice .

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(a) ELISA of serum IL-1β and IL-18 at 24 h after intraperitoneal injection of LPS (12 mg per kg body weight) into male Map1lc3b+/+ and Map1lc3b−/− mice.


( b ) Survival of Map1lc3b + / + mice ( n = 11 ) and Map1lc3b − / − mice ( n = 10 ) after LPS challenge ( 100 mg per kg body weight ) , assessed over a period of 2 weeks .

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(b) Survival of Map1lc3b+/+ mice (n = 11) and Map1lc3b−/− mice (n = 10) after LPS challenge (100 mg per kg body weight), assessed over a period of 2 weeks.


( c , d ) ELISA of serum IL - 1β and IL - 18 in male Map1lc3b + / + and Map1lc3b − / − mice ( c ) and Becn1 + / + and Becn1 + / − mice ( d ) at 24 h after CLP or sham laparotomy . In a , c , d , each symbol represents an individual mouse ; small horizontal lines indicate the mean . P values , unpaired two - tailed Student ' s t - test ( a , c , d ) or log - rank test ( b ) . Data are representative of two experiments .

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(c,d) ELISA of serum IL-1β and IL-18 in male Map1lc3b+/+ and Map1lc3b−/− mice (c) and Becn1+/+ and Becn1+/− mice (d) at 24 h after CLP or sham laparotomy. In a,c,d, each symbol represents an individual mouse; small horizontal lines indicate the mean. P values, unpaired two-tailed Student's t-test (a,c,d) or log-rank test (b). Data are representative of two experiments.


B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2 - KO PSCs , NPCs , and neurons . Scale bar = 10 µm .

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B Immunofluorescent staining confirmed the absence of MeCP2 in MECP2-KO PSCs, NPCs, and neurons. Scale bar = 10 µm.


C RT - qPCR array revealed differential gene expression between control and MECP2 - KO 28 - day neurons . The P values are calculated based on a Student ' s t - test of the replicate 2 - ΔΔCt values for each gene in the control group and KO groups ( WT83 / Q83X cell lines were used ; N = 3 clones from each genotype ) .

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C RT-qPCR array revealed differential gene expression between control and MECP2-KO 28-day neurons. The P values are calculated based on a Student's t-test of the replicate 2-ΔΔCt values for each gene in the control group and KO groups (WT83/Q83X cell lines were used; N = 3 clones from each genotype).


D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function ( days 14 and 28 post differentiation ; normalized by control NPC expression ) .

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D Overtime quantitative gene expression profile of many of the involved genes relevant to synaptic function (days 14 and 28 post differentiation; normalized by control NPC expression).


E Single - cell RT - qPCR analysis distinguished populations of control and KO 28 - day neurons . Principal Component Analysis ( PCA ) of 441 cells projected onto the first two components . Overlaid populations of WT83 / Q83X and WT82 / K82fs neurons are shown .

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E Single-cell RT-qPCR analysis distinguished populations of control and KO 28-day neurons. Principal Component Analysis (PCA) of 441 cells projected onto the first two components. Overlaid populations of WT83/Q83X and WT82/K82fs neurons are shown.


F Violin plots of selected genes showing the comparison between control and MECP2 - KO neurons from the single - cell analyses ( log2 ( expression ) values ) that overlapped with the results obtained via RT - PCR array .

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F Violin plots of selected genes showing the comparison between control and MECP2-KO neurons from the single-cell analyses (log2(expression) values) that overlapped with the results obtained via RT-PCR array.


G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single - cell analyses . The dotted lines represent differentially expressed genes between the groups at P < 0 . 05 ( unpaired Student ' s t - test ) .

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G Volcano plot illustrates differences in expression patterns of target genes of neurons from the qPCR single-cell analyses. The dotted lines represent differentially expressed genes between the groups at P < 0.05 (unpaired Student's t-test).


H Left : Schematic of laminar cortical markers . Right : Top : Quantitation of laminar cell markers based on the single - cell RT - qPCR analysis ( two - way analysis of variance ( ANOVA ) , N = 3 replicates per genotype , F3 , 24 = 3 . 31 , * P = 0 . 04 , with difference in the proportion of cortical layers V and VI , 95 % confidence interval ( CI ) [ 8 . 31 , 42 . 41 ] ) ; Bottom : altered proportions of neurotransmitter markers ( two - way ANOVA , N = 3 replicates per genotype , F5 , 24 = 27 . 33 , * * * P < 0 . 0001 , with differences in the proportions of glutamatergic , 95 % CI [ 22 . 33 , 33 . 60 ] , and cholinergic , 95 % CI [ 0 . 166 , 11 . 43 ] , neurons ) .

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H Left: Schematic of laminar cortical markers. Right: Top: Quantitation of laminar cell markers based on the single-cell RT-qPCR analysis (two-way analysis of variance (ANOVA), N = 3 replicates per genotype, F3,24 = 3.31, *P = 0.04, with difference in the proportion of cortical layers V and VI, 95% confidence interval (CI) [8.31, 42.41]); Bottom: altered proportions of neurotransmitter markers (two-way ANOVA, N = 3 replicates per genotype, F5,24 = 27.33, ***P < 0.0001, with differences in the proportions of glutamatergic, 95% CI [22.33, 33.60], and cholinergic, 95% CI [0.166, 11.43], neurons).


D , E Western blot quantification showed decreased presynaptic Synapsin1 ( D ) and postsynaptic PSD - 95 ( E ) in untreated 6 - week MECP2 - KO neurons that can be increased by drug treatment ( Kruskal - Wallis test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; full Western blot , Fig EV4A ) ( WT83 / Q83X cell lines were used ; N = 3 clones from each genotype ) .

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D, E Western blot quantification showed decreased presynaptic Synapsin1 (D) and postsynaptic PSD-95 (E) in untreated 6-week MECP2-KO neurons that can be increased by drug treatment (Kruskal-Wallis test, *P < 0.05, **P < 0.01, ***P < 0.001; full Western blot, Fig EV4A) (WT83/Q83X cell lines were used; N = 3 clones from each genotype).


F , G 6 - week MECP2 - KO neurons ( F ) showed a pharmacologically rescuable reduction of co - localized synaptic puncta ( G ; one - way ANOVA , F16 , 118 = 9 . 148 , * P < 0 . 05 ; Dunnet ' s multiple comparisons test vs untreated KO ( WT83 / Q83X cell lines were used ; N = 7 - 8 neurons / condition ) . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Z scores relative to KO untreated : Control = 3 . 903 ; Nefiracetam = 3 . 560 ; Carbamoylcholine = 2 . 705 ; Pirenzepine = 0 . 0448 ; PHA543613 = 2 . 121 ; Acamprosate = 3 . 339 ; Baclofen = 0 . 5672 ; GR73632 = 2 . 237 ; Hyperforin = 3 . 016 , and IGF - 1 = 3 . 791 . Scale bar = 5 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 O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR 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

F, G 6-week MECP2-KO neurons (F) showed a pharmacologically rescuable reduction of co-localized synaptic puncta (G; one-way ANOVA, F16,118 = 9.148, *P < 0.05; Dunnet's multiple comparisons test vs untreated KO (WT83/Q83X cell lines were used; N = 7-8 neurons/condition). *P < 0.05, **P < 0.01, ***P < 0.001. Z scores relative to KO untreated: Control = 3.903; Nefiracetam = 3.560; Carbamoylcholine = 2.705; Pirenzepine = 0.0448; PHA543613 = 2.121; Acamprosate = 3.339; Baclofen = 0.5672; GR73632 = 2.237; Hyperforin = 3.016, and IGF-1 = 3.791. Scale bar = 5 µm.


