Human striatal organoids were injected with AAV - Con ( GFP ) or AAV - mtHSF1 at D42 . Scatterplot showed the percentage of NeuN + cells ( red ) among Flag + or GFP + cells ( green ) after injection for 2 weeks . The data were obtained from 3 independent biological experiments . Con : n = 11 organoids ; Flag - mtHSF1 : n = 13 organoids . The scale bar represents 100 µm .

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Human striatal organoids were injected with AAV-Con (GFP) or AAV-mtHSF1 at D42. Scatterplot showed the percentage of NeuN+ cells (red) among Flag+ or GFP+ cells (green) after injection for 2 weeks. The data were obtained from 3 independent biological experiments. Con: n=11 organoids; Flag-mtHSF1: n=13 organoids. The scale bar represents 100 µm.


Representative images of immunostaining with DARPP32 ( red ) and Flag ( green ) . The DARPP32 fluorescence density was analyzed in Flag + or Flag - sections by confocal microscopy and showed by scatterplot . Thickness : 30 µm . The scale bar is 2 mm , and the enlarged image scale bar is 40 µm . ( n = 12 mice / group ) .

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Representative images of immunostaining with DARPP32 (red) and Flag (green). The DARPP32 fluorescence density was analyzed in Flag+ or Flag- sections by confocal microscopy and showed by scatterplot. Thickness: 30 µm. The scale bar is 2 mm, and the enlarged image scale bar is 40 µm. (n=12 mice/group).


The striatum of AAV - Con - or AAV - mtHSF1 - injected mice were harvested . The DARPP32 protein levels were tested by WB analysis . ( n = 6 mice / group ) .

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The striatum of AAV-Con- or AAV-mtHSF1-injected mice were harvested. The DARPP32 protein levels were tested by WB analysis. (n=6 mice/group).


The percentage of NeuN + cells in Flag + or Flag - sections was determined by confocal microscopy and showed by scatterplot . Thickness : 10 µm . ( n = 9 mice / group ) .

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The percentage of NeuN+ cells in Flag+ or Flag- sections was determined by confocal microscopy and showed by scatterplot. Thickness: 10 µm. (n=9 mice/group).


Mouse weight and motor activity were recorded post AAV injection . ( n = 7 - 13 mice / group ) .

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Mouse weight and motor activity were recorded post AAV injection. (n=7-13 mice/group).


Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti - HSF1 antibody followed by immunoblotting . ( n = 3 biological replicates ) .

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Total lysates of HdhQ7 or HdhQ111 cells were subjected to immunoprecipitation with an anti-HSF1 antibody followed by immunoblotting. (n=3 biological replicates).


Drp1 and HSF1 binding in vitro was analyzed by WB analysis . ( n = 3 biological replicates ) .

Drp1 and HSF1 binding in vitro was analyzed by WB analysis. (n=3 biological replicates).


Mitochondrial fractions were isolated from WT or Drp1 - KO MEFs . The protein levels of Drp1 and HSF1 were tested by immunoblotting . ( n = 3 biological replicates ) .

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Mitochondrial fractions were isolated from WT or Drp1-KO MEFs. The protein levels of Drp1 and HSF1 were tested by immunoblotting. (n=3 biological replicates).


GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1 . ( n = 3 biological replicates ) .

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GST pulldown assay of Drp1 and HSF1 in the presence or absence of DH1. (n=3 biological replicates).


HdhQ7 or HdhQ111 cells ( 1 μM / day , 3 days ) , were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

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HdhQ7 or HdhQ111 cells (1 μM/day, 3 days), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


H . YAC128 or age - matched littermates ( 3 mg / kg / day , 3 months ) , were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

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H. YAC128 or age-matched littermates (3 mg/kg/day, 3 months), were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


I . striatal organoids differentiated from patient iPSCs or control iPSCs ( 1 μM / day , 7 days ) were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

I. striatal organoids differentiated from patient iPSCs or control iPSCs (1 μM/day, 7 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


J . patient fibroblasts ( 1 μM / day , 5 days ) were treated with DH1 or TAT . mtHSF1 was measured by immunoblotting . ( n = 3 - 5 biological replicates ) .

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J. patient fibroblasts (1 μM/day, 5 days) were treated with DH1 or TAT. mtHSF1 was measured by immunoblotting. (n=3-5 biological replicates).


HdhQ7 or HdhQ111 were treated with DH1 or TAT ( 1 μM / day , 3 days ) . SSBP1 oligomers were analyzed by immunoblotting . ( n = 3 - 6 biological replicates ) .

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HdhQ7 or HdhQ111 were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).


L . patient fibroblasts were treated with DH1 or TAT ( 1 μM / day , 3 days ) . SSBP1 oligomers were analyzed by immunoblotting . ( n = 3 - 6 biological replicates ) .

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L. patient fibroblasts were treated with DH1 or TAT (1 μM/day, 3 days). SSBP1 oligomers were analyzed by immunoblotting. (n=3-6 biological replicates).


Immunostaining for mitochondria ( Tom20 , green ) and mtDNA ( anti - DNA , red ) in striatal organoids treated with DH1 or TAT at D60 . Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria . The data were obtained from 3 independent biological experiments . Left , n = 222 - 311 mitochondria per group ; Right , n = 14 - 20 organoids per group . Images were taken using Structured Illumination Microscopy ( SIM ) . The scale bar represents 10 μm .

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Immunostaining for mitochondria (Tom20, green) and mtDNA (anti-DNA, red) in striatal organoids treated with DH1 or TAT at D60. Scatterplots with bar showed the length of mitochondria and the number of mtDNA colocalized with mitochondria. The data were obtained from 3 independent biological experiments. Left, n=222-311 mitochondria per group; Right, n=14-20 organoids per group. Images were taken using Structured Illumination Microscopy (SIM). The scale bar represents 10 μm.


Immunostaining for mitochondria ( Tom20 , red ) and p - Drp1 S616 ( green ) in striatal treated with DH1 or TAT at D60 . Scatterplot with bar showed the number of p - Drp1 S616 per cell in organoids . The data were obtained from 3 independent biological experiments . n = 29 - 34 cells from at least 10 organoids . The scale bar represents 5 μm .

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Immunostaining for mitochondria (Tom20, red) and p-Drp1 S616 (green) in striatal  treated with DH1 or TAT at D60. Scatterplot with bar showed the number of p-Drp1 S616 per cell in organoids. The data were obtained from 3 independent biological experiments. n=29-34 cells from at least 10 organoids. The scale bar represents 5 μm.


Scatterplot with bar showed the ratio of MSNs ( DARPP32 , green ) in striatal organoids in the presence or absence of DH1 . The data were obtained from 3 independent biological experiments . n = 30 organoids per group .

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Scatterplot with bar showed the ratio of MSNs (DARPP32, green) in striatal organoids in the presence or absence of DH1. The data were obtained from 3 independent biological experiments. n=30 organoids per group.


Representative tracing images of DARPP32 + neurons treated with DH1 or TAT at D45 . Scatterplot with bar showed the results of longest neurites length . The data were obtained from 3 independent biological experiments . n = 45 - 92 neurons per group .

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Representative tracing images of DARPP32+ neurons treated with DH1 or TAT at D45. Scatterplot with bar showed the results of longest neurites length. The data were obtained from 3 independent biological experiments. n=45-92 neurons per group.


Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1 . The arrows ( white ) mark fragmented nuclei . The data were obtained from 3 independent biological experiments . n = 7 - 10 organoids per group . The scale bar represents 20 μm .

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Representative images and scatterplot with bar of nuclear fragmentation in striatal organoids treated with DH1. The arrows (white) mark fragmented nuclei. The data were obtained from 3 independent biological experiments. n=7-10 organoids per group. The scale bar represents 20 μm.


