( B ) At 24h after inoculation of H1N1 / pdm or mock inoculation in triplicate , 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH . Data represent the mean and SD of a representative experiment performed three times . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(B) At 24h after inoculation of H1N1/pdm or mock inoculation in triplicate, 2D human airway organoids were harvested to detect mRNA expression levels of the indicated genes normalized with GAPDH. Data represent the mean and SD of a representative experiment performed three times. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( E ) 2D airway organoids were applied to fluorescence assay after incubation with α - SPINK6 or isotypic IgG in triplicate for 4 hours ( left ) . Data present mean and SD of triplicated samples ( right ) . Data present the mean and SD of triplicated samples . ( F ) After inoculation of H1N1 / pdm , 2D human airway organoids were incubated with an α - SPINK6 or isotype IgG in triplicate for 24 hours ( left ) . Cell - free culture media were harvested for viral load detection ( right ) and examination of HA cleavage by WB after 10 - fold concentration ( middle ) . Viral load data present the mean and SD of triplicated samples . Data information : * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(E) 2D airway organoids were applied to fluorescence assay after incubation with α-SPINK6 or isotypic IgG in triplicate for 4 hours (left). Data present mean and SD of triplicated samples (right). Data present the mean and SD of triplicated samples. (F) After inoculation of H1N1/pdm, 2D human airway organoids were incubated with an α-SPINK6 or isotype IgG in triplicate for 24 hours (left). Cell-free culture media were harvested for viral load detection (right) and examination of HA cleavage by WB after 10-fold concentration (middle). Viral load data present the mean and SD of triplicated samples. Data information: *, P<0.05; **, P<0.01; ***, P<0.001. Student's t test.


( B ) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel - Cox test .

(B) Survival rates of mice treated with wtSPINK6 and mutSPINK6 was analyzed with Mantel-Cox test.


( C ) Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6 . Data represent mean ± SD . * * , P < 0 . 01 ; * * * , P < 0 . 001 . Student ' s t test .

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(C) Viral load and viral titer in the lung homogenates of mice treated with wtSPINK6 and mutSPINK6. Data represent mean ± SD. **, P < 0.01; ***, P< 0.001. Student's t test.


( D ) Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP positive cells ( green ) . Nuclei are counterstained with DAPI ( blue ) . Representative confocal images of virus - infected cells in the indicated mice . Scale bar , 10 µm .

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(D) Mouse lung tissues are applied to immunofluorescence staining to identify the viral NP positive cells (green). Nuclei are counterstained with DAPI (blue). Representative confocal images of virus-infected cells in the indicated mice. Scale bar, 10 µm.


( A ) The association SNP rs1432689 genotypes are correlated to differential SPINK6 mRNA expression in over 1000 human lung tissues from three centers . The box denotes interquartile range ; the thick line and diamond within the box are the median and mean respectively ; whiskers are the minimum and maximum and open dots are outliers . Linear regression was used for eQTL data analysis . Data of three cohorts were applied to meta - analysis and resulted in the P - value of the association SNP .

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(A) The association SNP rs1432689 genotypes are correlated to differential SPINK6 mRNA expression in over 1000 human lung tissues from three centers. The box denotes interquartile range; the thick line and diamond within the box are the median and mean respectively; whiskers are the minimum and maximum and open dots are outliers. Linear regression was used for eQTL data analysis. Data of three cohorts were applied to meta-analysis and resulted in the P-value of the association SNP.


( B ) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database . Sample sizes for each genotype are shown in the x - axis . The y - axis shows the normalized expression of SPINK6 ( Norm . expression ) in the indicated trusses . Median and interquartile range are represented by a white line and dark box respectively . Data distribution is colored with light blue ; a wider section of the violin plot indicates the data on the section have a higher frequency . Linear regression was used for data analysis .

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(B) rs1432689 genotypes are correlated to SPINK6 expression in human lung tissues and esophagus mucosa from GTEx database. Sample sizes for each genotype are shown in the x-axis. The y-axis shows the normalized expression of SPINK6 (Norm. expression) in the indicated trusses. Median and interquartile range are represented by a white line and dark box respectively. Data distribution is colored with light blue; a wider section of the violin plot indicates the data on the section have a higher frequency. Linear regression was used for data analysis.


C38 and IB3 - 1 cells transfected with GFP - LC3 plasmid in nutrient - rich or starvation medium . ( a ) Top : confocal microscopy of GFP . N , nucleus . Scale bar , 10 μm . Bottom : percentage of cells containing more than five GFP - LC3 punctate dots per cell . Means ± s . d . ( n = 5 ) . Asterisk , P 0 . 001 versus C38 cells ; analysis of variance ( ANOVA ) .

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C38 and IB3-1 cells transfected with GFP-LC3 plasmid in nutrient-rich or starvation medium. (a) Top: confocal microscopy of GFP. N, nucleus. Scale bar, 10 μm. Bottom: percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. (n = 5). Asterisk, P 0.001 versus C38 cells; analysis of variance (ANOVA).


( b ) Left : electron microscopy of starved C38 and IB3 - 1 cells . The right - hand images are higher magnifications showing areas enriched in autophagosomes in C38 cells ( arrows ) and perinuclear mitochondria in IB3 - 1 cells . N , nucleus . Scale bar , 250 nm . Right : numbers of autophagosomes per cell counted in 20 cells per experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus C38 cells ; ANOVA .

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(b) Left: electron microscopy of starved C38 and IB3-1 cells. The right-hand images are higher magnifications showing areas enriched in autophagosomes in C38 cells (arrows) and perinuclear mitochondria in IB3-1 cells. N, nucleus. Scale bar, 250 nm. Right: numbers of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus C38 cells; ANOVA.


( c ) Fluorescence - activated cell sorting ( FACS ) analysis after staining with fluorescent dye DiOC6 in starved C38 and IB3 - 1 cells .

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(c) Fluorescence-activated cell sorting (FACS) analysis after staining with fluorescent dye DiOC6 in starved C38 and IB3-1 cells.


( d , e ) Immunoblot of LC3 in starved C38 and IB3 - 1 cells ( d ) and IB3 - 1 cells ( e ) after incubation with E64d and pepstatin A .

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(d, e) Immunoblot of LC3 in starved C38 and IB3-1 cells (d) and IB3-1 cells (e) after incubation with E64d and pepstatin A.