H Drug treatment increased calcium transient frequency in 6 - week neurons . Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest ( one - way ANOVA , F4 , 52 = 20 . 28 , Dunnett ' s multiple comparisons test vs untreated KO : Nefiracetam , * * P < 0 . 01 and Z = 2 . 364 ; PHA 543613 , * * * P < 0 . 001 and Z = 3 . 391 ; Acamprosate , * * * P < 0 . 001 and Z = 3 . 153 ; WT83 / Q83X cell lines were used ; N = 10 - 14 neurons / condition ) and the percentage of active neurons ( one - way ANOVA , F4 , 52 = 23 . 11 ; Dunnett ' s multiple comparison test vs untreated KO : Nefiracetam , * * * P < 0 . 001 and Z = 3 . 144 ; PHA 543613 , * * P < 0 . 01 and Z = 2 . 567 ; * * Acamprosate , P < 0 . 01 and Z = 2 . 285 ; N = 10 - 14 neurons / condition ) .

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

H Drug treatment increased calcium transient frequency in 6-week neurons. Fluorescence intensity changes reflecting intracellular calcium transients in neurons in different regions of interest (one-way ANOVA, F4,52 = 20.28, Dunnett's multiple comparisons test vs untreated KO: Nefiracetam, **P < 0.01 and Z = 2.364; PHA 543613, ***P < 0.001 and Z = 3.391; Acamprosate, ***P < 0.001 and Z = 3.153; WT83/Q83X cell lines were used; N = 10-14 neurons/condition) and the percentage of active neurons (one-way ANOVA, F4,52 = 23.11; Dunnett's multiple comparison test vs untreated KO: Nefiracetam, ***P < 0.001 and Z = 3.144; PHA 543613, **P < 0.01 and Z = 2.567; **Acamprosate, P < 0.01 and Z = 2.285; N = 10-14 neurons/condition).


I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2 - KO neurons on MEA ( two - sided unpaired Student ' s t - test compared to KO untreated : Nefiracetam , * P = 0 . 016 and Z = 2 . 418 ; PHA 543613 , * P = 0 . 027 and Z = 2 . 147 ; Acamprosate , P = 0 . 39 and Z = 0 . 477 ; WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 4 - 11 MEA wells / condition ) .

O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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

I Treatment with either Nefiracetam or PHA 543613 increased network spiking activity in MECP2-KO neurons on MEA (two-sided unpaired Student's t-test compared to KO untreated: Nefiracetam, *P = 0.016 and Z = 2.418; PHA 543613, *P = 0.027 and Z = 2.147; Acamprosate, P = 0.39 and Z = 0.477; WT83/Q83X and WT82/K82fs cell lines were used; N = 4-11 MEA wells/condition).


B , C Immunofluorescent staining ( B ) and quantitation ( C ) confirmed the graded decrease in MeCP2 expression from control to 50 % MECP2 mosaic to full MECP2 - KO neurospheres . Scale bar = 50 µm .

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B, C Immunofluorescent staining (B) and quantitation (C) confirmed the graded decrease in MeCP2 expression from control to 50% MECP2 mosaic to full MECP2-KO neurospheres. Scale bar = 50 µm.


D 8 - week MECP2 - KO neurospheres ( N = 44 ) exhibited decreased size ( diameter ; * * P < 0 . 01 ) , but 50 % MECP2 - mosaic neurospheres ( CTR / KO ; N = 44 ) were unchanged from controls ( one - way ANOVA , F2 , 150 = 6 . 03 , P = 0 . 003 ; Dunnett ' s multiple comparisons test ; N = 65 ) . Left : representative brightfield images of neurospheres ; Right : neurosphere size quantification . Scale bar = 200 µm .

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D 8-week MECP2-KO neurospheres (N = 44) exhibited decreased size (diameter; **P < 0.01), but 50% MECP2-mosaic neurospheres (CTR/KO; N = 44) were unchanged from controls (one-way ANOVA, F2,150 = 6.03, P = 0.003; Dunnett's multiple comparisons test; N = 65). Left: representative brightfield images of neurospheres; Right: neurosphere size quantification. Scale bar = 200 µm.


E Calcium imaging analysis showing calcium transients normalized by control ( one - way ANOVA , F2 , 72 = 15 . 62 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs control : MECP2 - mosaic , * * * P < 0 . 001 ; MECP2 - KO , * * * P < 0 . 001 ) . WT83 / Q83X cell lines were used ; N = 44 Q83X neurospheres and 65 WT83 neurospheres .

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

E Calcium imaging analysis showing calcium transients normalized by control (one-way ANOVA, F2,72 = 15.62, P < 0.0001; Dunnett's multiple comparisons test vs control: MECP2-mosaic, ***P < 0.001; MECP2-KO, ***P < 0.001). WT83/Q83X cell lines were used; N = 44 Q83X neurospheres and 65 WT83 neurospheres.


F 8 - week neurosphere size ( diameter ) remained unchanged by treatment with either drug ( one - way ANOVA , F6 , 344 = 0 . 52 , P = 0 . 79 ; N = 44 Q83X neurospheres and 65 WT83 neurospheres ) .

F 8-week neurosphere size (diameter) remained unchanged by treatment with either drug (one-way ANOVA, F6,344 = 0.52, P = 0.79; N = 44 Q83X neurospheres and 65 WT83 neurospheres).


G Cell viability was improved or unharmed in treated 8 - week neurospheres ( one - way ANOVA , F9 , 104 = 6 . 81 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : Nefi + PHA 1 µM , * * P < 0 . 01 and Z = 2 . 974 ; Nefi + PHA 10 µM , * * * P < 0 . 001 and Z = 3 . 733 ; N = 8 - 24 neurospheres per condition ) .

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G Cell viability was improved or unharmed in treated 8-week neurospheres (one-way ANOVA, F9,104 = 6.81; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi + PHA 1 µM, **P < 0.01 and Z = 2.974; Nefi + PHA 10 µM, ***P < 0.001 and Z = 3.733; N = 8-24 neurospheres per condition).


H , I Calcium transient frequency ( number of peaks in 10 mins recording ) normalized by CTR / KO untreated ( H ; one - way ANOVA , F9 , 239 = 6 . 18 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : Nefi 1 µM , * P < 0 . 05 and Z = 0 . 780 ; Nefi + PHA 10 µM , * * * P < 0 . 001 and Z = 1 . 972 ; N = 15 - 61 neurospheres per condition ) and calcium transient amplitude normalized by CTR / KO untreated ( I ; one - way ANOVA , F9 , 241 = 1 . 27 , P = 0 . 255 ; N = 16 - 62 neurospheres per condition ) .