H . PGC1α protein levels were determined by immunoblotting . ( n = 3 biological replicates ) .

H. PGC1α protein levels were determined by immunoblotting. (n=3 biological replicates).


I . DARPP32 protein levels were determined by immunoblotting . ( n = 3 biological replicates ) .

I. DARPP32 protein levels were determined by immunoblotting. (n=3 biological replicates).


Mouse behavior was tested after DH1 treatment . ( n = 9 - 12 mice / group ) .

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Mouse behavior was tested after DH1 treatment. (n=9-12 mice/group).


b In HEK293 cells , recombinantly expressed GluN1 / GluN2A and GluN1 / GluN2A ( N615S ) channels were activated by fast glutamate application ( 1 mM ; in the continuous presence of the co - agonist glycine , 10 µM ) at holding potentials from - 100 to + 40 mV in different extracellular Mg2 + concentrations . NMDAR - mediated peak currents were normalized to those obtained at + 40 mV . Data points represent mean ± SEM for n = 4 - 7 different HEK293 cells . Representative current traces evoked in 0 mM Mg2 + at - 60 mV , with 20 and 600 ms applications , are shown below the IV plots and were used to determine the current kinetics ( Supplementary Table 1 ) .

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b In HEK293 cells, recombinantly expressed GluN1/GluN2A and GluN1/GluN2A(N615S) channels were activated by fast glutamate application (1 mM; in the continuous presence of the co-agonist glycine, 10 µM) at holding potentials from -100 to +40 mV in different extracellular Mg2+ concentrations. NMDAR-mediated peak currents were normalized to those obtained at +40 mV. Data points represent mean ± SEM for n = 4-7 different HEK293 cells. Representative current traces evoked in 0 mM Mg2+ at - 60 mV, with 20 and 600 ms applications, are shown below the IV plots and were used to determine the current kinetics (Supplementary Table 1).


d Reverse transcription PCR ( RT - PCR ) - sequence analyses of total brain mRNA show the A - to - G mutation in pos . 0 and two diagnostic silent mutations at pos . - 11 and - 14 in the pore loop encoding gene segment in Grin2a + / + , Grin2aS / S , Grin2a + / S mice . In Grin2a + / S mice , the overlay of two different colored " nucleotide " peaks , at position 0 , - 11 , and - 14 indicate equimolar amounts of mRNA from the Grin2a + and the targeted Grin2aS alleles .

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d Reverse transcription PCR (RT-PCR)-sequence analyses of total brain mRNA show the A-to-G mutation in pos. 0 and two diagnostic silent mutations at pos. -11 and -14 in the pore loop encoding gene segment in Grin2a+/+, Grin2aS/S, Grin2a+/S mice. In Grin2a+/S mice, the overlay of two different colored "nucleotide" peaks, at position 0, -11, and -14 indicate equimolar amounts of mRNA from the Grin2a+ and the targeted Grin2aS alleles.


e Immunoblots of forebrain protein lysates of 4 - week - old mice ( Supplementary Fig . 2 ) indicate no genotype - specific differences of GluN1 , GluN2A , and the AMPAR subunit GluA1 expression relative to the β - actin levels ( p > 0 . 05 ) . The levels of PSD95 and αCaMKII ( in its phosphorylated state , pCaMKII ) are also comparable between genotypes relative to the GAPDH expression . The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS / S mice when compared to Grin2a + / + and Grin2a + / S mice but not in the levels of total protein lysates ( for statistics : Supplementary Statistics to Fig . 1 ) . The number of mice is given in brackets . Error bars represent mean ± SEM .

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e Immunoblots of forebrain protein lysates of 4-week-old mice (Supplementary Fig. 2) indicate no genotype-specific differences of GluN1, GluN2A, and the AMPAR subunit GluA1 expression relative to the β-actin levels (p > 0.05). The levels of PSD95 and αCaMKII (in its phosphorylated state, pCaMKII) are also comparable between genotypes relative to the GAPDH expression. The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS/S mice when compared to Grin2a+/+ and Grin2a+/S mice but not in the levels of total protein lysates (for statistics: Supplementary Statistics to Fig. 1). The number of mice is given in brackets. Error bars represent mean ± SEM.


a ( left ) In the absence of extracellular Mg2 + the synaptic AMPA / NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a + / S and Grin2aS / S mice compared to control littermates . ( right ) In the presence of extracellular Mg2 + the strong reduction of the AMPA / NMDA ratio in Grin2aS / S mice relates to the increased NMDA currents at CA1 synapses ( Tukey ' s test ) . Example traces are depicted to the right of each bar graph . Data from the same experimental group were pooled across animals and are presented as mean ± SEM ( see also ref . 35 ) with p < 0 . 05 being designated as statistically significant . Numbers in bar graphs indicate the number of slices .

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a (left) In the absence of extracellular Mg2+ the synaptic AMPA/NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a+/S and Grin2aS/S mice compared to control littermates. (right) In the presence of extracellular Mg2+ the strong reduction of the AMPA/NMDA ratio in Grin2aS/S mice relates to the increased NMDA currents at CA1 synapses (Tukey's test). Example traces are depicted to the right of each bar graph. Data from the same experimental group were pooled across animals and are presented as mean ± SEM (see also ref. 35) with p < 0.05 being designated as statistically significant. Numbers in bar graphs indicate the number of slices.


b Paired - pulse facilitation at CA3 - to - CA1 synapses excluded strong alterations of presynaptic function in mutant mice . The stimulation strengths ( in nC ) necessary to elicit a pre - volley of 1 . 0 and 1 . 5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1 . 5 mV pre - volley amplitudes in Grin2aS / S mice . The fEPSP amplitudes necessary to elicit a just detectable population spike ( 1 ) and a population spike of 2 mV amplitude ( 2 ) and the paired - pulse facilitation ratio ( PPF ) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS / S and Grin2a + / S mice compared to WT littermates . The number of slices is indicated in the bar graphs or in brackets .

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b Paired-pulse facilitation at CA3-to-CA1 synapses excluded strong alterations of presynaptic function in mutant mice. The stimulation strengths (in nC) necessary to elicit a pre-volley of 1.0 and 1.5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1.5 mV pre-volley amplitudes in Grin2aS/S mice. The fEPSP amplitudes necessary to elicit a just detectable population spike (1) and a population spike of 2 mV amplitude (2) and the paired-pulse facilitation ratio (PPF) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS/S and Grin2a+/S mice compared to WT littermates. The number of slices is indicated in the bar graphs or in brackets.


c Field LTP ( fLTP ) at CA3 - to - CA1 synapses , induced by tetanic stimulation in slices ( 1 s ; 100 Hz ; arrow ) , was comparable in all three genotypes . The inset in c gives a schematic view of the stimulating ( white arrow ) and the recording ( green arrow ) electrode positions in str . radiatum ( r ) and str . oriens ( o ) . d Similarly , CA1 - to - CA3 fLTP ( induction : 2 × 1 s ; 100 Hz ; arrow ) in freely moving mice was comparable between Grin2aS / S and Grin2a + / + mice . The inset shows a Nissl - stained slice of one recorded mouse post mortem . e In mutant mice , but not in control littermates , the GluN2B - containing NMDAR contributes significantly to the magnitude of LTP , since LTP was significantly reduced by the GluN2B - specific antagonist CP101 , 106 ( CP ) . Recordings of the non - tetanized control pathway in c and e are given as dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 2 ) .