( f , g ) Immunoblots ( IB ) of polyubiquitin ( f ) in starved C38 and IB3 - 1 cells . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(f, g) Immunoblots (IB) of polyubiquitin (f) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( f , g ) Immunoblots ( IB ) of p62 ( g ) in starved C38 and IB3 - 1 cells . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

(f, g) Immunoblots (IB) of p62 (g) in starved C38 and IB3-1 cells. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( h ) Immunoblot of LC3 and p62 in control ( n = 10 ) and CF ( n = 10 ) human nasal mucosa . ( i ) Number of LC3 - positive dots ( per 104 mm − 2 of mucosa ) in control ( n = 10 ) and CF ( n = 10 ) human nasal mucosa . Means ± s . d . Asterisk , P 0 . 01 versus controls ; ANOVA .

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(h) Immunoblot of LC3 and p62 in control (n = 10) and CF (n = 10) human nasal mucosa. (i) Number of LC3-positive dots (per 104 mm−2 of mucosa) in control (n = 10) and CF (n = 10) human nasal mucosa. Means ± s.d. Asterisk, P 0.01 versus controls; ANOVA.


( j ) Confocal microscopy of p62 in control and CF human nasal mucosa . Representative images from ten patients with CF . Nuclei counterstained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar , 10 μm .

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(j) Confocal microscopy of p62 in control and CF human nasal mucosa. Representative images from ten patients with CF. Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 10 μm.


( k ) Number of LC3 - positive dots in lung tissues from wild - type ( WT ) and CftrF508del mice . Means ± s . d . for lung tissues from ten mice per group . Asterisk , P 0 . 001 versus WT mice ; ANOVA . ( l ) Confocal microscopy of p62 in WT and CftrF508del homozygous mice . Representative staining from at least five airway epithelial areas per mouse ( n = 10 mice per group ) . L , lumen . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm .

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(k) Number of LC3-positive dots in lung tissues from wild-type (WT) and CftrF508del mice. Means ± s.d. for lung tissues from ten mice per group. Asterisk, P 0.001 versus WT mice; ANOVA. (l) Confocal microscopy of p62 in WT and CftrF508del homozygous mice. Representative staining from at least five airway epithelial areas per mouse (n = 10 mice per group). L, lumen. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm.


( a - d ) Starved C38 and IB3 - 1 cells . ( a - c ) Confocal microscopy images of beclin 1 ( green ) and hVps34 ( red ) ( a ) , calnexin ( green ) and hVps34 ( red ) ( b ) , and beclin 1 ( green ) and HDAC6 ( red ) ( c , left ) or hVps34 ( green ) and HDAC6 ( red ) ( c , right ) . N , nucleus . Scale bar , 10 μm . Yellow indicates co - localization .

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(a-d) Starved C38 and IB3-1 cells. (a-c) Confocal microscopy images of beclin 1 (green) and hVps34 (red) (a), calnexin (green) and hVps34 (red) (b), and beclin 1 (green) and HDAC6 (red) (c, left) or hVps34 (green) and HDAC6 (red) (c, right). N, nucleus. Scale bar, 10 μm. Yellow indicates co-localization.


( d ) Immunoblot of Ambra1 , hVps15 , hVps34 , Atg14L and beclin 1 . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(d) Immunoblot of Ambra1, hVps15, hVps34, Atg14L and beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( e ) Confocal microscopy images of HA ( red ) and calnexin ( green ) ( top ) or hVps34 ( red ) and calnexin ( green ) ( bottom ) . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(e) Confocal microscopy images of HA (red) and calnexin (green) (top) or hVps34 (red) and calnexin (green) (bottom). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( f ) Left : confocal images of LC3 - positive dots . N , nucleus . Scale bar , 10 μm . Right : percentage of cells containing more than five LC3 - positive dots . At least 60 cells were counted in each experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus cells transfected with the empty vector ; ANOVA .

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(f) Left: confocal images of LC3-positive dots. N, nucleus. Scale bar, 10 μm. Right: percentage of cells containing more than five LC3-positive dots. At least 60 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA.


( g ) Left : electron microscopy of HA - beclin 1 - transfected IB3 - 1 cells shows areas enriched in autophagosomes ( arrows ) . Scale bar , 250 nm . Right : number of autophagosomes per cell ; 20 cells were counted in each experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus cells transfected with the empty vector ; ANOVA .

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(g) Left: electron microscopy of HA-beclin 1-transfected IB3-1 cells shows areas enriched in autophagosomes (arrows). Scale bar, 250 nm. Right: number of autophagosomes per cell; 20 cells were counted in each experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus cells transfected with the empty vector; ANOVA.


( h ) Immunoblot of HA and LC3 . ( i ) Immunoblot of p62 . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

(h) Immunoblot of HA and LC3. (i) Immunoblot of p62. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( j ) Immunoblot of beclin 1 in WT and CftrF508del mice . Representative of seven mice for each group .

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(j) Immunoblot of beclin 1 in WT and CftrF508del mice. Representative of seven mice for each group.


( k , l ) CftrF508del mice ( n = 3 per group ) were administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( k ) LC3 - positive dots per mm2 of lung tissues . Means ± s . d . for lung tissues from three mice per group . ( l ) Confocal microscopy micrographs of p62 . L , lumen . Representative stainings from at least five airway epithelial areas per mouse . Scale bar , 10 μm .

(k, l) CftrF508del mice (n = 3 per group) were administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (k) LC3-positive dots per mm2 of lung tissues. Means ± s.d. for lung tissues from three mice per group. (l) Confocal microscopy micrographs of p62. L, lumen. Representative stainings from at least five airway epithelial areas per mouse. Scale bar, 10 μm.


( a ) IB3 - 1 cells cultured with 250 μM cystamine or transfected with either 50 nM human TG2 siRNA or scrambled oligonucleotides . IP , immunoprecipitation ; IB , immunoblot .

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(a) IB3-1 cells cultured with 250 μM cystamine or transfected with either 50 nM human TG2 siRNA or scrambled oligonucleotides. IP, immunoprecipitation; IB, immunoblot.


( b ) IB3 - 1 cells cultured with the proteasome inhibitor MG132 ( 50 μM ) .

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(b) IB3-1 cells cultured with the proteasome inhibitor MG132 (50 μM).


( c , d ) IB3 - 1 cells transfected with either human TG2 siRNA or scrambled oligonucleotides followed by incubation with MG132 . ( c ) Cells immunostained with anti - beclin 1 ( green ) and anti - HDAC - 6 ( red ) antibodies . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(c, d) IB3-1 cells transfected with either human TG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (c) Cells immunostained with anti-beclin 1 (green) and anti-HDAC-6 (red) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( c , d ) IB3 - 1 cells transfected with either humanTG2 siRNA or scrambled oligonucleotides followed by incubation with MG132 . ( d ) FRET analysis of beclin 1 - Alexa 546 ( Al546 ) fluorescence after HDAC6 - Cy5 photobleaching . F / F0 indicates the post - bleaching / pre - bleaching fluorescence ratio of Al546 / Cy5 .