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

H, I Calcium transient frequency (number of peaks in 10 mins recording) normalized by CTR/KO untreated (H; one-way ANOVA, F9,239 = 6.18; Dunnett's multiple comparisons test vs CTR/KO untreated: Nefi 1 µM, *P < 0.05 and Z = 0.780; Nefi + PHA 10 µM, ***P < 0.001 and Z = 1.972; N = 15-61 neurospheres per condition) and calcium transient amplitude normalized by CTR/KO untreated (I; one-way ANOVA, F9,241 = 1.27, P = 0.255; N = 16-62 neurospheres per condition).


J , K MEA spike frequency heatmap ( J ) and quantification ( K ) normalized by CTR / KO untreated ( one - way ANOVA , F5 , 38 = 1 . 21 , P = 0 . 321 ; Dunnett ' s multiple comparisons test vs CTR / KO untreated : PHA , P = 0 . 65 and Z = 1 . 134 ; Nefi , P = 0 . 98 and Z = 0 . 06 ; PHA + Nefi , P = 0 . 86 and Z = 0 . 94 ; N = 7 - 8 wells per condition ) . ' X ' on activity heatmap signifies absence of a neurosphere in that position .

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

J, K MEA spike frequency heatmap (J) and quantification (K) normalized by CTR/KO untreated (one-way ANOVA, F5,38 = 1.21, P = 0.321; Dunnett's multiple comparisons test vs CTR/KO untreated: PHA, P = 0.65 and Z = 1.134; Nefi, P = 0.98 and Z = 0.06; PHA + Nefi, P = 0.86 and Z = 0.94; N = 7-8 wells per condition). 'X' on activity heatmap signifies absence of a neurosphere in that position.


B 2 - month old cortical organoid diameter ( one - way ANOVA , F2 , 173 = 91 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs untreated : Nefiracetam , * * * P < 0 . 001 and Z = 5 . 414 ; PHA 543613 , * * * P < 0 . 001 and Z = 5 . 351 ; WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 28 - 90 organoids per condition ) . Left : representative brightfield images of cortical organoids ; Right : cortical organoid size quantification . Scale bar = 200 µm .

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B 2-month old cortical organoid diameter (one-way ANOVA, F2,173 = 91.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, ***P < 0.001 and Z = 5.414; PHA 543613, ***P < 0.001 and Z = 5.351; WT83/Q83X and WT82/K82fs cell lines were used; N = 28-90 organoids per condition). Left: representative brightfield images of cortical organoids; Right: cortical organoid size quantification. Scale bar = 200 µm.


C Ki67 + proliferative area ( Student ' s t - test , t10 = 0 . 36 , P = 0 . 73 , N = 6 each ) . Left : representative immunofluorescence images of progenitor , proliferative , and neuronal markers in cortical organoids ; Right : quantification of Ki67 + proliferation in cortical organoids . Scale bar = 50 µm .

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C Ki67+ proliferative area (Student's t-test, t10 = 0.36, P = 0.73, N = 6 each). Left: representative immunofluorescence images of progenitor, proliferative, and neuronal markers in cortical organoids; Right: quantification of Ki67+ proliferation in cortical organoids. Scale bar = 50 µm.


RNA sequencing ( D , left : heatmap ) and GO analysis ( D , right ) of untreated MECP2 - KO organoids vs Nefiracetam ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated genetic expression in synapse - relevant pathways .

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RNA sequencing (D, left: heatmap) and GO analysis (D, right) of untreated MECP2-KO organoids vs Nefiracetam (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated genetic expression in synapse-relevant pathways.


E , center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles ; whiskers are minimum and maximum log2 normalized expression values .

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E, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.


RNA sequencing ( F , left : heatmap ) and GO analysis ( F , right ) of untreated MECP2 - KO organoids vs PHA 543613 ( N = 4 samples each of ~ 10 - 15 pooled organoids ) indicated that treatment upregulated expression of genes in synapse - relevant pathways .

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

RNA sequencing (F, left: heatmap) and GO analysis (F, right) of untreated MECP2-KO organoids vs PHA 543613 (N = 4 samples each of ~10-15 pooled organoids) indicated that treatment upregulated expression of genes in synapse-relevant pathways.


G , center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles ; whiskers are minimum and maximum log2 normalized expression values .

G, center band of the box is median log2 normalized expression and edges are 25th and 75th percentiles; whiskers are minimum and maximum log2 normalized expression values.


H Synapsin1 + puncta quantitation ( one - way ANOVA , F3 , 34 = 10 . 07 , P < 0 . 0001 ; Dunnett ' s multiple comparisons test vs untreated : Nefiracetam , * * P < 0 . 01 and Z = 2 . 998 ; PHA 543613 , P > 0 . 05 and Z = 1 . 266 ; WT83 / Q83X cell lines were used ; N = 8 - 10 each ) . Left : representative image of Synapsin1 + immunofluorescent staining ; Right : Synapsin1 + puncta quantification . Scale bar = 20 µm .

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H Synapsin1+ puncta quantitation (one-way ANOVA, F3,34 = 10.07, P < 0.0001; Dunnett's multiple comparisons test vs untreated: Nefiracetam, **P < 0.01 and Z = 2.998; PHA 543613, P > 0.05 and Z = 1.266; WT83/Q83X cell lines were used; N = 8-10 each). Left: representative image of Synapsin1+ immunofluorescent staining; Right: Synapsin1+ puncta quantification. Scale bar = 20 µm.


MECP2 - KO and control organoids had proportions of cells positive for the neuronal marker NeuN ( I ; Student ' s t - test , t14 = 0 . 26 , P = 0 . 80 , N = 8 each )

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MECP2-KO and control organoids had proportions of cells positive for the neuronal marker NeuN (I; Student's t-test, t14 = 0.26, P = 0.80, N = 8 each)


J MECP2 - KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 ( J ; one - way ANOVA , F3 , 40 = 2 . 489 , P = 0 . 074 and Z < 1 , WT83 / Q83X and WT82 / K82fs cell lines were used ; N = 8 - 14 organoids each ) . J , left : representative image of CTIP2 + immunofluorescent staining in cortical organoids ; J , right : quantification of CTIP2 + neurons . Scale bar = 50 µm .

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J MECP2-KO and control organoids had proportions of cells positive for cortical layer V and VI neuronal marker CTIP2 (J; one-way ANOVA, F3,40 = 2.489, P = 0.074 and Z < 1, WT83/Q83X and WT82/K82fs cell lines were used; N = 8-14 organoids each). J, left: representative image of CTIP2+ immunofluorescent staining in cortical organoids; J, right: quantification of CTIP2+ neurons. Scale bar = 50 µm.


L ; left and center : representative population spiking traces of control and MECP2 - KO organoids ; right : quantification of organoid population spiking activity , N = 6 replicates each ) .

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L; left and center: representative population spiking traces of control and MECP2-KO organoids; right: quantification of organoid population spiking activity, N = 6 replicates each).


Drug treatment increased spiking network activity of MECP2 - KO cortical organoids to a level not significantly different from that of control organoids ( M ; one - way ANOVA , F3 , 31 = 3 . 775 , Dunnett ' s multiple comparisons test vs control : untreated KO , * * P < 0 . 01 and Z = 3 . 192 ; Nefiracetam , P = 0 . 98 and Z = 2 . 117 ; PHA 543613 , P = 0 . 12 and Z = 1 . 101 , WT83 / Q83X cell lines were used ; N = 7 - 10 MEA wells per condition ) .