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c Field LTP (fLTP) at CA3-to-CA1 synapses, induced by tetanic stimulation in slices (1 s; 100 Hz; arrow), was comparable in all three genotypes. The inset in c gives a schematic view of the stimulating (white arrow) and the recording (green arrow) electrode positions in str. radiatum (r) and str. oriens (o). d Similarly, CA1-to-CA3 fLTP (induction: 2 × 1 s; 100 Hz; arrow) in freely moving mice was comparable between Grin2aS/S and Grin2a+/+ mice. The inset shows a Nissl-stained slice of one recorded mouse post mortem. e In mutant mice, but not in control littermates, the GluN2B-containing NMDAR contributes significantly to the magnitude of LTP, since LTP was significantly reduced by the GluN2B-specific antagonist CP101,106 (CP). Recordings of the non-tetanized control pathway in c and e are given as dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 2).


aGrin2aS / S mice had a reduced body weight ( by 18 % in adults ) , significantly lower score in nesting and in overnight burrowing activity ( filled circles : 30 - week - old mice , white dots : 18 - week - old mice ) .

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aGrin2aS/S mice had a reduced body weight (by 18% in adults), significantly lower score in nesting and in overnight burrowing activity (filled circles: 30-week-old mice, white dots: 18-week-old mice).


b Grin2aS / S mice showed the clasping reflex . c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS / S mice .

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b Grin2aS/S mice showed the clasping reflex. c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS/S mice.


d Nissl staining revealed unaltered cell density throughout the brain ( top ) and layering of hippocampal subfields , including CA1 ( bottom ) , in Grin2aS / S mice compared to Grin2a + / + . Error bars represent mean ± SEM . e In the TUNEL assay no apoptotic cells could be detected in the hippocampus of Grin2aS / S mice , compared to the staining of the nuclease - treated positive control slice . f No aberrant mossy fiber sprouting in either genotype in the dentate gyrus inner ( IML ) and outer molecular layer ( OML ) can be found by Timm staining of coronal sections .

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d Nissl staining revealed unaltered cell density throughout the brain (top) and layering of hippocampal subfields, including CA1 (bottom), in Grin2aS/S mice compared to Grin2a+/+. Error bars represent mean ± SEM. e In the TUNEL assay no apoptotic cells could be detected in the hippocampus of Grin2aS/S mice, compared to the staining of the nuclease-treated positive control slice. f No aberrant mossy fiber sprouting in either genotype in the dentate gyrus inner (IML) and outer molecular layer (OML) can be found by Timm staining of coronal sections.


g The distribution of NeuN - positive neurons and of GFAP - positive glial cells are indistinguishable between Grin2aS / S and Grin2a + / + mice . Mossy fiber projections visualized by anti - calbindin ( CB ) staining , as well as numbers of parvalbumin ( PV ) - positive interneurons are similar between both genotypes . Anti - GluA1 immunosignal in all hippocampal layers is comparable between brain sections of Grin2aS / S mice and control littermates . CA1 cornu ammonis region 1 , CA3 cornu ammonis region 3 , DG dentate gyrus , mol stratum moleculare , ori stratum oriens , rad , stratum radiatum . Scale bars in d - g are in mm . The number of animals is given below the bars . For the Nissl stain , Tunnel test and Timm stain 3 mice were used per genotype . For the immunohistological analysis of glial fibrillary acidic protein ( GFAP ) , neuronal nuclear antigen ( NeuN ) , Calbindin ( CB ) , and Parvalbumin ( PV ) five mice and for the GluA1 immunofluorescence stain , three mice were used ( for statistics : Supplementary Statistics to Fig . 3 ) .

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g The distribution of NeuN-positive neurons and of GFAP-positive glial cells are indistinguishable between Grin2aS/S and Grin2a+/+ mice. Mossy fiber projections visualized by anti-calbindin (CB) staining, as well as numbers of parvalbumin (PV)-positive interneurons are similar between both genotypes. Anti-GluA1 immunosignal in all hippocampal layers is comparable between brain sections of Grin2aS/S mice and control littermates. CA1 cornu ammonis region 1, CA3 cornu ammonis region 3, DG dentate gyrus, mol stratum moleculare, ori stratum oriens, rad, stratum radiatum. Scale bars in d-g are in mm. The number of animals is given below the bars. For the Nissl stain, Tunnel test and Timm stain 3 mice were used per genotype. For the immunohistological analysis of glial fibrillary acidic protein (GFAP), neuronal nuclear antigen (NeuN), Calbindin (CB), and Parvalbumin (PV) five mice and for the GluA1 immunofluorescence stain, three mice were used (for statistics: Supplementary Statistics to Fig. 3).


a Mortality curve showing that none of the Grin2a + / + controls were affected , while audiogenic seizures ( AGSs ) followed by respiratory arrest ( RA ) were induced in all Grin2aS / S mice and in a subset of heterozygous Grin2a + / S mutants during the 11 kHz tone exposure [ 4 repetitions × 20 s tone , 2 s brake ; pink squares ; p < 0 . 0001 by Log - rank ( Mantel - Cox ) test ] .

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a Mortality curve showing that none of the Grin2a+/+ controls were affected, while audiogenic seizures (AGSs) followed by respiratory arrest (RA) were induced in all Grin2aS/S mice and in a subset of heterozygous Grin2a+/S mutants during the 11 kHz tone exposure [4 repetitions × 20 s tone, 2 s brake; pink squares; p < 0.0001 by Log-rank (Mantel-Cox) test].


b The c - Fos immunoreactivity was specifically increased in the hypothalamus ( VMH ) , the inferior colliculus ( IC ) , and periaqueductal gray ( PAG ) but not in the hippocampus ( HPC ) of a resuscitated Grin2aS / S mouse 90 min after AGS when compared to tone - exposed Grin2a + / + littermates .

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b The c-Fos immunoreactivity was specifically increased in the hypothalamus (VMH), the inferior colliculus (IC), and periaqueductal gray (PAG) but not in the hippocampus (HPC) of a resuscitated Grin2aS/S mouse 90 min after AGS when compared to tone-exposed Grin2a+/+ littermates.


c Memantine i . p . injection in Grin2aS / S mice , 3 h before tone exposure , rescued AGS susceptibility in Grin2aS / S mice . The rescue effect could still be observed in four out of eight , and in two out of eight , mice 27 and 51 h after memantine treatment , respectively .

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c Memantine i.p. injection in Grin2aS/S mice, 3 h before tone exposure, rescued AGS susceptibility in Grin2aS/S mice. The rescue effect could still be observed in four out of eight, and in two out of eight, mice 27 and 51 h after memantine treatment, respectively.


d Increased c - Fos immunofluorescence ( in grayscale ) in the medial amygdala ( MeA ) and the VMH of a Grin2aS / S animal with AGS ( saline injection ) compared to a memantine rescued Grin2aS / S littermate . In the paraventricular nucleus of the thalamus ( PVT ) , the hippocampus ( HPC ) and cortex ( Cx ) there was no difference in c - Fos expression between memantine - injected and saline - injected animals ( for fluorescence images , see Supplementary Fig . 5 ) .

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d Increased c-Fos immunofluorescence (in grayscale) in the medial amygdala (MeA) and the VMH of a Grin2aS/S animal with AGS (saline injection) compared to a memantine rescued Grin2aS/S littermate. In the paraventricular nucleus of the thalamus (PVT), the hippocampus (HPC) and cortex (Cx) there was no difference in c-Fos expression between memantine-injected and saline-injected animals (for fluorescence images, see Supplementary Fig. 5).


e As in Fig . 4c but now MK - 801 and saline are used for pre - treatment . The MK - 801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK - 801 injection .

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e As in Fig. 4c but now MK-801 and saline are used for pre-treatment. The MK-801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK-801 injection.


f Decreased c - Fos DAB immunosignal in an MK - 801 AGS - rescued Grin2aS / S animal ( bottom ) in the medial amygdala ( MeA ) , the VMH and the PVT when compared to saline - injected Grin2aS / S littermate ( top ) . Scale bars are in mm ( for statistics : Supplementary Statistics to Fig . 4 ) .