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(c, d) IB3-1 cells transfected with either humanTG2 siRNA or scrambled oligonucleotides followed by incubation with MG132. (d) FRET analysis of beclin 1-Alexa 546 (Al546) fluorescence after HDAC6-Cy5 photobleaching. F/F0 indicates the post-bleaching/pre-bleaching fluorescence ratio of Al546/Cy5.


( e ) IB3 - 1 cells were transfected with either human TG2 siRNA or scrambled oligonucleotides . Immunoblot of TG2 , Ambra - 1 , hVps15 , hVps34 , Atg14L and beclin 1 in IB3 - 1 cells grown in starvation . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(e) IB3-1 cells were transfected with either human TG2 siRNA or scrambled oligonucleotides. Immunoblot of TG2, Ambra-1, hVps15, hVps34, Atg14L and beclin 1 in IB3-1 cells grown in starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( f ) IB3 - 1 and C38 cells were transfected with either 1 μg of pLPCX - WT - TG or pLPCX - Cys277Ser - TG or the empty vector . Immunoblot of beclin 1 . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(f) IB3-1 and C38 cells were transfected with either 1 μg of pLPCX-WT-TG or pLPCX-Cys277Ser-TG or the empty vector. Immunoblot of beclin 1. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( g , h ) IB3 - 1 cells were treated with cystamine or 10 mM EUK - 134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides . ( g ) Percentage of cells containing more than five GFP - LC3 punctate dots . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus control ; ANOVA .

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(g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides. (g) Percentage of cells containing more than five GFP-LC3 punctate dots. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA.


( g , h ) IB3 - 1 cells were treated with cystamine or 10 mM EUK - 134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides . ( h ) Left : electron micrographs of IB3 - 1 cells showing areas enriched in autophagosomes ( arrows ) on PIASy gene silencing , EUK - 134 or cystamine . Scale bar , 100 nm . Right : number of autophagosomes per cell counted in 20 cells per experiment . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus control ; ANOVA .

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(g, h) IB3-1 cells were treated with cystamine or 10 mM EUK-134 or transfected with either 50 nM humanPIASy siRNA or scrambled oligonucleotides. (h) Left: electron micrographs of IB3-1 cells showing areas enriched in autophagosomes (arrows) on PIASy gene silencing, EUK-134 or cystamine. Scale bar, 100 nm. Right: number of autophagosomes per cell counted in 20 cells per experiment. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus control; ANOVA.


( i ) IB3 - 1 cells treated with cystamine . Immunoblot of p62 .

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(i) IB3-1 cells treated with cystamine. Immunoblot of p62.


( j ) IB3 - 1 cells treated with cystamine or EUK - 134 or transfected with human PIASy siRNA or scrambled oligonucleotides . Immunoblot of beclin 1 ( left ) and PIASy ( right ) . Uncropped images of blots are shown in Supplementary Information , Fig . S10 . The experiments were repeated three times . β - actin was used as loading control for all blots . Densitometric analysis of blots and quantitative measurement of co - localizations are shown in Supplementary Information , Fig . S9 .

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(j) IB3-1 cells treated with cystamine or EUK-134 or transfected with human PIASy siRNA or scrambled oligonucleotides. Immunoblot of beclin 1 (left) and PIASy (right). Uncropped images of blots are shown in Supplementary Information, Fig. S10. The experiments were repeated three times. β-actin was used as loading control for all blots. Densitometric analysis of blots and quantitative measurement of co-localizations are shown in Supplementary Information, Fig. S9.


( a , b ) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides ( a , b ) or cultured with CFTRinh - 172 ( b ) in the presence or absence of cystamine ( 250 μM ) or EUK - 134 ( 10 mM ) . ( a ) Immunoblot of beclin 1 , LC3 and p62 in C38 cells on starvation . Uncropped images of blots are shown in Supplementary Information , Fig . S10 . ( b ) Percentage of cells containing more than five GFP - LC3 punctate dots per cell . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 versus scrambled oligonucleotides ; circle , P 0 . 01 versus CFTR siRNA or CFTRinh - 172 ; ANOVA .

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(a, b) C38 cells were transfected with either 50 nM human CFTR siRNA or scrambled oligonucleotides (a, b) or cultured with CFTRinh-172 (b) in the presence or absence of cystamine (250 μM) or EUK-134 (10 mM). (a) Immunoblot of beclin 1, LC3 and p62 in C38 cells on starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10. (b) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001 versus scrambled oligonucleotides; circle, P 0.01 versus CFTR siRNA or CFTRinh-172; ANOVA.


( c - g ) A549 cells were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in the presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( c ) Immunoblot of TG2 and PIASy .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine. (c) Immunoblot of TG2 and PIASy.


( c - g ) A549 cells were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in the presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( d ) FRET analysis of TG2 - Alexa 546 fluorescence after SUMO - 1 Cy5 photobleaching .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(d) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching.


( c - g ) A549 cells were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in the presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( e ) Immunoblot of beclin 1 and LC3 in A549 cells after starvation . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(e) Immunoblot of beclin 1 and LC3 in A549 cells after starvation. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( c - g ) A549 cells were transfected with 1 μg of pGFP - - CFTRF508del or pGFP ( empty vector ) , in the presence or absence of 10 mM EUK - 134 or 250 μM cystamine . ( f ) Percentage of cells containing more than five GFP - LC3 punctate dots per cell . Means ± s . d . for three independent experiments . Asterisk , P 0 . 001 ; two asterisks , P 0 . 01 ; ANOVA . ( g ) Immunostaining with anti - p62 ( red ) and anti - GFP ( green ) antibodies in starved A549 cells . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm . Each experiment was repeated three times .

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(c-g) A549 cells were transfected with 1 μg of pGFP--CFTRF508del or pGFP (empty vector), in the presence or absence of 10 mM EUK-134 or 250 μM cystamine.(f) Percentage of cells containing more than five GFP-LC3 punctate dots per cell. Means ± s.d. for three independent experiments. Asterisk, P 0.001; two asterisks, P 0.01; ANOVA. (g) Immunostaining with anti-p62 (red) and anti-GFP (green) antibodies in starved A549 cells. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm. Each experiment was repeated three times.


( a , b ) IB3 - 1 cells were transfected with either HA - tagged human beclin 1 , empty vector , p62 siRNA or scrambled oligonucleotides followed by rosiglitazone ( 10 μM ) . ( a ) Cells were immunostained with anti - HDAC6 ( red ) and anti - PPAR - γ ( green ) antibodies . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM). (a) Cells were immunostained with anti-HDAC6 (red) and anti-PPAR-γ (green) antibodies. Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( a , b ) IB3 - 1 cells were transfected with either HA - tagged human beclin 1 , empty vector , p62 siRNA or scrambled oligonucleotides followed by rosiglitazone ( 10 μM ) . ( b ) FRET analysis of increase in PPAR - γ - Alexa 546 fluorescence after N - CoR Cy5 photobleaching .