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Drug treatment increased spiking network activity of MECP2-KO cortical organoids to a level not significantly different from that of control organoids (M; one-way ANOVA, F3,31 = 3.775, Dunnett's multiple comparisons test vs control: untreated KO, **P < 0.01 and Z = 3.192; Nefiracetam, P = 0.98 and Z = 2.117; PHA 543613, P = 0.12 and Z = 1.101, WT83/Q83X cell lines were used; N = 7-10 MEA wells per condition).


Pre - enriched thymocytes from WT BALB / c thymi as in ( a ) were labeled by CFSE ( CFSE + ) , mixed with pre - enriched thymocytes from SKG thymi ( CFSE - ) , loaded with Indo - 1 , and stimulated as in ( a ) . Ratio of Indo ( Violet ) and Indo ( Blue ) was analyzed by flow cytometry . Comparison of cytoplasmic Ca2 + profiles between WT and SKG NKT1 ( c ) and NKT2 ( d ) cells in the same test tube are shown . Data are representative from one of at least three independent experiments . Graphs represent the average Ca2 + levels .

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

Pre-enriched thymocytes from WT BALB/c thymi as in (a) were labeled by CFSE (CFSE+), mixed with pre-enriched thymocytes from SKG thymi (CFSE-), loaded with Indo-1, and stimulated as in (a). Ratio of Indo (Violet) and Indo (Blue) was analyzed by flow cytometry. Comparison of cytoplasmic Ca2+ profiles between WT and SKG NKT1 (c) and NKT2 (d) cells in the same test tube are shown. Data are representative from one of at least three independent experiments. Graphs represent the average Ca2+ levels.


Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet , and activated by anti - CD3ε / CD28 Dynabeads for 2 days , in the presence of different [ EGTA ] in the medium . CellTrace Violet / CTV signal in living cells at the end of the culture is shown . Cell proliferation was quantified as division index based on CTV signals in ( a ) .

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Sorted thymic NKT1 and NKT2 cells were labeled with CellTrace Violet, and activated by anti-CD3ε/CD28 Dynabeads for 2 days, in the presence of different [EGTA] in the medium. CellTrace Violet/CTV signal in living cells at the end of the culture is shown. Cell proliferation was quantified as division index based on CTV signals in (a).


Thymic lobes from new born ( day 0 ) BALB / c mice were cultured in vitro for 6 days . EGTA was added 48h after the start of culture ( day 2 ) , and the expression of CXCR3 ( g ) , PLZF ( h ) and PD1 ( i ) in total iNKT cells are shown .

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Thymic lobes from new born (day 0) BALB/c mice were cultured in vitro for 6 days. EGTA was added 48h after the start of culture (day 2), and the expression of CXCR3 (g), PLZF (h) and PD1 (i) in total iNKT cells are shown.


oxygen consumption rate ( OCR ) from sorted thymic T cell populations measured in a Seahorse Mitostress test ;

oxygen consumption rate (OCR) from sorted thymic T cell populations measured in a Seahorse Mitostress test;


Confocal images of Tim 23 ( Alexa Fluor 488 , green ) and MitoTracker deep red ( Alexa Fluor 647 , red ) in freshly isolated thymic NKT1 ( c1 , 2 ) and NKT2 ( c5 , 6 ) cells using Airyscan technology . 3D images reconstructed by Imaris software in NKT1 ( c3 , 4 ) and NKT2 cells ( c7 , 8 ) . Dotted line in panel ( 5 - 8 ) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells .

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Confocal images of Tim 23 (Alexa Fluor 488, green) and MitoTracker deep red (Alexa Fluor 647, red) in freshly isolated thymic NKT1 (c1, 2) and NKT2 (c5, 6) cells using Airyscan technology. 3D images reconstructed by Imaris software in NKT1 (c3, 4) and NKT2 cells(c7, 8). Dotted line in panel (5-8) indicates juxtaposition of two NKT2 cell images taken with the same resolution and magnification as NKT1 cells.


Representative images of electron microscopy in NKT1 and NKT2 cells ; ( f ) individual mitochondria size and ( g ) cristae perimeters normalized to mitochondria perimeter based on EM in ( e ) .

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Representative images of electron microscopy in NKT1 and NKT2 cells; (f) individual mitochondria size and (g) cristae perimeters normalized to mitochondria perimeter based on EM in (e).


Maximal cytoplasmic [ Ca2 + ] in single , freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 ( 1uM ) ;

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Maximal cytoplasmic [Ca2+] in single, freshly sorted NKT1 and NKT2 cells following TCR stimulation in the presence or absence of BTP2 (1uM);


BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of Tbet in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) .

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BTP2 (1uM) was added 48h after the start of culture, and the expression of Tbet in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).


BTP2 ( 1uM ) was added 48h after the start of culture , and the expression of CXCR3 ( d ) , PLZF ( e ) , and PD1 ( f ) in total iNKT cells is shown . Data are representative from one of three independent experiments . Graphs represent mean ± SD with symbols representing individual cells ( a ) , or individual thymic lobes ( b - f ) . * * p < 0 . 01 ; * * * * p < 0 . 0001 ; n . s . not significant ( unpaired two - tailed Student ' s t test ) .

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BTP2 (1uM) was added 48h after the start of culture, and the expression of CXCR3 (d), PLZF (e), and PD1 (f) in total iNKT cells is shown. Data are representative from one of three independent experiments. Graphs represent mean ± SD with symbols representing individual cells (a), or individual thymic lobes (b-f). **p < 0.01; ****p < 0.0001; n.s. not significant (unpaired two-tailed Student's t test).


Profiles showing average Ca2 + influx induced by Thapsigargin ( TG ) in freshly sorted NKT1 ( Red ) and NKT2 ( Black ) cells . The timing of TG ( 1uM ) , ionomycin ( Iono ) ( 2uM ) and CCCP ( 1uM ) addition , and extracellular [ Ca2 + ] in mM are shown .

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Profiles showing average Ca2+ influx induced by Thapsigargin (TG) in freshly sorted NKT1 (Red) and NKT2 (Black) cells. The timing of TG (1uM), ionomycin (Iono) (2uM) and CCCP (1uM) addition, and extracellular [Ca2+] in mM are shown.


( B ) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs , and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA . The efficiency of each TR ' s knockdown ( KD ) does not correlate with its effect in reprogramming efficiency . The knockdown of 9 ( Class III ) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner ( unpaired two - tailed Student ' s t - test , p - value < 0 . 05 ) . Data are shown as mean ± SEM of at least two independent experiments . Class I corresponds to one TR inhibiting reprogramming , whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming . Shown are the effects of 28 out of 30 TRs , because we did not succeed in knocking down Cphx and Phox2a .

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(B) Bar graph summarizing the efficiency of single RNAi knockdowns for 28 out of the 30 TRs, and their effects in the reprogramming efficiency in comparison to control cells expressing scramble shRNA. The efficiency of each TR's knockdown (KD) does not correlate with its effect in reprogramming efficiency. The knockdown of 9 (Class III) out of the 28 TRs decreased the efficiency of the reprogramming in a statistically significant manner (unpaired two-tailed Student's t-test, p-value <0.05). Data are shown as mean ± SEM of at least two independent experiments. Class I corresponds to one TR inhibiting reprogramming, whereas Class II corresponds to 18 TRs having a weak or no effect in repogramming. Shown are the effects of 28 out of 30 TRs, because we did not succeed in knocking down Cphx and Phox2a.