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f Decreased c-Fos DAB immunosignal in an MK-801 AGS-rescued Grin2aS/S animal (bottom) in the medial amygdala (MeA), the VMH and the PVT when compared to saline-injected Grin2aS/S littermate (top). Scale bars are in mm (for statistics: Supplementary Statistics to Fig. 4).


C - Fos expression 120 min after i . p . injection of MK - 801 . a C - Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated . The original images , including the cortical regions and results of PBS injected animals , are presented in Supplementary Fig . 6 . The number of mice analyzed is given in brackets . b For quantitative comparisons the total numbers of c - Fos - positive cells in the cellular layers of the hippocampus were counted . C - Fos - positive cells in extra - hippocampal regions are given per mm2 . Slices from six mice per genotype were used for the statistical analyses ( two - tailed t - tests ) and error bars represent mean ± SEM . CA1 and CA3 cornu ammonis regions 1 and 3 , DG dentate gyrus , PC piriform cortex , RSC retrosplenial cortex , Neo neocortex , MTN midline thalamic nuclei , BLA basolateral amygdala . All scale bars : 0 . 2 mm .

O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-Fos expression 120 min after i.p. injection of MK-801. a C-Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated. The original images, including the cortical regions and results of PBS injected animals, are presented in Supplementary Fig. 6. The number of mice analyzed is given in brackets. b For quantitative comparisons the total numbers of c-Fos-positive cells in the cellular layers of the hippocampus were counted. C-Fos-positive cells in extra-hippocampal regions are given per mm2. Slices from six mice per genotype were used for the statistical analyses (two-tailed t-tests) and error bars represent mean ± SEM. CA1 and CA3 cornu ammonis regions 1 and 3, DG dentate gyrus, PC piriform cortex, RSC retrosplenial cortex, Neo neocortex, MTN midline thalamic nuclei, BLA basolateral amygdala. All scale bars: 0.2 mm.


a ( left ) Peak frequencies during exploration and rapid eye movement ( REM ) sleep for theta ( 0 - 15 Hz , left diagrams ) and gamma oscillations ( 15 - 200 Hz , right diagrams ) in all genotypes . During exploration , the average theta range peak frequencies were at 8 . 61 ± 0 . 07 and 8 . 75 ± 0 . 28 Hz for Grin2a + / + mice and Grin2a + / S mice , respectively . In Grin2aS / S mice the theta range peak frequency was significantly slower , measuring 7 . 43 ± 0 . 24 Hz ( Kruskal Wallis test , p = 0 . 0159 ) . During REM sleep the average frequency ranges of Grin2a + / + and Grin2a + / S mice were around 7 . 00 ± 0 . 20 Hz whereas Grin2aS / S mice had a lower but not significantly different range 6 . 71 ± 0 . 22 Hz . a ( right ) No significant differences on the low and high gamma frequency bands - ranges between 20 - 100 Hz and 100 - 200 Hz , respectively111 - could be detected between genotypes , neither during exploration nor in REM sleep . The number of mice used in this experiment are given with the name of the genotypes .

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a (left) Peak frequencies during exploration and rapid eye movement (REM) sleep for theta (0-15 Hz, left diagrams) and gamma oscillations (15-200 Hz, right diagrams) in all genotypes. During exploration, the average theta range peak frequencies were at 8.61 ± 0.07 and 8.75 ± 0.28 Hz for Grin2a+/+ mice and Grin2a+/S mice, respectively. In Grin2aS/S mice the theta range peak frequency was significantly slower, measuring 7.43 ± 0.24 Hz (Kruskal Wallis test, p = 0.0159). During REM sleep the average frequency ranges of Grin2a+/+ and Grin2a+/S mice were around 7.00 ± 0.20 Hz whereas Grin2aS/S mice had a lower but not significantly different range 6.71 ± 0.22 Hz. a (right) No significant differences on the low and high gamma frequency bands-ranges between 20-100 Hz and 100-200 Hz, respectively111-could be detected between genotypes, neither during exploration nor in REM sleep. The number of mice used in this experiment are given with the name of the genotypes.


b ( left ) Grin2aS / S and Grin2a + / S mice show reduced theta - gamma phase - amplitude coupling as determined by the modulation index ( MI ) in the low gamma component in CA1 ( white arrowheads ) during awake and REM sleep states . b ( right ) For Grin2aS / S the MI value dropped significantly during exploration and REM sleep although the MI reduction reached significance only during exploration ( one - way ANOVA , Kruskal - Wallis test ) . Numbers below bars of bar graphs indicate the number of animals . Error bars represent mean ± SEM .

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b (left) Grin2aS/S and Grin2a+/S mice show reduced theta-gamma phase-amplitude coupling as determined by the modulation index (MI) in the low gamma component in CA1 (white arrowheads) during awake and REM sleep states. b (right) For Grin2aS/S the MI value dropped significantly during exploration and REM sleep although the MI reduction reached significance only during exploration (one-way ANOVA, Kruskal-Wallis test). Numbers below bars of bar graphs indicate the number of animals. Error bars represent mean ± SEM.


a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS / S mice .

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a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS/S mice.


b Similarly , the exploratory behavior of Grin2aS / S mice remained high in all three successive 6 min exposure sessions to five novel objects ( ITI = 4 min ) , as exemplified by traces of the animal ' s movement on the right .

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b Similarly, the exploratory behavior of Grin2aS/S mice remained high in all three successive 6 min exposure sessions to five novel objects (ITI = 4 min), as exemplified by traces of the animal's movement on the right.


c ( left ) The time of first fall ( Cliff Avoidance Reflex , CAR ) was significantly reduced for Grin2aS / S and Grin2a + / S mice . c ( right ) In a 60 - min session Grin2aS / S mice showed a significantly increased number of falls compared to wild - type mice .

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c (left) The time of first fall (Cliff Avoidance Reflex, CAR) was significantly reduced for Grin2aS/S and Grin2a+/S mice. c (right) In a 60-min session Grin2aS/S mice showed a significantly increased number of falls compared to wild-type mice.


d ( left ) In the three - chamber sociability test Grin2aS / S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a + / S and Grin2a + / + littermates . d ( right ) Occupancy heat maps in the three - chamber sociability test show a representative example of a Grin2aS / S mouse with a reduced preference for another mouse versus an inanimate object . The social preference is visible for Grin2a + / + and Grin2a + / S mice . The occupancy is color - coded separately for each group and translates to a % given in white numbers on the key for each genotype .

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d (left) In the three-chamber sociability test Grin2aS/S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a+/S and Grin2a+/+ littermates. d (right) Occupancy heat maps in the three-chamber sociability test show a representative example of a Grin2aS/S mouse with a reduced preference for another mouse versus an inanimate object. The social preference is visible for Grin2a+/+ and Grin2a+/S mice. The occupancy is color-coded separately for each group and translates to a % given in white numbers on the key for each genotype.


f During the sample run in the Y - maze Grin2aS / S mice made significantly more total arm entries . In the test run , Grin2a + / + and Grins2a + / S showed a preference for the unexplored novel Y - maze arm , whereas Grin2aS / S mice showed a lack of novelty preference . The numbers of mice are shown in brackets or below the bars of bar graphs . Chance levels ( Ch . ) are indicated by dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 7 ) .

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f During the sample run in the Y-maze Grin2aS/S mice made significantly more total arm entries. In the test run, Grin2a+/+ and Grins2a+/S showed a preference for the unexplored novel Y-maze arm, whereas Grin2aS/S mice showed a lack of novelty preference. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 7).


c Morris watermaze . In the standard Morris watermaze task ( left graph ) the path length to reach the hidden platform decreased across training blocks for Grin2a + / + and Grin2a + / S mice but not for Grin2aS / S mice . Two probe trials were conducted after 12 and 24 training trials ( P1 and P2 , respectively ) during which the platform was removed from the pool . At both P1 and P2 ( right bar graphs ) the Grin2aS / S mice failed to search for the platform in the target quadrant . AdjL adjacent left , Target fixed location of the hidden escape platform during acquisition , AdjR adjacent right , Opp opposite of target quadrant . Dashed lines indicate chance levels .