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(a, b) IB3-1 cells were transfected with either HA-tagged human beclin 1, empty vector, p62 siRNA or scrambled oligonucleotides followed by rosiglitazone (10 μM).(b) FRET analysis of increase in PPAR-γ-Alexa 546 fluorescence after N-CoR Cy5 photobleaching.


( c ) IB3 - 1 cells were transfected with either HA - tagged human beclin 1 or the empty vector under nutrient starvation . Cells were immunostained with anti - LC - 3 ( red ) and anti - CFTR ( green ) . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(c) IB3-1 cells were transfected with either HA-tagged human beclin 1 or the empty vector under nutrient starvation. Cells were immunostained with anti-LC-3 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( d ) IB3 - 1 cells were cultured under nutrient starvation with cystamine ( 250 μM ) in the presence or absence of the PI ( 3 ) K inhibitor 3 - MA . Immunostaining was with anti - LAMP - 1 ( red ) and anti - CFTR ( green ) . Yellow indicates co - localization . N , nucleus . Scale bar , 10 μm .

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(d) IB3-1 cells were cultured under nutrient starvation with cystamine (250 μM) in the presence or absence of the PI(3)K inhibitor 3-MA. Immunostaining was with anti-LAMP-1 (red) and anti-CFTR (green). Yellow indicates co-localization. N, nucleus. Scale bar, 10 μm.


( e ) IB3 - 1 cells were transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of 250 μM cystamine under nutrient starvation . Cells were immunostained with anti - p62 ( red ) and anti - CFTR ( green ) antibodies . Yellow indicates co - localization . N , cell nucleus . Scale bar , 10 μm .

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(e) IB3-1 cells were transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of 250 μM cystamine under nutrient starvation. Cells were immunostained with anti-p62 (red) and anti-CFTR (green) antibodies. Yellow indicates co-localization. N, cell nucleus. Scale bar, 10 μm.


( f , g ) IB3 - 1 cells were transfected with 1 μg of pGFP - CFTRF508del or the empty vector , in the presence or absence of 250 μM cystamine . ( f ) Cells were immunostained with anti - GM - 130 ( red ) and anti - GFP ( green ) antibodies . Yellow indicates co - localization ( arrows ) . Scale bar , 10 μm . ( g ) FRET analysis of GFP fluorescence after HDAC6 Cy3 photobleaching . Each experiment was repeated three times . Quantitative measurement of co - localizations is shown in Supplementary Information , Fig . S9 .

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(f, g) IB3-1 cells were transfected with 1 μg of pGFP-CFTRF508del or the empty vector, in the presence or absence of 250 μM cystamine. (f) Cells were immunostained with anti-GM-130 (red) and anti-GFP (green) antibodies. Yellow indicates co-localization (arrows). Scale bar, 10 μm. (g) FRET analysis of GFP fluorescence after HDAC6 Cy3 photobleaching. Each experiment was repeated three times. Quantitative measurement of co-localizations is shown in Supplementary Information, Fig. S9.


( a - d ) IB3 - 1 cells transfected for 24 h at 37 ° C with 1 μg of pGFP - CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides . ( a ) Left : CFTR revealed with anti - GFP primary antibody in IB3 - 1 cell lysate . Top arrow , complex - glycosylated form ( apparent Mr of band C about 170K , revealed as 210K with GFP tag ) ; bottom arrow , core - glycosylated form ( apparent Mr of band B about 150K , revealed as 190K with GFP tag ) . Uncropped images of blots are shown in Supplementary Information , Fig . S10 . Right : quantification of bands B ( black ) and C ( white ) normalized to β - actin levels . Values are means ± s . d . for three independent experiments .

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(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM human p62 siRNA or scrambled oligonucleotides. (a) Left: CFTR revealed with anti-GFP primary antibody in IB3-1 cell lysate. Top arrow, complex-glycosylated form (apparent Mr of band C about 170K, revealed as 210K with GFP tag); bottom arrow, core-glycosylated form (apparent Mr of band B about 150K, revealed as 190K with GFP tag). Uncropped images of blots are shown in Supplementary Information, Fig. S10. Right: quantification of bands B (black) and C (white) normalized to β-actin levels. Values are means ± s.d. for three independent experiments.


( a - d ) IB3 - 1 cells transfected for 24 h at 37 ° C with 1 μg of pGFP - CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides . ( b ) Cells immunostained with an anti - GFP antibody under non - permeabilizing conditions . Serial confocal sections were collected from top to bottom of cell . ( c ) Side view ( X - Z ) of a z - stack of confocal images taken in b . ( c ) Side view ( X - Z ) of a z - stack of confocal images taken in b .

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(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(b) Cells immunostained with an anti-GFP antibody under non-permeabilizing conditions. Serial confocal sections were collected from top to bottom of cell. (c) Side view (X-Z) of a z-stack of confocal images taken in b. (c) Side view (X-Z) of a z-stack of confocal images taken in b.


( a - d ) IB3 - 1 cells transfected for 24 h at 37 ° C with 1 μg of pGFP - CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides . ( d ) Surface biotinylation assays was performed with membrane - impermeable sulpho - NHS - LC - biotin . The cells were fractioned to obtain the plasma membrane fraction , and biotinylated proteins were precipitated with streptavidin beads . Immunoblot of GFP to reveal the C form . E - cadherin and β - actin were used as positive and negative controls , respectively .

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(a-d) IB3-1 cells transfected for 24 h at 37 °C with 1 μg of pGFP-CFTRF508del or empty vector in the presence or absence of 250 μM cystamine or with either 50 nM humanp62 siRNA or scrambled oligonucleotides.(d) Surface biotinylation assays was performed with membrane-impermeable sulpho-NHS-LC-biotin. The cells were fractioned to obtain the plasma membrane fraction, and biotinylated proteins were precipitated with streptavidin beads. Immunoblot of GFP to reveal the C form. E-cadherin and β-actin were used as positive and negative controls, respectively.


( e ) IB3 - 1 cells transfected with either HA - tagged human beclin 1 or empty vector . Immunoblot of PIASy , TG2 , phospho - p42 / 44 and PPAR - γ proteins .

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(e) IB3-1 cells transfected with either HA-tagged human beclin 1 or empty vector. Immunoblot of PIASy, TG2, phospho-p42/44 and PPAR-γ proteins.