( C ) Left : Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs . The data were plotted after normalization with the endogenous Gapdh using the " ΔCt method " . The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks . Notice that the y - axis is broken to accommodate the large spread of expression levels . Right : Shown are western blots using antibodies specific for Irf6 , Tead4 , Taf1c , Rcan1 and Tfap4 . Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side - by - side with extracts from control samples ( day 0 ) . Gapdh , mTOR and b - Tubulin were used as loading controls .

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(C) Left: Shown is a line graph depicting normalized mRNA expression levels of the 9 TRs at the indicated time points during the reprogramming of MEFs. The data were plotted after normalization with the endogenous Gapdh using the "ΔCt method". The grey vertical dashed line depicts day 6 in which the expression levels of nearly all TRs peaks. Notice that the y-axis is broken to accommodate the large spread of expression levels. Right: Shown are western blots using antibodies specific for Irf6, Tead4, Taf1c, Rcan1 and Tfap4. Whole cell extracts were prepared from MEFs at day 6 of reprogramming and run side-by-side with extracts from control samples (day 0). Gapdh, mTOR and b-Tubulin were used as loading controls.


( D ) Same as in ( B ) except MEFs were co - transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs . Left : Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency ( RE % ) . Shown are the average REs values from at least two independent experiments . The REs of the double KDs were evaluated against the RE of the corresponding single KDs . Right : Alkaline Phosphatase ( AP ) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA - based screens .

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(D) Same as in (B) except MEFs were co-transduced with lentiviruses expressing all possible pairwise combinations of shRNAs for the nine TRs. Left: Heat map depicting the effects of paired KD combinations in the Reprogramming Efficiency (RE%). Shown are the average REs values from at least two independent experiments. The REs of the double KDs were evaluated against the RE of the corresponding single KDs. Right: Alkaline Phosphatase (AP) stained cultures of terminal reprogrammed MEFs upon representative single and double KDs of our shRNA-based screens.


( A ) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming . The boundaries of the early - iPSCs formations are indicated by a blue dashed line . Scale bars : 100 μm .

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(A) RNA in situ hybridization depicting the expression patterns of the nine TRs in days 3 and 6 of MEFs reprogramming. The boundaries of the early-iPSCs formations are indicated by a blue dashed line. Scale bars: 100 μm.


( C ) Bar graph showing the percentage of cells isolated from early - iPSCs colonies or outside of the colonies ( rest of population ) expressing each of the 9 TRs . Data are shown as mean ± SEM of at least two independent experiments . 0 . 05 < ( * ) p < 0 . 1 , * p < 0 . 05 , * * p < 0 . 01 by unpaired Student ' s t - test or Welch ' s t - test , when appropriate .

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(C) Bar graph showing the percentage of cells isolated from early-iPSCs colonies or outside of the colonies (rest of population) expressing each of the 9 TRs. Data are shown as mean ± SEM of at least two independent experiments. 0.05 < (*)p < 0.1, *p < 0.05, **p < 0.01 by unpaired Student's t-test or Welch's t-test, when appropriate.


( D ) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs . Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments . Data are shown as mean ± SEM of at least two independent experiments . Anova test p < 0 . 0001 .

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(D) Scatter plot depicting a visual unbiased representation of the percentile of cells expressing each of the nine TRs. Each dot depicts the percentage of cells expressing the indicated TRs in individual experiments. Data are shown as mean ± SEM of at least two independent experiments. Anova test p<0.0001.


( E ) Schematic representation of the statistical analysis estimating the probability of co - expression of the nine TRs per cell within the early - iPSCs colonies . Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs ( shown with different color ) in 282 cells ( right side legend ) . The co - expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure . The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells , while the right part depicts the co - expression of combinations of the nine TRs based on double and triple single - cell experiments .

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(E) Schematic representation of the statistical analysis estimating the probability of co-expression of the nine TRs per cell within the early-iPSCs colonies. Each row consists of 9 tiles representing a single cell and each column shows the expression status of each of the nine TRs (shown with different color) in 282 cells (right side legend). The co-expression of gradually increasing numbers of TRs is grouped from the bottom to the top of the figure. The left part has been generated by estimating the percentages of expression of any single TR per cell as derived from single gene expression in single cells, while the right part depicts the co-expression of combinations of the nine TRs based on double and triple single-cell experiments.


( A - I ) Shown are ChIP - seq big wig files in the IGV browser depicting the binding of Oct4 ( Red ) , Sox2 ( Blue ) , Klf4 ( Green ) and c - Myc ( Black ) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming ( 18 hrs , D3 and D5 ) and in control MEFs , mESCs and miPSCs . All peaks have been normalized against input DNA . The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated . The side color bar depicts the expression levels of each TR at the indicated time points .

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(A-I) Shown are ChIP-seq big wig files in the IGV browser depicting the binding of Oct4 (Red), Sox2 (Blue), Klf4 (Green) and c-Myc (Black) to putative regulatory regions in the nine TRs genes in MEFs undergoing reprogramming (18 hrs, D3 and D5) and in control MEFs, mESCs and miPSCs. All peaks have been normalized against input DNA. The relative sizes of the represented genomic loci and the corresponding TSSs are also indicated. The side color bar depicts the expression levels of each TR at the indicated time points.


( C ) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6 ; The white color denotes no change in expression levels .

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(C) Heatmap summarizing the effects of each of the nine TRs single knockdowns on the expression of the other TRs on day 6; The white color denotes no change in expression levels.


( D ) Scatter plots depicting the extent of co - expression of either the broadly - or early - iPSCs - specifically expressed TRs in individual cells obtained from the early - iPSCs colonies or from the rest of the population ( left and right panel respectively ) . Each dot depicts the percentage of cells co - expressing a unique combination of two or three TRs . Data are shown as mean ± SEM of at least two independent experiments .

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(D) Scatter plots depicting the extent of co-expression of either the broadly- or early-iPSCs-specifically expressed TRs in individual cells obtained from the early-iPSCs colonies or from the rest of the population (left and right panel respectively). Each dot depicts the percentage of cells co-expressing a unique combination of two or three TRs. Data are shown as mean ± SEM of at least two independent experiments.


( E ) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers ; the white color indicates no change in expression levels . The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming ; Lin28a is the latest upregulated gene .

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(E) Heatmap summarizing the most striking effects of double nine TRs KDs on the expression of the other TRs and on the expression of the Epcam and Lin28a pluripotency markers; the white color indicates no change in expression levels. The genes examined have been ranked in the heatmap according to their order of upregulation during reprogramming; Lin28a is the latest upregulated gene.


( B ) Shown is a bar graph depicting the effect of overexpression of the indicated TRs in reprogramming efficiency . Data are shown as mean ± SEM of at least two independent experiments . The y axis labeled as % reprogramming efficiency refers to the AP + colonies scored as iPSCs .

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(B) Shown is a bar graph depicting the effect of overexpression of the indicated TRs in reprogramming efficiency. Data are shown as mean ± SEM of at least two independent experiments. The y axis labeled as % reprogramming efficiency refers to the AP+ colonies scored as iPSCs.