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c Morris watermaze. In the standard Morris watermaze task (left graph) the path length to reach the hidden platform decreased across training blocks for Grin2a+/+ and Grin2a+/S mice but not for Grin2aS/S mice. Two probe trials were conducted after 12 and 24 training trials (P1 and P2, respectively) during which the platform was removed from the pool. At both P1 and P2 (right bar graphs) the Grin2aS/S mice failed to search for the platform in the target quadrant. AdjL adjacent left, Target fixed location of the hidden escape platform during acquisition, AdjR adjacent right, Opp opposite of target quadrant. Dashed lines indicate chance levels.


d Rewarded alternation . ( left ) In the T - maze rewarded alternation task ( right ) spatial working memory performance was substantially impaired in Grin2aS / S mice .

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d Rewarded alternation. (left) In the T-maze rewarded alternation task (right) spatial working memory performance was substantially impaired in Grin2aS/S mice.


e Contiguous task . In the contiguous version of the conditional T - maze task ( with floor inserts covering the entire T - maze ; white Perspex versus gray wire mesh ) , Grin2a + / + and Grin2aS / S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm .

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e Contiguous task. In the contiguous version of the conditional T-maze task (with floor inserts covering the entire T-maze; white Perspex versus gray wire mesh), Grin2a+/+ and Grin2aS/S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm.


f Discontiguous task . Separate groups of mice were trained in the discontiguous version of this conditional task , in which the floor insert cues were now limited to the start arm only . Grin2a + / + mice readily acquired the task , but Grin2aS / S mice failed to learn . The numbers of mice are shown in brackets or below the bars of bar graphs . Chance levels ( Ch . ) are indicated by dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 8 ) .

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f Discontiguous task. Separate groups of mice were trained in the discontiguous version of this conditional task, in which the floor insert cues were now limited to the start arm only. Grin2a+/+ mice readily acquired the task, but Grin2aS/S mice failed to learn. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 8).


A , HeLa cells were treated with H2O2 ( 5 mM , 1 min ) or PR - 619 ( 10 µM , 10 min ) and analysed by immunoblotting for endogenous p62 , NDP52 and OPTN in either reducing ( 2 . 5 % β - mE ) or non - reducing conditions . Data information : # , non - specific band or a post - translational modification .

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A, HeLa cells were treated with H2O2 (5 mM, 1 min) or PR-619 (10 µM, 10 min) and analysed by immunoblotting for endogenous p62, NDP52 and OPTN in either reducing (2.5% β-mE) or non-reducing conditions. Data information: #, non-specific band or a post-translational modification.


B , NDP52 and OPTN are reducible by the thioredoxin antioxidant system . HeLa cells were pre - treated with TrxR inhibitors , curcumin ( 50 μM ) or auranofin ( 5 μM ) for 30 min , then treated with H2O2 ( 500 μM ) at different time points as indicated . Immunoblotting was performed to detect NDP52 , OPTN and PRX - 3 ( dimer formation reflecting oxidation of the protein ) in non - reduced conditions and PRX - SO3 ( marker of oxidative stress and actin in reduced conditions .

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B, NDP52 and OPTN are reducible by the thioredoxin antioxidant system. HeLa cells were pre-treated with TrxR inhibitors, curcumin (50 μM) or auranofin (5 μM) for 30 min, then treated with H2O2 (500 μM) at different time points as indicated. Immunoblotting was performed to detect NDP52, OPTN and PRX-3 (dimer formation reflecting oxidation of the protein) in non-reduced conditions and PRX-SO3 (marker of oxidative stress and actin in reduced conditions.


C , D , Immunoblotting for NDP52 in either reducing or non - reducing conditions of whole cell samples ( C ) and mitochondrial fractions ( D ) from HeLa cells treated with the combination of 4 µM antimycin A1 and 10 µM oligomycin ( AO ) in the presence or absence of 400 nM bafilomycin A1 ( Baf ) for 2 h . NDP52 DLC in mitochondrial fraction was quantified ( D ) . Data are mean ± s . e . m ( D ) . P values were calculated by one - way ANOVA followed by Sidak test on four independent experiments ( D ) . * * * , p < 0 . 001 ; ns ( non - significant ) . Data information : # , non - specific band or a post - translational modification .

O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O 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, D, Immunoblotting for NDP52 in either reducing or non-reducing conditions of whole cell samples (C) and mitochondrial fractions (D) from HeLa cells treated with the combination of 4 µM antimycin A1 and 10 µM oligomycin (AO) in the presence or absence of 400 nM bafilomycin A1 (Baf) for 2 h. NDP52 DLC in mitochondrial fraction was quantified (D). Data are mean ± s.e.m (D). P values were calculated by one-way ANOVA followed by Sidak test on four independent experiments (D). ***, p<0.001; ns (non-significant). Data information: #, non-specific band or a post-translational modification.


A - D , Single ( A ) , double ( B ) , triple ( C ) , and quadruple ( D ) cysteine mutant NDP52 plasmids were generated and transfected into HeLa PentaKO cells . Cells were treated with 20 µM PR - 619 for 10 min and analysed by immunoblotting for NDP52 in either reducing or non - reducing conditions . The constructs highlighted in red were subjected to the next round of Cys - Ala screen or further analyses .

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A-D, Single (A), double (B), triple (C), and quadruple (D) cysteine mutant NDP52 plasmids were generated and transfected into HeLa PentaKO cells. Cells were treated with 20 µM PR-619 for 10 min and analysed by immunoblotting for NDP52 in either reducing or non-reducing conditions. The constructs highlighted in red were subjected to the next round of Cys-Ala screen or further analyses.


B , Recombinant proteins of NDP52 WT and C18 , 153 , 163 , 321S mutant ( Mut ) were exposed to the indicated concentrations of H2O2 for 5 min and subjected to SDS - PAGE and CBB gel staining .

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B, Recombinant proteins of NDP52 WT and C18,153,163,321S mutant (Mut) were exposed to the indicated concentrations of H2O2 for 5 min and subjected to SDS-PAGE and CBB gel staining.


C , Analytical size - exclusion chromatography ( SEC ) of the NDP52 WT and Mut proteins .

C, Analytical size-exclusion chromatography (SEC) of the NDP52 WT and Mut proteins.


D , Recombinant NDP52 WT and Mut proteins ( 5 ng ) were subjected to NATIVE - PAGE analysis . S . E , short exposure ; L . E . long exposure .

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D, Recombinant NDP52 WT and Mut proteins (5 ng) were subjected to NATIVE-PAGE analysis. S.E, short exposure; L.E. long exposure.


A , B , Fluorescence microscopy images ( A ) and quantification ( B ) of mitophagy of HeLa wild - type ( WT ) , PentaKO + empty vector , PentaKO + NDP52 WT or NDP52 C18 , 153 , 163 , 321S ( Mut ) cells , stably expressing YFP - Parkin and mt - mKeima were pre - treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times . Data information Data are mean ± s . e . m . ( B or displayed as cell popular violin plots ( B ) . P values were calculated by one - way ANOVA followed by Sidak test on three independent experiments ( B * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § , p < 0 . 05 , § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O

A, B, Fluorescence microscopy images (A) and quantification (B) of mitophagy of HeLa wild-type (WT), PentaKO + empty vector, PentaKO + NDP52 WT or NDP52 C18,153,163,321S (Mut) cells, stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without MitoQ for 22 h and treated with 4 µM / 10 µM AO for the indicated times. Data information Data are mean ± s.e.m. (B or displayed as cell popular violin plots (B). P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments (B *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm (A


C , HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132 ( MG ) and 400 nM Baf , followed by a mitochondrial fractionation and immunoblotting for NDP52 in non - reducing conditions . The mitochondrial protein UQCRC2 was used as a loading control . Data information : # , non - specific band or a post - translational modification .