( f ) IB3 - 1 cells transfected with either 50 nM human p62 siRNA or scrambled oligonucleotides . Immunoblot of p62 and phospho - p42 / 44 . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(f) IB3-1 cells transfected with either 50 nM human p62 siRNA or scrambled oligonucleotides. Immunoblot of p62 and phospho-p42/44. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( g ) IB3 - 1 cells transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of cystamine . Immunoblot of phospho - p42 / 44 .

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(g) IB3-1 cells transfected with either 50 nM human beclin 1 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. Immunoblot of phospho-p42/44.


( h ) IB3 - 1 cells transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine . TNF - α secretion . Each bar represents the mean ± s . d . for n = 3 experiments . Asterisk , P 0 . 01 ; ANOVA .

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(h) IB3-1 cells transfected with either 50 nM humanbeclin 1 siRNA or 50 nM humanATG5 siRNA or scrambled oligonucleotides in the presence or absence of cystamine. TNF-α secretion. Each bar represents the mean ± s.d. for n = 3 experiments. Asterisk, P 0.01; ANOVA.


CF human nasal mucosae cultured with cystamine or NAC ( patients 4 - 10 in Supplementary Information , Table S1 ) ( a - c ) ( a ) Immunoblot of beclin 1 .

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CF human nasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) (a) Immunoblot of beclin 1.


CF humannasal mucosae cultured with cystamine or NAC ( patients 4 - 10 in Supplementary Information , Table S1 ) ( a - c ) in the presence or absence of 3 - MA ( patients 6 - 10 in Supplementary Information , Table S1 ) ( b , c ) . ( b ) Fluorescence microscopy quantification of LC3 - positive dots . Results represent means ± s . d . Asterisk , P 0 . 05 between groups of treatment ; ANOVA .

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CF humannasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) in the presence or absence of 3-MA (patients 6-10 in Supplementary Information, Table S1) (b, c). (b) Fluorescence microscopy quantification of LC3-positive dots. Results represent means ± s.d. Asterisk, P 0.05 between groups of treatment; ANOVA.


CF humannasal mucosae cultured with cystamine or NAC ( patients 4 - 10 in Supplementary Information , Table S1 ) ( a - c ) in the presence or absence of 3 - MA ( patients 6 - 10 in Supplementary Information , Table S1 ) ( b , c ) . ( c ) Confocal microscopy micrographs of p62 . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm . Representative of five patients per group .

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CF humannasal mucosae cultured with cystamine or NAC (patients 4-10 in Supplementary Information, Table S1) (a-c) in the presence or absence of 3-MA (patients 6-10 in Supplementary Information, Table S1) (b, c). (c) Confocal microscopy micrographs of p62. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative of five patients per group.


( d - i ) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( d ) Immunoblot of beclin 1 .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (d) Immunoblot of beclin 1.


( d - i ) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( e ) Quantification of LC3 dots ( per mm2 of lung tissue ) . Means ± s . d . Asterisk , P 0 . 01 ; ANOVA .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (e) Quantification of LC3 dots (per mm2 of lung tissue). Means ± s.d. Asterisk, P 0.01; ANOVA.


( d - i ) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( f ) Immunoblot of TG2 . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (f) Immunoblot of TG2. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( d - i ) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( g ) Confocal microscopy of SUMO - 1 in lung tissues . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (g) Confocal microscopy of SUMO-1 in lung tissues. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm.


( d - i ) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( h ) Number of CD68 + macrophages counted in 15 - 20 different randomly taken sections . Means ± s . d . per mm2 of lung tissue from seven mice in each group . Asterisk , P 0 . 01 ; ANOVA .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (h) Number of CD68+ macrophages counted in 15-20 different randomly taken sections. Means ± s.d. per mm2 of lung tissue from seven mice in each group. Asterisk, P 0.01; ANOVA.


( d - i ) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3 - MA ( e - i ) ( n = 7 per group ) . ( i ) MPO activity in lung homogenates ( expressed as 10 − 3 × optical density ( OD ) s − 1 ) . Means ± s . d . for lung tissues from seven mice in each group . Asterisk , P 0 . 05 ; ANOVA .

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(d-i) CftrF508del mice treated with cystamine or PBS in the presence or absence of 3-MA (e-i) (n = 7 per group). (i) MPO activity in lung homogenates (expressed as 10−3 × optical density (OD) s−1). Means ± s.d. for lung tissues from seven mice in each group. Asterisk, P 0.05; ANOVA.


( j - m ) CftrF508del mice ( n = 3 per group ) administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( j ) TG2 Immunoblot in lung tissues . Uncropped images of blots are shown in Supplementary Information , Fig . S10 .

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(j-m) CftrF508del mice (n = 3 per group) administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (j) TG2 Immunoblot in lung tissues. Uncropped images of blots are shown in Supplementary Information, Fig. S10.


( j - m ) CftrF508del mice ( n = 3 per group ) administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( k ) Confocal microscopy of TG2 activity . Representative stainings from at least five airway epithelial areas per mouse from three mice per group . L , lumen . Scale bar , 10 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

(j-m) CftrF508del mice (n = 3 per group) administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (k) Confocal microscopy of TG2 activity. Representative stainings from at least five airway epithelial areas per mouse from three mice per group. L, lumen. Scale bar, 10 μm.


( j - m ) CftrF508del mice ( n = 3 per group ) administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( l ) Number of CD68 + macrophages counted in 15 - 20 different randomly taken sections . Means ± s . d . per mm2 of lung tissue . Asterisk , P 0 . 01 ; ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-m) CftrF508del mice (n = 3 per group) administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (l) Number of CD68+ macrophages counted in 15-20 different randomly taken sections. Means ± s.d. per mm2 of lung tissue. Asterisk, P 0.01; ANOVA.


( j - m ) CftrF508del mice ( n = 3 per group ) administered intranasally with a lentiviral vector encoding beclin 1 ( LV - beclin 1 ) or GFP ( LV - GFP ) . ( m ) MPO activity in lung homogenates ( expressed as 10 − 3 × OD s − 1 ) . Means ± s . d . for lung tissues . Asterisk , P 0 . 05 ; ANOVA . Anti - β - Tubulin was used as loading control for immunoblot analysis . Densitometric analysis of blots is shown in Supplementary Information , Fig . S9 .

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(j-m) CftrF508del mice (n = 3 per group) administered intranasally with a lentiviral vector encoding beclin 1 (LV-beclin 1) or GFP (LV-GFP). (m) MPO activity in lung homogenates (expressed as 10−3 × OD s−1). Means ± s.d. for lung tissues. Asterisk, P 0.05; ANOVA. Anti-β-Tubulin was used as loading control for immunoblot analysis. Densitometric analysis of blots is shown in Supplementary Information, Fig. S9.