( C ) Shown is a bar graph depicting the effects of rescue experiments in reprogramming efficiency . MEFs undergoing reprogramming were knocked down using shRNAs for the endogenous Nanog or Irf6 expression and were co - infected with a vector expressing an shRNA - resistant human homologue of Nanog or Irf6 ( hNanog OE , hIrf6 OE ) . Data are shown as mean ± SEM of two independent experiments .

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(C) Shown is a bar graph depicting the effects of rescue experiments in reprogramming efficiency. MEFs undergoing reprogramming were knocked down using shRNAs for the endogenous Nanog or Irf6 expression and were co-infected with a vector expressing an shRNA-resistant human homologue of Nanog or Irf6 (hNanog OE, hIrf6 OE). Data are shown as mean ± SEM of two independent experiments.


( A ) Protein abundance is the normalized signal intensity ( LFQ ) for a protein divided by its molecular weight . Specificity ( enrichment ) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples . The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes . LFQ , Label - Free - Quantification ; M . W . , molecular weight .

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(A) Protein abundance is the normalized signal intensity (LFQ) for a protein divided by its molecular weight. Specificity (enrichment) is the ratio of the protein LFQ intensity in the MIA40FLAG fraction to control samples. The LFQ for proteins that were not detected in the control samples was arbitrarily set to 1 for calculation purposes. LFQ, Label-Free-Quantification; M.W., molecular weight.


( B ) Flp - In T - REx 293 cells with induced MIA40FLAG expression were solubilized , and the affinity purification of MIA40FLAG was performed . Fractions were analyzed by SDS - PAGE and Western blot . Load : 2 . 5 % . Eluate : 100 % . Unbound : 2 . 5 % .

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(B) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Fractions were analyzed by SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %. Unbound: 2.5 %.


( C ) Flp - In T - REx 293 cells with induced MIA40FLAG expression were solubilized , and the affinity purification of MIA40FLAG was performed . Eluate fractions were solubilized under reducing ( DTT ) or non - reducing ( IAA ) conditions and analyzed by SDS - PAGE and Western blot . DTT , dithiothreitol ; IAA , iodoacetamide . Eluate : 100 % .

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(C) Flp-In T-REx 293 cells with induced MIA40FLAG expression were solubilized, and the affinity purification of MIA40FLAG was performed. Eluate fractions were solubilized under reducing (DTT) or non-reducing (IAA) conditions and analyzed by SDS-PAGE and Western blot. DTT, dithiothreitol; IAA, iodoacetamide. Eluate: 100 %.


( D ) Cellular protein extracts from Flp - In T - REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification . Load and eluate fractions were analyzed by reducing SDS - PAGE and Western blot . Load : 2 . 5 % . Eluate : 100 % .

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(D) Cellular protein extracts from Flp-In T-REx 293 cells with induced expression of wildtype or mutant MIA40FLAG were subjected to affinity purification. Load and eluate fractions were analyzed by reducing SDS-PAGE and Western blot. Load: 2.5 %. Eluate: 100 %.


( A ) Schematic representation of the thiol trapping assay . Mitochondria were solubilized in sample buffer with either dithiothreitol ( DTT ) , iodoacetamide ( IAA ) , or 4 - acetamido - 4 - maleimidylstilbene - 2 , 2 - disulfonic acid ( AMS ) . The samples were analyzed by SDS - PAGE and Western blot .

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(A) Schematic representation of the thiol trapping assay. Mitochondria were solubilized in sample buffer with either dithiothreitol (DTT), iodoacetamide (IAA), or 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS). The samples were analyzed by SDS-PAGE and Western blot.


( B ) Indirect thiol trapping assay . Mitochondria were pretreated with IAA as indicated to block free cysteine residues , and disulfide bonds were subsequently reduced by DTT . Mitochondria were solubilized in sample buffer with AMS .

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(B) Indirect thiol trapping assay. Mitochondria were pretreated with IAA as indicated to block free cysteine residues, and disulfide bonds were subsequently reduced by DTT. Mitochondria were solubilized in sample buffer with AMS.


( C ) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria ( 250 mM sucrose ) , mitoplasts ( 5 mM sucrose ) , and mitochondrial lysates ( 1 % Triton X - 100 ) . The samples were analyzed by SDS - PAGE and Western blot . Mitos , mitochondria ; Sup , post - mitochondria supernatant ; OM , outer membrane ; IM , inner membrane ; IMS , intermembrane space .

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(C) Localization of mitochondrial proteins analyzed by limited degradation by proteinase K in intact mitochondria (250 mM sucrose), mitoplasts (5 mM sucrose), and mitochondrial lysates (1 % Triton X-100). The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria; Sup, post-mitochondria supernatant; OM, outer membrane; IM, inner membrane; IMS, intermembrane space.


( D ) N - SIM super - resolution micrographs of one Z - stack ( 0 . 15 μm ) orthogonal section ( XYZ ) of HeLa cells or HeLa cells that stably expressed COA7 - HA transfected with different markers TOMM20 - DsRed ( OM ) , COX8A - DsRed ( IM ) , and matrix targeted photoactivatable GFP ( Matrix target ) labeled with anti - HA , anti - COA7 and Smac / Diablo ( IMS ) antibodies . The picture represents the majority population of cells from three independent experiments . Scale bar = 2 μm , scale bar in the magnified insert = 0 . 5 μm . The panel shows Pearson ' s coefficient in a co - localized volume of different subcompartment combinations with anti - HA or anti - COA7 . The data are expressed as a mean ± SD ( n = 5 ) . * * * p < 0 . 001 [ IMSxEnd vs . IMxEnd p = 0 . 0001 ; IMSxEnd vs . MxEnd p = 0 . 0002 ] , * * * * p < 0 . 0001 [ OMxHA vs . IMSxHA p < 0 . 0001 ; IMSxHA vs . IMxHA p < 0 . 0001 ] ( one - way ANOVA ) . M , matrix ; End , endogenous COA7 ; HA , COA7 - HA .

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(D) N-SIM super-resolution micrographs of one Z-stack (0.15 μm) orthogonal section (XYZ) of HeLa cells or HeLa cells that stably expressed COA7-HA transfected with different markers TOMM20-DsRed (OM), COX8A-DsRed (IM), and matrix targeted photoactivatable GFP (Matrix target) labeled with anti-HA, anti-COA7 and Smac/Diablo (IMS) antibodies. The picture represents the majority population of cells from three independent experiments. Scale bar = 2 μm, scale bar in the magnified insert = 0.5 μm. The panel shows Pearson's coefficient in a co-localized volume of different subcompartment combinations with anti-HA or anti-COA7. The data are expressed as a mean ± SD (n=5). ***p<0.001 [IMSxEnd vs. IMxEnd p=0.0001; IMSxEnd vs. MxEnd p=0.0002], ****p<0.0001 [OMxHA vs. IMSxHA p<0.0001; IMSxHA vs. IMxHA p<0.0001] (one-way ANOVA). M, matrix; End, endogenous COA7; HA, COA7-HA.


( A ) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector . Mitochondria were solubilized and analyzed by reducing SDS - PAGE and Western blot . Mitos , mitochondria .

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(A) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. Mitochondria were solubilized and analyzed by reducing SDS-PAGE and Western blot. Mitos, mitochondria.