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

C, HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the absence or presence of 10 μM MG132 (MG) and 400 nM Baf, followed by a mitochondrial fractionation and immunoblotting for NDP52 in non-reducing conditions. The mitochondrial protein UQCRC2 was used as a loading control. Data information: #, non-specific band or a post-translational modification.


D , HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima were treated with 1 mM DFP for 24 h ( D ) , or pre - treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h ( D Cells were then analysed by MitoSOX staining ( D ) Data information : # , Data are mean ± s . e . m . D ) P values were calculated by one - way ANOVA followed by Sidak test on three independent experiments D ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § , p < 0 . 05 , § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm D ) .

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

D, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 1 mM DFP for 24 h (D), or pre-treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h (D Cells were then analysed by MitoSOX staining (D) Data information: #, Data are mean ± s.e.m. D) P values were calculated by one-way ANOVA followed by Sidak test on three independent experiments D). *, p<0.05; **, p<0.01; ***, p<0.001; §, p<0.05, §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm D).


E , HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima were treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h E ) in the presence or absence of 400 nM Baf ( E ) . immunoblotting for NDP52 ( E ) . Data information : # , non - specific band or a post - translational modification .

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

E, HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 500 nM MitoQ for 22 h and treated with 4 µM / 10 µM AO for 2 h E) in the presence or absence of 400 nM Baf (E). immunoblotting for NDP52 (E). Data information: #, non-specific band or a post-translational modification.


A , Fluorescence microscopy images and quantification of mitophagy in HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM rotenone ( R ) , 4 µM A , 10 µM O , or the combination of RO or AO for 3h . Data information Data are mean ± s . e . m . ( A or displayed as cell popular violin plots ( A , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated ( A , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Fluorescence microscopy images and quantification of mitophagy in HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM rotenone (R), 4 µM A, 10 µM O, or the combination of RO or AO for 3h. Data information Data are mean ± s.e.m. (A or displayed as cell popular violin plots (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated (A, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm (A


B , Fluorescence microscopy images and quantification of MitoSOX staining in HeLa PentaKO + NDP52 WT cells in the same conditions as ( A ) . Data information Data are mean ± s . e . m . B , or displayed as cell popular violin plots B , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated B , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm B ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Fluorescence microscopy images and quantification of MitoSOX staining in HeLa PentaKO + NDP52 WT cells in the same conditions as (A). Data information Data are mean ± s.e.m. B, or displayed as cell popular violin plots B, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated B, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm B,


HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima ( C , were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h , followed by mitophagy measurement ( C ) Data information Data are mean ± s . e . m . C , or displayed as cell popular violin plots C , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated C , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20

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

HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima (C, were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by mitophagy measurement (C) Data information Data are mean ± s.e.m. C, or displayed as cell popular violin plots C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated C, *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm


HeLa PentaKO + NDP52 WT cells ( D ) were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h , followed by MitoSOX staining ( D ) Data information Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm ,

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

HeLa PentaKO + NDP52 WT cells (D) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by MitoSOX staining (D) Data information Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm , D


HeLa PentaKO + NDP52 WT cells stably expressing YFP - Parkin and mt - mKeima E ) were pre - treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h , followed by immunoblotting for NDP52 in non - reducing conditions ( E ) . Data information : # , non - specific band or a post - translational modification .

O O 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 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

HeLa PentaKO + NDP52 WT cells stably expressing YFP-Parkin and mt-mKeima E) were pre-treated with or without 10 µM S1QEL or 20 µM S3QEL for 30 min and treated with AO for 2 h or with RO for 3 h, followed by immunoblotting for NDP52 in non-reducing conditions (E). Data information: #, non-specific band or a post-translational modification.


F , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin and mt - mKeima were pre - treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by mitophagy measurements ( F ) Data information Data are mean ± s . e . m . F ) or displayed as cell popular violin plots F ) . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on at least three independent experiments as indicated F ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; ns ( non - significant ) . Scale bars : 20 μm F ) .

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

F, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by mitophagy measurements (F) Data information Data are mean ± s.e.m. F) or displayed as cell popular violin plots F). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on at least three independent experiments as indicated F). *, p<0.05; **, p<0.01; ***, p<0.001; ns (non-significant). Scale bars: 20 μm F).


G , HeLa PentaKO + NDP52 WT stably expressing YFP - Parkin and mt - mKeima were pre - treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by immunoblotting for NDP52 in non - reducing conditions ( G ) . Data information : # , non - specific band or a post - translational modification .

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

G, HeLa PentaKO + NDP52 WT stably expressing YFP-Parkin and mt-mKeima were pre-treated with or without 10 mM DTT for 30 min and treated with AO for 2 h followed by immunoblotting for NDP52 in non-reducing conditions (G). Data information: #, non-specific band or a post-translational modification.


HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by immunofluorescence analyses ( A ) The number of foci of the indicated proteins colocalised with NDP52 , or foci of NDP52 colocalised with Parkin , was quantified . Data information : Data are mean ± s . e . m . ( A , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments ( A * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) . Scale bars : 20 μm ( A ,

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

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunofluorescence analyses (A) The number of foci of the indicated proteins colocalised with NDP52, or foci of NDP52 colocalised with Parkin, was quantified. Data information: Data are mean ± s.e.m. (A, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments (A *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant). Scale bars: 20 μm (A,


HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by immunoblotting for LC3 and NDP52 in mitochondrial fraction ( B ) or whole cell lysate ( C ) in reducing conditions Data information : Data are mean ± s . e . m . B , C , P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments B , C * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) .

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

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by immunoblotting for LC3 and NDP52 in mitochondrial fraction (B) or whole cell lysate (C) in reducing conditions Data information: Data are mean ± s.e.m. B, C, P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments B, C *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).


HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP - Parkin and mt - mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf , followed by co - immunoprecipitation assay to analyse the interaction of NDP52 with FIP200 , ULK1 and ATG13 ( D ) . Data information : Data are mean ± s . e . m . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments D , * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ns ( non - significant ) .

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

HeLa PentaKO + NDP52 WT or NDP52 Mut cells stably expressing YFP-Parkin and mt-mKeima were treated with 4 µM / 10 µM AO for 2 h in the presence or absence of 400 nM Baf, followed by co-immunoprecipitation assay to analyse the interaction of NDP52 with FIP200, ULK1 and ATG13 (D). Data information: Data are mean ± s.e.m. P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments D, *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 ns (non-significant).


E , MEFs stably expressing YFP - Parkin , mt - mKeima and empty , human NDP52 ( hNDP52 ) WT or hNDP52 Mut were pre - treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement . Data information : Data are mean ± s . e . m . E ) or displayed as cell popular violin plots ( E ) . P values were calculated by one - way ANOVA followed by Sidak test ( for multiple groups ) or unpaired two - tailed Student ' s t - test ( for two groups ) on three independent experiments E ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; § § , p < 0 . 01 ; § § § , p < 0 . 001 ( relative to MitoQ - untreated condition ) ; ns ( non - significant ) . Scale bars : 20 μm E ) .