CftrF508del mice ( a - d ) ( a ) Confocal microscopy of LC3 ( top ) and p62 ( bottom ) in lung tissues from CftrF508del mice . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm . Representative images of ten PBS - treated and ten NAC - treated mice . ( b ) Quantification of LC3 dots ( per mm2 of tissue ) in lung tissues from CftrF508del mice . Means ± s . d . Asterisk , P 0 . 001 versus PBS - treated mice ; ANOVA .

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CftrF508del mice (a-d)(a) Confocal microscopy of LC3 (top) and p62 (bottom) in lung tissues from CftrF508del mice. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. Representative images of ten PBS-treated and ten NAC-treated mice. (b) Quantification of LC3 dots (per mm2 of tissue) in lung tissues from CftrF508del mice. Means ± s.d. Asterisk, P 0.001 versus PBS-treated mice; ANOVA.


CftrF508del mice ( a - d ) ( c ) Number of macrophages per mm2 of lung tissue from CftrF508del mice ( CD68 - positive cells in 15 - 20 different randomly taken sections for each mouse lung for each condition ) ; means ± s . d . for three separate experiments . Asterisk , P 0 . 05 versus PBS - treated mice ; ANOVA .

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CftrF508del mice (a-d)(c) Number of macrophages per mm2 of lung tissue from CftrF508del mice (CD68-positive cells in 15-20 different randomly taken sections for each mouse lung for each condition); means ± s.d. for three separate experiments. Asterisk, P 0.05 versus PBS-treated mice; ANOVA.


CftrF508del mice ( a - d ) ( d ) MPO activity in lung homogenates from CftrF508del mice ( expressed as 10 − 3 × OD s − 1 ) . Means ± s . d . Asterisk , P 0 . 05 versus PBS - treated mice ; ANOVA .

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CftrF508del mice (a-d)(d) MPO activity in lung homogenates from CftrF508del mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA.


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( e ) Confocal microscopy of LC3 - positive dots in lung tissues from PBS - treated or NAC - treated mice - Tg LC3 . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm . ( f ) Number of LC3 - positive dots in Scnn1b tissues from mice - Tg PBS . Means ± s . d . Asterisk , P 0 . 01 versus mice - treated mice ; ANOVA .

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Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (e) Confocal microscopy of LC3-positive dots in lung tissues from PBS-treated or NAC-treated mice-Tg LC3. Nuclei counterstained with DAPI (blue). Scale bar, 10 μm. (f) Number of LC3-positive dots in Scnn1b tissues from mice-Tg PBS. Means ± s.d. Asterisk, P 0.01 versus mice-treated mice; ANOVA.


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( g ) FRET analysis of TG2 - Alexa 546 fluorescence after SUMO - 1 Cy5 photobleaching in lung tissues from PBS - treated WT mice and PBS - treated or NAC - treated Scnn1b - Tg mice .

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Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (g) FRET analysis of TG2-Alexa 546 fluorescence after SUMO-1 Cy5 photobleaching in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice.


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( h ) Immunoblot of TG2 and phospho - p42 / 44 in lung tissues from PBS - treated WT mice and PBS - treated or NAC - treated Scnn1b - Tg mice . αβ - Tubulin was used as loading control .

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Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (h) Immunoblot of TG2 and phospho-p42/44 in lung tissues from PBS-treated WT mice and PBS-treated or NAC-treated Scnn1b-Tg mice. αβ-Tubulin was used as loading control.


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( i ) Lung histology of PBS - treated and NAC - treated Scnn1b - Tg mice . Haematoxylin staining . Scale bar , 150 μm . Black arrows indicate decrease in lung infiltration in NAC - treated in comparison with PBS - treated mice ( arrowheads ) .

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Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (i) Lung histology of PBS-treated and NAC-treated Scnn1b-Tg mice. Haematoxylin staining. Scale bar, 150 μm. Black arrows indicate decrease in lung infiltration in NAC-treated in comparison with PBS-treated mice (arrowheads).


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( j ) Left : confocal microscopy of COX - 2 staining ( left ) in PBS - treated and NAC - treated Scnn1b - Tg mice . Nuclei counterstained with DAPI ( blue ) . Scale bar , 150 μm . Right ; number of COX - 2 - positive cells per mm2 of lung tissue in NAC - treated and Scnn1b - treated mice - Tg PBS . Means ± s . d . Asterisk , P 0 . 05 versus mice - treated mice ; ANOVA . 1 versus PBS - treated mice ; ANOVA .

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 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 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

Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (j) Left: confocal microscopy of COX-2 staining (left) in PBS-treated and NAC-treated Scnn1b-Tg mice. Nuclei counterstained with DAPI (blue). Scale bar, 150 μm. Right; number of COX-2-positive cells per mm2 of lung tissue in NAC-treated and Scnn1b-treated mice-Tg PBS. Means ± s.d. Asterisk, P 0.05 versus mice-treated mice; ANOVA.1 versus PBS-treated mice; ANOVA.


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( k ) MPO activity in lung homogenates from PBS - treated and NAC - treated Scnn1b - Tg mice ( expressed as 10 − 3 × OD s − 1 ) . Means ± s . d . Asterisk , P 0 . 05 versus PBS - treated mice ; ANOVA . Densitometric analysis of the blot is shown in Supplementary Information , Fig . S9 .

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Scnn1b-Tg mice (e-k) treated with NAC or PBS (n = 10 per group). (k) MPO activity in lung homogenates from PBS-treated and NAC-treated Scnn1b-Tg mice (expressed as 10−3 × OD s−1). Means ± s.d. Asterisk, P 0.05 versus PBS-treated mice; ANOVA. Densitometric analysis of the blot is shown in Supplementary Information, Fig. S9.


IFM images of KIF13A - YFP transfected control and Rab - knockdown HeLa cells . TN : average tubule number ( mean ± SEM , n = 3 ) . B , C Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 1A ( mean ± SEM ) . n = 3 . nc : total number of cells . nt : total number of tubules . arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10 μm .

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IFM images of KIF13A-YFP transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean±SEM, n=3). B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean± SEM). n=3. nc: total number of cells. nt: total number of tubules.arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm.


IFM images of KIF13A - YFP and mCherry - Rab7A / 11A / 22A transfected HeLa cells arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A transfected HeLa cells arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Live cell imaging of GFP / mCherry - Rab22A with respect to mCherry - Rab11A or GFP - Rab7A in HeLa cells . Magnified view of insets ( at 0 , 20 , 40 seconds ) are shown separately ) , arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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Live cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 seconds) are shown separately ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Pull - down of different His - KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins . The bead bound His - KIF13A in each pull - down was shown as coomassie gel . * , non - specific bands .