( B ) Radiolabeled [ 35S ] TIMM8A and [ 35S ] COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 30 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

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(B) Radiolabeled [35S]TIMM8A and [35S]COX19 precursors were imported into mitochondria that were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 30 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( C ) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing ( DTT ) and non - reducing ( IAA ) conditions and analyzed for levels of MIA40 by Western blot .

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(C) Mitochondria were isolated from cells that were transfected with a plasmid that encoded COA7HIS or an empty vector under reducing (DTT) and non-reducing (IAA) conditions and analyzed for levels of MIA40 by Western blot.


( D ) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG - PCMal . Control cells were pretreated with the thiol oxidizing agent diamide ( DAM ) or the reductant DTT . The samples were analyzed by SDS - PAGE and Western blot .

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(D) Proteins from HEK293 cells transfected with empty plasmid or RESA1HIS were modified with PEG-PCMal. Control cells were pretreated with the thiol oxidizing agent diamide (DAM) or the reductant DTT. The samples were analyzed by SDS-PAGE and Western blot.


( E ) Flp - In T - REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector . The affinity purification of MIA40FLAG was performed , and eluate fractions were analyzed by SDS - PAGE and Western blot .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flp-In T-REx 293 cells induced to express MIA40FLAG were transfected with a plasmid that encoded COA7HIS or an empty vector. The affinity purification of MIA40FLAG was performed, and eluate fractions were analyzed by SDS-PAGE and Western blot.


Radiolabeled [ 35S ] COA7 ( A ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express MIA40FLAG . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

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

Radiolabeled [35S]COA7 (A) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


Radiolabeled [ 35S ] TIMM8A ( B ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express MIA40FLAG . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

Radiolabeled [ 35S]TIMM8A (B) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( C ) Mitochondria were isolated from Flp - In T - REx 293 cells induced to express MIA40FLAG and control cells . The samples were analyzed by SDS - PAGE and Western blot . Mitos , mitochondria .

(C) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express MIA40FLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot. Mitos, mitochondria.


Radiolabeled [ 35S ] COA7 ( D ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express ALRFLAG and control cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological experiments were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) .

Radiolabeled [35S] COA7 (D) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).


Radiolabeled [ 35S ] TIMM8A ( E ) precursors were imported into mitochondria that were isolated from Flp - In T - REx 293 cells induced to express ALRFLAG and control cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological experiments were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) .

Radiolabeled [35S] TIMM8A (E) precursors were imported into mitochondria that were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological experiments were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3).


( F ) Mitochondria were isolated from Flp - In T - REx 293 cells induced to express ALRFLAG and control cells . The samples were analyzed by SDS - PAGE and Western blot .

O O O O O O O O O O O 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) Mitochondria were isolated from Flp-In T-REx 293 cells induced to express ALRFLAG and control cells. The samples were analyzed by SDS-PAGE and Western blot.


( A ) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides . The samples were analyzed by reducing SDS - PAGE and Western blot .

(A) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of MIA40 mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.


( B ) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides . The samples were analyzed by reducing SDS - PAGE and Western blot .

(B) Cellular protein extracts were isolated from HeLa cells transfected with oligonucleotides that targeted different regions of ALR mRNA or with control oligonucleotides. The samples were analyzed by reducing SDS-PAGE and Western blot.


( D ) Cellular protein extracts were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells . The samples were analyzed by SDS - PAGE and Western blot .

O O O O O O O O O O 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) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by SDS-PAGE and Western blot.


( E ) Cellular protein extracts were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells transfected with a plasmid that encoded MIA40 or an empty vector . The samples were subjected to reducing SDS - PAGE and Western blot .

O O O O O O O O O O O O O O O O O O O O O O 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) Cellular protein extracts were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells transfected with a plasmid that encoded MIA40 or an empty vector. The samples were subjected to reducing SDS-PAGE and Western blot.


Radiolabeled [ 35S ] COA7 ( F ) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

Radiolabeled [35S]COA7 (F) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


Radiolabeled [ [ 35S ] TIMM8A ( G ) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT / WT and WT / Del53 - 60 cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to control mitochondria at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

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

Radiolabeled [ [35S]TIMM8A (G) precursors were imported into mitochondria that were isolated from HEK293 MIA40 WT/WT and WT/Del53-60 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to control mitochondria at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( B ) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7 . The samples were analyzed by reducing SDS - PAGE and Western blot .

O O O O O O O O O O O O O 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) Cellular protein extracts were isolated from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The samples were analyzed by reducing SDS-PAGE and Western blot.


( C ) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7 . The fractions were analyzed by reducing SDS - PAGE and Western blot . T , total ; C , cytosol ; M , mitochondria .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Cellular fractions were prepared from HEK293 cells that were transfected with a plasmid that encoded wildtype or mutant COA7. The fractions were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.


( D ) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized , and the affinity purification of COA7HIS was performed . The samples were analyzed by reducing and non - reducing SDS - PAGE and Western blot . Load : 3 % . Eluate : 100 % .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were solubilized, and the affinity purification of COA7HIS was performed. The samples were analyzed by reducing and non-reducing SDS-PAGE and Western blot. Load: 3 %. Eluate: 100 %.


( E ) Equal amounts of wildtype and mutant radiolabeled [ 35S ] COA7 precursors were imported into mitochondria isolated from HEK293 cells . The samples were analyzed by reducing SDS - PAGE and autoradiography . The results of three biological replicates were analyzed , quantified , and normalized to wildtype COA7 at 45 min . The data are expressed as a mean ± SEM ( n = 3 ) . IAA , iodoacetamide .

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

(E) Equal amounts of wildtype and mutant radiolabeled [35S]COA7 precursors were imported into mitochondria isolated from HEK293 cells. The samples were analyzed by reducing SDS-PAGE and autoradiography. The results of three biological replicates were analyzed, quantified, and normalized to wildtype COA7 at 45 min. The data are expressed as a mean ± SEM (n=3). IAA, iodoacetamide.


( B ) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times , and protein extracts were isolated . The samples were analyzed by reducing SDS - PAGE and Western blot .

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) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot.


( D ) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and / or MG132 for the indicated times , and protein extracts were isolated . The samples were analyzed by reducing SDS - PAGE and Western blot . CHX , cycloheximide .

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

(D) HEK293 cells that transiently expressed wildtype or mutant COA7HIS were treated with CHX and/or MG132 for the indicated times, and protein extracts were isolated. The samples were analyzed by reducing SDS-PAGE and Western blot. CHX, cycloheximide.


( F ) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132 . The samples were analyzed by reducing SDS - PAGE and Western blot .

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

(F) Cellular fractions were prepared from HEK293 cells that transiently expressed mutant COA7HIS and were treated with MG132. The samples were analyzed by reducing SDS-PAGE and Western blot.


( A ) Cellular protein extracts were isolated from immortalized patient - derived skin fibroblasts that were treated with the indicated concentrations of MG132 , bortezomib , or carfilzomib . The samples were analyzed by reducing SDS - PAGE and Western blot .

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

(A) Cellular protein extracts were isolated from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot.


( B ) Cellular fractions were prepared from immortalized patient - derived skin fibroblasts that were treated with the indicated concentrations of MG132 , bortezomib , or carfilzomib . The samples were analyzed by reducing SDS - PAGE and Western blot . T , total ; C , cytosol ; M , mitochondria .