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

E, MEFs stably expressing YFP-Parkin, mt-mKeima and empty, human NDP52 (hNDP52) WT or hNDP52 Mut were pre-treated with or without 500 nM MitoQ for 21 h and treated with AO for 3 h followed by mitophagy measurement. Data information: Data are mean ± s.e.m. E) or displayed as cell popular violin plots (E). P values were calculated by one-way ANOVA followed by Sidak test (for multiple groups) or unpaired two-tailed Student's t-test (for two groups) on three independent experiments E). *, p<0.05; **, p<0.01; ***, p<0.001; §§, p<0.01; §§§, p<0.001 (relative to MitoQ-untreated condition); ns (non-significant). Scale bars: 20 μm E).


( B ) Serum CRAMP determination by ELISA ( n = 8 ) . Data information : Data in B were representative and were the mean ± SD from three independent experiments . P values were calculated by unpaired two - tailed t - test for comparison of two

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Serum CRAMP determination by ELISA (n = 8). Data information: Data in B were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( E ) Localization and expression of CRAMP ( red ) , E - cadherin ( green ) , F4 / 80 ( green ) and Ly6G ( green ) in duodenum by immunofluorescent staining . Representative photomicrographs of individual and merged staining were shown . Nuclei were stained with DAPI ( blue ) . Scale bar : 50 μm . Data information : Data were representative from three independent experiments .

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

(E) Localization and expression of CRAMP (red), E-cadherin (green), F4/80 (green) and Ly6G (green) in duodenum by immunofluorescent staining. Representative photomicrographs of individual and merged staining were shown. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Data information: Data were representative from three independent experiments.


( C ) Left , representative images of duodenal damage by H & E staining . Scale bar : 200 μm . Right , graph depicted the ratio of the morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( D ) Western blot and densitometry analyses of duodenal tight junction proteins ( claudin - 1 , occludin , ZO - 1 and ZO - 2 ; n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . .

(D) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( E ) Western blot and densitometry analysis of duodenal zonulin ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons . .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons..


( D ) Left , representative images of duodenal damage by H & E staining . Scale bar : 200 μm . Right , graph depicted the ratio of the morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left, representative images of duodenal damage by H&E staining. Scale bar: 200 μm. Right, graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E ) Western blot and densitometry analyses of duodenal tight junction proteins ( claudin - 1 , occludin , ZO - 1 and ZO - 2 ; n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot and densitometry analyses of duodenal tight junction proteins (claudin-1, occludin, ZO-1 and ZO-2; n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( F ) Western blot and densitometry analysis of duodenal zonulin ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

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(F) Western blot and densitometry analysis of duodenal zonulin (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( A ) Western blot and densitometry analyses of gliadin - triggered inflammatory signaling [ p - EGFR ( Tyr1068 ) , p - Akt , Akt , MyD88 , TRAF6 , p - p65 and p65 ] in epithelial cells ex vivo ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot and densitometry analyses of gliadin-triggered inflammatory signaling [p-EGFR (Tyr1068), p-Akt, Akt, MyD88, TRAF6, p-p65 and p65] in epithelial cells ex vivo (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E - G ) The percentages of ( E ) Foxp3 + Treg , ( F ) T - bet + Th1 and ( G ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative and were the median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) The percentages of (E) Foxp3+ Treg, (F) T-bet+ Th1 and (G) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( D ) Duodenal colonizing P . aeruginosa and A . muciniphila as determined by RT - qPCR using bacterial - specific species gene primers ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by unpaired two - tailed t - test for comparison of two

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Duodenal colonizing P. aeruginosa and A. muciniphila as determined by RT-qPCR using bacterial-specific species gene primers (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( E ) The production of LasB ( a protease of P . aeruginosa ) was examined in duodenum ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by unpaired two - tailed t - test for comparison of two

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The production of LasB (a protease of P. aeruginosa) was examined in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by unpaired two-tailed t-test for comparison of two groups


( H ) Duodenal colonizing PA14 and PAO1 as determined by RT - qPCR using P . aeruginosa - specific species gene primer ( n = 9 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Duodenal colonizing PA14 and PAO1 as determined by RT-qPCR using P. aeruginosa-specific species gene primer (n = 9). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons


( C ) The levels of CRAMP in duodenum ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

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(C) The levels of CRAMP in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( D ) Representative images of intestinal damage by H & E staining . Scale bar : 200 μm . The graph depicted the ratio of the morphometric assessment of villus height to crypt depth ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative images of intestinal damage by H&E staining. Scale bar: 200 μm. The graph depicted the ratio of the morphometric assessment of villus height to crypt depth (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( E ) Western blot and densitometry analyses of tight junction proteins ( claudin - 1 and occludin ) and zonulin in duodenum ( n = 6 ) . Data information : Data were representative and were the mean ± SD from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot and densitometry analyses of tight junction proteins (claudin-1 and occludin) and zonulin in duodenum (n = 6). Data information: Data were representative and were the mean ± SD from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


( I ) The percentages of three subsets of CD45 + CD11c + DCs ( n = 8 ) . ( J - L ) The percentages of ( J ) Foxp3 + Treg , ( K ) T - bet + Th1 and ( L ) RORγt + Th17 among CD3 + CD4 + population in lamina propria were shown by flow cytometry ( n = 8 ) . Data information : Data were representative and were the median ± interquartile range from three independent experiments . P values were calculated by one - way ANOVA followed by Tukey ' s post hoc test for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The percentages of three subsets of CD45+CD11c+ DCs (n = 8). (J-L) The percentages of (J) Foxp3+ Treg, (K) T-bet+ Th1 and (L) RORγt+ Th17 among CD3+CD4+ population in lamina propria were shown by flow cytometry (n = 8). Data information: Data were representative and were the median ± interquartile range from three independent experiments. P values were calculated by one-way ANOVA followed by Tukey's post hoc test for multiple comparisons.


A , B Representative size - exclusion chromatography profiles of the MICU1 - MICU2 complex and chimaera in 2 mM EGTA ( A ) and 2 mM CaCl2 ( B ) conditions .

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A, B Representative size-exclusion chromatography profiles of the MICU1-MICU2 complex and chimaera in 2 mM EGTA (A) and 2 mM CaCl2 (B) conditions.


A The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA . B The binding of fluorescently ( GFP ) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2 + .

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A The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide shown by MST in the presence of EGTA. B The binding of fluorescently (GFP) labelled MICU1 and mutants to EMRE peptide were analysed with MST in the presence of Ca2+.


C Comparison of binding of MICU1 and EMRE peptide in both conditions .

C Comparison of binding of MICU1 and EMRE peptide in both conditions.


D MICU1 - MICU2 heterodimer interacts with the EMRE peptide at a 1 : 1 ratio .

D MICU1-MICU2 heterodimer interacts with the EMRE peptide at a 1:1 ratio.


E Carbachol ( CCh ) - induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt - EMRE and scramble peptides . Solid lines , mean ; shaded regions , ± SEM , n = 3 .

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E Carbachol (CCh)-induced representative traces of mitochondrial calcium uptake in HEK cells treated with mt-EMRE and scramble peptides. Solid lines, mean; shaded regions, ± SEM, n = 3.


F Resting mitochondrial Ca2 + levels in mt - EMRE and scramble peptides treated cells , evaluated via ratiometric imaging of the mitochondrial targeted mt - riG6m . Mean ± s . d . , student t - test , * * p < 0 . 01 , n = 3 . Data information : Error bars for some points are too small to be visible .

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F Resting mitochondrial Ca2+ levels in mt-EMRE and scramble peptides treated cells, evaluated via ratiometric imaging of the mitochondrial targeted mt-riG6m. Mean ± s.d., student t-test, **p < 0.01, n = 3. Data information: Error bars for some points are too small to be visible.


G Mean mitochondrial Ca2 + peaks response to 100 μM CCh . Mean ± s . d . , student t - test , * * * p ≤ 0 . 001 , n = 3 . Data information : Error bars for some points are too small to be visible .