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Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands.


IFM images of KIF13A - YFP and mCherry - Rab22AWT / Q64L / S19N cotransfected HeLa cells . Arrowheads and arrows point to the KIF13A - positive tubular REs and E / SEs respectively . The colocalization coefficient ( r , in mean ± SEM ) between two markers was indicated separately . Scale bars : 10 μm Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 2A . n = 3 . nc : total number of cells . nt : total number of

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IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs respectively. The colocalization coefficient (r, in mean±SEM) between two markers was indicated separately. Scale bars: 10 μm Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A. n=3. nc: total number of cells. nt: total number of tubules


Subcellular fractionation of HeLa cells to probe the localization of Rab22A ( red box ) with respect to other organelle specific proteins . * , non - specific bands .

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Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle specific proteins. *, non-specific bands.


IFM images of KIF13A - YFP transfected control and Rab22A - , BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa cells . Cells were stained for AP - 1 ( γ ) or internalized with Tf - Alexa fluor 594 arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

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IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594 arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


Graphs represent the measurement of KIF13A - positive TN ( B ) and TL ( C ) in HeLa cells of Fig 3A . n = 3 . nc : total number of cells . nt : total number of

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Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A. n=3. nc: total number of cells. nt: total number of tubules


Immunoblotting analysis of proteins in control and Rab22A - , BLOC - 1 - , BLOC - 2 - depleted HeLa cells . * , non - specific bands . Protein band intensities were quantified and indicated on the gels .

Immunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels.


IFM images of mCherry - Rab22A and KIF13A - YFP cotransfected control and BLOC - 1Mu - , BLOC - 2HPS6 - knockdown HeLa cells ) , arrowheads and arrows point to the KIF13A - / Rab22A - positive tubular REs and E / SEs respectively . Scale bars : 10

IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells ), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm


BF and IFM images of control and Rab22A - depleted melanocytes . The colocalization coefficient ( r , in mean ± SEM ) between the two markers indicated separately . Nuclei are stained with Hoechst33258 ) , arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1 / GFP - Rab22A . Scale bars : 10

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BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258 ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm


Graph represents the quantified melanin content in control sh , Rab22A sh and BLOC - 1 - melanocytes . n = 3 . The fold - change in melanin content ( mean ± SEM ) indicated

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Graph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separately


Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ) , protein band intensities were quantified and indicated on the

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Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes ), protein band intensities were quantified and indicated on the gels


EM of control and Rab22A - knockdown MNT - 1 melanocytes . Arrowheads indicate PMEL fibrils . Scale bars : 1 μm . I , II , III , IV : stages of melanosomes . M : mitochondria . Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions . Note : the presence of hybrid stage I / II melanosomes in Rab22A - knockdown cells . Insets ( b ) - ( d ) and ( g ) - ( h ) are from different cells of respective condition . Scale bars : 200 nm .

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EM of control and Rab22A-knockdown MNT-1 melanocytes. Arrowheads indicate PMEL fibrils. Scale bars: 1 μm. I, II, III, IV: stages of melanosomes. M: mitochondria. Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions. Note: the presence of hybrid stage I/II melanosomes in Rab22A-knockdown cells. Insets (b)-(d) and (g)-(h) are from different cells of respective condition. Scale bars: 200 nm.


BF and IFM images of GFP - Rab22A transfected BLOC - 1 - ( Mu ) melanocytes ) , arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1 / GFP - Rab22A . Scale bars : 10

BF and IFM images of GFP-Rab22A transfected BLOC-1-(Mu) melanocytes ), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm


Immunoblotting analysis of proteins in BLOC - 1 - and BLOC - 1R melanocytes protein band intensities were quantified and indicated on the

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Immunoblotting analysis of proteins in BLOC-1- and BLOC-1R melanocytes protein band intensities were quantified and indicated on the gels


Subcellular membrane fractionation of control and Rab22A - , BLOC - 1 - , BLOC - 2 - knockdown HeLa cells and probed the fractions for pallidin , dysbindin and muted . Red colored box emphasizes BLOC - 1 membrane association in the respective cell types .

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Subcellular membrane fractionation of control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red colored box emphasizes BLOC-1 membrane association in the respective cell types.


Cytosol - membrane fractionation of homogenates from HeLa cells ( B as indicated . Protein band intensities were quantified and indicated the percentage membrane association on the

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Cytosol-membrane fractionation of homogenates from HeLa cells (B as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels


Cytosol - membrane fractionation of homogenates fro melanocyte as indicated . Protein band intensities were quantified and indicated the percentage membrane association on the

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

Cytosol-membrane fractionation of homogenates fro melanocyte as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels


Pull - down of His - Rab22AWT using HeL lysate the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

Pull-down of His-Rab22AWT using HeL lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Pull - down of His - Rab22AWT usin melanocyte ( E ) lysate . In E , the beads were preloaded with GTPγS or GDP the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

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Pull-down of His-Rab22AWT usin melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


Pull - down of different His - KIF13A domains using HeLa cell lysate the bead bound His - Rab22A / His - KIF13A domains were shown as coomassie stained gel

Pull-down of different His-KIF13A domains using HeLa cell lysate the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separatel


C - E , boxplots showing the serum level of phenylacetylglutamine , mandelic acid and homovanillic acid in different CHF subgroups in the REM - HF cohort . NGT : n = 23 ; NGT + CHF : n = 48 ; Prediabetes + CHF : n = 83 ; T2D + CHF : n = 56 ; Prediabetes + CHF + CKD : n = 34 ; T2D + CHF + CKD : n = 16 ( biological replicates ) . Data information : the central band in each box represents the median , the top and bottom of the box the 25th and 75th percentiles , and the whiskers 1 . 5 times the interquartile range . Wilcoxon rank - sum test was used for all group comparisons .

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C-E, boxplots showing the serum level of phenylacetylglutamine, mandelic acid and homovanillic acid in different CHF subgroups in the REM-HF cohort. NGT: n = 23; NGT+CHF: n = 48; Prediabetes+CHF: n = 83; T2D+CHF: n = 56; Prediabetes+CHF+CKD: n = 34; T2D+CHF+CKD: n = 16 (biological replicates). Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.


F , the boxplot showing the serum level of ImP in the Swedish prediabetes versus the Chinese REM - HF cohorts . For Swedish prediabetes cohort , NGT : n = 125 ; prediabetes : n = 262 ; T2D : n = 17 ( biological replicates ) . For Chinese REM - HF cohort : NGT : n = 23 ; NGT + CHF : n = 48 ; Prediabetes + CHF : n = 83 ; T2D + CHF : n = 56 ; Prediabetes + CHF + CKD : n = 34 ; T2D + CHF + CKD : n = 16 . Data information : the central band in each box represents the median , the top and bottom of the box the 25th and 75th percentiles , and the whiskers 1 . 5 times the interquartile range . Wilcoxon rank - sum test was used for all group comparisons .