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 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

(B) Cellular fractions were prepared from immortalized patient-derived skin fibroblasts that were treated with the indicated concentrations of MG132, bortezomib, or carfilzomib. The samples were analyzed by reducing SDS-PAGE and Western blot. T, total; C, cytosol; M, mitochondria.


( A ) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient - derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified , and normalized to control fibroblasts . Data are expressed as a mean ± SEM ( n = 3 , * p = 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from the immortalized patient-derived skin fibroblasts and control healthy fibroblasts The results of three biological replicates were quantified, and normalized to control fibroblasts. Data are expressed as a mean ± SEM (n=3, *p=0.01).


( B ) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient - derived skin fibroblasts treated with MG132 ( 1 μM ) , bortezomib ( 20 nM ) , or carfilzomib ( 50 nM ) for 12 h and recovered for another 6 h . The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM ( n = 3 , * p = 0 . 04 [ DMSO vs MG132 ] , * * p = 0 . 0025 [ DMSO vs Bortezomib ] , * p = 0 . 02 [ DMSO vs Carfilzomib ] ) .

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

(B) Complex IV activity was assessed by oxidation of cytochrome c in digitonized cellular extracts from immortalized patient-derived skin fibroblasts treated with MG132 (1 μM), bortezomib (20 nM), or carfilzomib (50 nM) for 12 h and recovered for another 6 h. The results of three biological replicates were quantified and normalized to DMSO treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.04 [DMSO vs MG132], **p=0.0025 [DMSO vs Bortezomib], *p=0.02 [DMSO vs Carfilzomib]).


( C ) Immortalized patient - derived skin fibroblasts transiently expressing wildtype or mutant COA7 ( COA7 - Y137C and COA7 - Ex2 ∆ ) for 48 h were treated with bortezomib ( 20 nM ) during the last 12 h . The fibroblasts were harvested , and complex IV activity was measured in digitonized cellular extracts . The results of three biological replicates were quantified and normalized to empty vector - bortezomib treated samples and the data are expressed as a mean ± SEM ( n = 3 , * p = 0 . 01 [ Empty vector vs COA7 - Wt ] , * p = 0 . 005 [ Empty vector vs COA7 - Y137C ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immortalized patient-derived skin fibroblasts transiently expressing wildtype or mutant COA7 (COA7-Y137C and COA7-Ex2∆) for 48 h were treated with bortezomib (20 nM) during the last 12 h. The fibroblasts were harvested, and complex IV activity was measured in digitonized cellular extracts. The results of three biological replicates were quantified and normalized to empty vector-bortezomib treated samples and the data are expressed as a mean ± SEM (n=3, *p=0.01 [Empty vector vs COA7- Wt], *p=0.005 [Empty vector vs COA7 - Y137C]).


( D ) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4 - 13 % gel BN - PAGE and Western blot . SC , supercomplexes .

(D) Mitochondria isolated from control and patient fibroblast were solubilized in digitonin buffer and analyzed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.


( E ) Immortalized patient - derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7 - Y137CHIS were solubilized in DDM buffer and analyzed by 4 - 13 % gel BN - PAGE and Western blot .

O O O O O O O O O O O O O O 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) Immortalized patient-derived skin fibroblasts transfected with plasmid encoding wtCOA7HIS and COA7-Y137CHIS were solubilized in DDM buffer and analyzed by 4-13 % gel BN-PAGE and Western blot.


( F ) Mitochondria were isolated from immortalized patient - derived skin fibroblasts treated with bortezomib ( 10 nM ) for 12 h and recovered for another 6 h . Mitochondria were solubilized in digitonin buffer and analysed by 4 - 13 % gel BN - PAGE and Western blot . SC , supercomplexes .

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

(F) Mitochondria were isolated from immortalized patient-derived skin fibroblasts treated with bortezomib (10 nM) for 12 h and recovered for another 6 h. Mitochondria were solubilized in digitonin buffer and analysed by 4-13 % gel BN-PAGE and Western blot. SC, supercomplexes.


Three - dimensional structure of 2019 - nCoV Mpro , in two different views . One protomer of the dimer is shown in light blue , the other one in orange . Amino - acid residues of the catalytic site are indicated as yellow and blue spheres , for Cys145 and His41 , respectively . Black spheres indicate the positions of Ala285 of each of the two domains III ( see text ) . Chain termini are labeled N and C for molecule A ( light blue ) and N * and C * for molecule B ( orange ) .

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

Three-dimensional structure of 2019-nCoV Mpro, in two different views. One protomer of the dimer is shown in light blue, the other one in orange. Amino-acid residues of the catalytic site are indicated as yellow and blue spheres, for Cys145 and His41, respectively. Black spheres indicate the positions of Ala285 of each of the two domains III (see text). Chain termini are labeled N and C for molecule A (light blue) and N* and C* for molecule B (orange).


Compound 13b in the substrate - binding cleft located between domains I and II of the Mpro , in the monoclinic crystal form ( space group C2 ) . 2Fo - Fc electron density is shown for the inhibitor ( contouring level : 1σ ) . Carbon atoms of the inhibitor are magenta , oxygens red , nitrogens blue , and sulfur yellow . Note the interaction between the N - terminal residue of chain B , S1 * , and E166 of chain A .

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

Compound 13b in the substrate-binding cleft located between domains I and II of the Mpro, in the monoclinic crystal form (space group C2). 2Fo-Fc electron density is shown for the inhibitor (contouring level: 1σ). Carbon atoms of the inhibitor are magenta, oxygens red, nitrogens blue, and sulfur yellow. Note the interaction between the N-terminal residue of chain B, S1*, and E166 of chain A.


( A ) Optically evoked auditory brainstem response in an AAV - CatCh - injected gerbil in response to a 1 ms laser pulse of ~ 35 mW ( mean of 1000 stimulus presentations at 10 Hz repetition rate ) .

O O O O O O O O O O O O O O O O O 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) Optically evoked auditory brainstem response in an AAV-CatCh-injected gerbil in response to a 1 ms laser pulse of ~35 mW (mean of 1000 stimulus presentations at 10 Hz repetition rate).


( B - D ) Catch - eYFP - expressing SGNs in the apical cochlear turn identified by parvalbumin expression ( B ) and CatCh - eYFP ( C ) . The inlay ( D ´ ) of the merged immunostaining ( D ) indicates the lack of CatCh - EYFP signal in inner hair cells ( dashed white line ) . Scale bar : 20 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

(B-D) Catch-eYFP-expressing SGNs in the apical cochlear turn identified by parvalbumin expression (B) and CatCh-eYFP (C). The inlay (D´) of the merged immunostaining (D) indicates the lack of CatCh-EYFP signal in inner hair cells (dashed white line). Scale bar: 20 µm.


( I ) oCI inserted into the cochlea ( dashed black lines ) via a cochleostomy in the basal cochlear turn ( black , solid line ) . The round window niche is highlighted by a dashed white line , the round window by a solid white line . SA : stapedial artery .

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

(I) oCI inserted into the cochlea (dashed black lines) via a cochleostomy in the basal cochlear turn (black, solid line). The round window niche is highlighted by a dashed white line, the round window by a solid white line. SA: stapedial artery.