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G Mean mitochondrial Ca2+ peaks response to 100 μM CCh. Mean ± s.d., student t-test, ***p ≤ 0.001, n = 3. Data information: Error bars for some points are too small to be visible.


H Representative [ Ca2 + ] c clearance traces of U2OS cells treated with PBS and mt - EMRE peptide .

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H Representative [Ca2+]c clearance traces of U2OS cells treated with PBS and mt-EMRE peptide.


I Quantification of mitochondria Ca2 + uptake rate in response to 100 μM histamine . Mean ± s . d . , student t - test , n . s . , not significant , n = 4 . Data information : Error bars for some points are too small to be visible .

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I Quantification of mitochondria Ca2+ uptake rate in response to 100 μM histamine. Mean ± s.d., student t-test, n.s., not significant, n = 4. Data information: Error bars for some points are too small to be visible.


( B ) Western blot analyses of whole - cell lysates of wild - type ( CC - 124 ) , bld10 , bld10 : : B10 - N - HA , bld10 : : B10 - M - HA , and bld10 : : B10 - C - HA cells using antibodies against the C - terminal region of Bld10p ( Anti - Bld10 - C antibody , upper blot ) and the HA antigen ( Anti - HA antibody , lower blot ) . The apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags .

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(B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, and bld10::B10-C-HA cells using antibodies against the C-terminal region of Bld10p (Anti-Bld10-C antibody, upper blot) and the HA antigen (Anti-HA antibody, lower blot). The apparent molecular weights of tagged Bld10ps are slightly larger than that of native Bld10p due to their HA tags.


( C ) Percentages of flagellated cells in wild - type ( CC - 124 ) , bld10 , bld10 : : B10 - N - HA , bld10 : : B10 - M - HA , bld10 : : B10 - C - HA , bld12 , bld10bld12 , bld10bld12 : : ∆ N3 , bld10bld12 : : ∆ C2 , bld10 : : ∆ N3 , and bld10 : : ∆ C2 cells . Cells with two ( red ) , one ( green ) , and zero ( blue ) flagella were counted . n , number of cells counted .

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(C) Percentages of flagellated cells in wild-type (CC-124), bld10, bld10::B10-N-HA, bld10::B10-M-HA, bld10::B10-C-HA, bld12, bld10bld12, bld10bld12::∆N3, bld10bld12::∆C2, bld10::∆N3, and bld10::∆C2 cells. Cells with two (red), one (green), and zero (blue) flagella were counted. n, number of cells counted.


( D ) Immunoelectron micrographs of wild type centrioles treated with anti - Bld10p ( B10 - C ) after embedding in the resin and sectioning . Localization of 16 gold particles in 13 centriole images are plotted on the triangular map ( the right panel ) . Data information : Scale bar , 100 nm .

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(D) Immunoelectron micrographs of wild type centrioles treated with anti-Bld10p (B10-C) after embedding in the resin and sectioning. Localization of 16 gold particles in 13 centriole images are plotted on the triangular map (the right panel). Data information: Scale bar, 100 nm.


( C ) A schematic diagram showing the positions of the observed filamentous structures ( L1 , L2 , and L3 ) . The distance between each filamentous structure and the inner side of the A - tubule is indicated in parentheses ( mean ± SEM ) . The numbers of observed L1 , L2 , and L3 are 25 , 19 , and 13 , respectively . ( D ) Slices from the reconstructed cryo - electron tomograms of probasal bodies . Lower panels show the regions with L1 , L2 and L3 , boxed from the upper panels . Red dots denote the connection points between the spokes and filamentous structures . Data information : Scale bar , 100 nm .

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(C) A schematic diagram showing the positions of the observed filamentous structures (L1, L2, and L3). The distance between each filamentous structure and the inner side of the A-tubule is indicated in parentheses (mean ± SEM). The numbers of observed L1, L2, and L3 are 25, 19, and 13, respectively. (D) Slices from the reconstructed cryo-electron tomograms of probasal bodies. Lower panels show the regions with L1, L2 and L3, boxed from the upper panels. Red dots denote the connection points between the spokes and filamentous structures. Data information: Scale bar, 100 nm.


( A ) Western blot analyses of whole - cell lysates of wild - type ( CC - 124 ) , bld12 , bld10 , bld10bld12 , and bld10bld12 : : ∆ N3 cells using an antibody against the C - terminal one - third of Bld10p ( Anti - Bld10 - C antibody ) . The band corresponding to truncated Bld10p ( Bld10p ∆ N3 ) overlaps with a non - specific band at 75 kDa . ( B ) Western blot analyses of whole - cell lysates of wild - type ( CC - 124 ) , bld12 , bld10 , bld10bld12 , and bld10bld12 : : ∆ C2 cells using an antibody raised against the N - terminal 120 amino - acid sequence of Bld10p ( Anti - Bld10 - N antibody ) .

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(A) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆N3 cells using an antibody against the C-terminal one-third of Bld10p (Anti-Bld10-C antibody). The band corresponding to truncated Bld10p (Bld10p ∆N3) overlaps with a non-specific band at 75 kDa. (B) Western blot analyses of whole-cell lysates of wild-type (CC-124), bld12, bld10, bld10bld12, and bld10bld12::∆C2 cells using an antibody raised against the N-terminal 120 amino-acid sequence of Bld10p (Anti-Bld10-N antibody).


( C ) Cross - sectional electron micrographs of centrioles in bld12 , bld10 : : ∆ N3 , bld10bld12 : : ∆ N3 , bld10 : : ∆ C2 , and bld10bld12 : : ∆ C2 cells . Scale bar , 100 nm . ( D ) Distribution of the number of triplets in centrioles of bld12 , bld10 : : ∆ N3 , bld10bld12 : : ∆ N3 , bld10 : : ∆ C2 , and bld10bld12 : : ∆ C2 cells . Red lines denote the normal number of microtubules in the centriole ( nine ) . n , number of centrioles observed .

(C) Cross-sectional electron micrographs of centrioles in bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Scale bar, 100 nm. (D) Distribution of the number of triplets in centrioles of bld12, bld10::∆N3, bld10bld12::∆N3, bld10::∆C2, and bld10bld12::∆C2 cells. Red lines denote the normal number of microtubules in the centriole (nine). n, number of centrioles observed.


( A and B ) Cross - sectional electron micrographs of the proximal ( A ) and distal ( B ) regions of centrioles containing eight or nine triplets in bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 cells . The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A - tubules in the centriole . Scale bar , 100 nm . ( C and D ) Distances between adjacent A - tubules at the proximal ( C ) and distal ( D ) regions of centrioles containing seven , eight , nine , and ten triplets in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 cells . The distances between the contact points of A - tubules with the inscribed circle were measured . These distances were ~ 10 % shorter in cells expressing truncated Bld10ps than in wild - type and bld12 cells . * * * , P < 0 . 0001 . n , number of centrioles measured . Data information : Data were analyzed by unpaired t - test . The graphs show mean ± SEM .

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(A and B) Cross-sectional electron micrographs of the proximal (A) and distal (B) regions of centrioles containing eight or nine triplets in bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The arrangement of triplets in images of axonemes were circularized using CentrioleJ program The colored circles represent the circles inscribed to the innermost positions of the A-tubules in the centriole. Scale bar, 100 nm. (C and D) Distances between adjacent A-tubules at the proximal (C) and distal (D) regions of centrioles containing seven, eight, nine, and ten triplets in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 cells. The distances between the contact points of A-tubules with the inscribed circle were measured. These distances were ~10% shorter in cells expressing truncated Bld10ps than in wild-type and bld12 cells. ***, P<0.0001. n, number of centrioles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.