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F, the boxplot showing the serum level of ImP in the Swedish prediabetes versus the Chinese REM-HF cohorts. For Swedish prediabetes cohort, NGT: n = 125; prediabetes: n = 262; T2D: n = 17 (biological replicates). For Chinese REM-HF cohort: NGT: n = 23; NGT+CHF: n = 48; Prediabetes+CHF: n = 83; T2D+CHF: n = 56; Prediabetes+CHF+CKD: n = 34; T2D+CHF+CKD: n = 16. Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.


G , differences in ImP when stratified by left ventricular ejection fraction ( LVEF ; two - tailed Wilcoxon rank - sum tests ) . NGT : n = 23 ; HFpEF : LVEF ≥ 50 , n = 66 ; HFmEF : 40 < LVEF < 50 , n = 68 ; HFrEF : LVEF ≤ 40 , n = 103 participants . Data information : the central band in each box represents the median , the top and bottom of the box the 25th and 75th percentiles , and the whiskers 1 . 5 times the interquartile range . Wilcoxon rank - sum test was used for all group comparisons .

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G, differences in ImP when stratified by left ventricular ejection fraction (LVEF; two-tailed Wilcoxon rank-sum tests). NGT: n = 23; HFpEF: LVEF ≥50, n = 66; HFmEF: 40<LVEF<50, n = 68; HFrEF: LVEF ≤40, n = 103 participants. Data information: the central band in each box represents the median, the top and bottom of the box the 25th and 75th percentiles, and the whiskers 1.5 times the interquartile range. Wilcoxon rank-sum test was used for all group comparisons.


B , representative JC - 1 staining images showing red fluorescence of JC - 1 aggregates and green signal of monomers . Scale bars : 20 μm .

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B, representative JC-1 staining images showing red fluorescence of JC-1 aggregates and green signal of monomers. Scale bars: 20 μm.


A , the Kaplan - Meier survival curve based on the Framingham and GWTG - HF risk scores . 240 participants with clinical variables for Framingham and GWTG - HF risk scores calculation were included in survival analysis ( biological replicates ) . P values were calculated based on the log - rank test .

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A, the Kaplan-Meier survival curve based on the Framingham and GWTG-HF risk scores. 240 participants with clinical variables for Framingham and GWTG-HF risk scores calculation were included in survival analysis (biological replicates). P values were calculated based on the log-rank test.


B , the Kaplan - Meier survival curve based on each microbially associated metabolite ( grey colors except for imidazole propionic acid ) and a combined metabolite risk score . 244 participants who completed the follow - up study were included in survival analysis ( biological replicates ) . P values were calculated based on the log - rank test .

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B, the Kaplan-Meier survival curve based on each microbially associated metabolite (grey colors except for imidazole propionic acid) and a combined metabolite risk score. 244 participants who completed the follow-up study were included in survival analysis (biological replicates). P values were calculated based on the log-rank test.


C , the time - dependent receiver operating characteristic ( ROC ) curves for the performance of GWTG - HF scores , metabolite - based risk score and the combination of both scores in CHF prognosis . The AUC values were determined by bootstrap resampling with 1 , 000 iterations .

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C, the time-dependent receiver operating characteristic (ROC) curves for the performance of GWTG-HF scores, metabolite-based risk score and the combination of both scores in CHF prognosis. The AUC values were determined by bootstrap resampling with 1,000 iterations.


Representative immunofluorescence of primary cilium in WT or Mecp2 null MEF cells . Cells were starved for 48 h before staining with anti - γ - tubulin ( green ) and anti - acetylated α - tubulin ( red ) antibodies . Merge of all channels and DAPI staining ( blue ) is depicted for both genotypes . Scale bar 20 µm , and 10 µm in the enlarged image . Arrows indicate not ciliated cells .

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

Representative immunofluorescence of primary cilium in WT or Mecp2 null MEF cells. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Merge of all channels and DAPI staining (blue) is depicted for both genotypes. Scale bar 20 µm, and 10 µm in the enlarged image. Arrows indicate not ciliated cells.


Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells ( n = 134 WT and n = 140 Mecp2 null cells ) ( B ) and the cilium length ( n = 104 WT and n = 43 Mecp2 null cells ) ( C ) . The graphs show the average of three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by Mann - Whitney test ; * * * p < 0 . 001 , by Student ' s t - test ) .

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Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=134 WT and n=140 Mecp2 null cells) (B) and the cilium length (n=104 WT and n=43 Mecp2 null cells) (C). The graphs show the average of three independent experiments (mean ± S.E., **p<0.01, by Mann-Whitney test; ***p<0.001, by Student's t-test).


Representative immunofluorescence of primary cilium in hTERT - RPE - 1 cells silenced with MECP2 - or control - siRNAs . Cells were starved for 48 h before staining with anti - γ - tubulin ( green ) and anti - acetylated α - tubulin ( red ) antibodies . Scale bar 10 µm , and 5 µm in the enlarged image . Arrows indicate not ciliated cells .

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

Representative immunofluorescence of primary cilium in hTERT-RPE-1 cells silenced with MECP2- or control-siRNAs. Cells were starved for 48 h before staining with anti-γ-tubulin (green) and anti-acetylated α-tubulin (red) antibodies. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells.


Representative Western blotting showing the MeCP2 expression in CTRL and silenced hTERT - RPE - 1 cells , with respect to GAPDH , used as loading control .

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Representative Western blotting showing the MeCP2 expression in CTRL and silenced hTERT-RPE-1 cells, with respect to GAPDH, used as loading control.


The percentage of MeCP2 positive cells was calculated by analysing n = 1000 CTRL and silenced hTERT - RPE - 1 cells ( mean ± S . E . , * * * p < 0 . 001 , by Student ' s t test ) .

The percentage of MeCP2 positive cells was calculated by analysing n=1000 CTRL and silenced hTERT-RPE-1 cells (mean ± S.E., ***p<0.001, by Student's t test).


Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells ( n = 191 CTRL and n = 197 siMeCP2 cells ) ( G ) and the cilium length ( n = 50 CTRL and n = 50 siMeCP2 cells ) ( H ) . The graphs show the mean ± S . E of three independent experiments ( * * * p < 0 . 001 , by Mann - Whitney test ( G ) or Student ' s t - test ( H ) ) .

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Cilia were counted and analysed with ImageJ to quantify the percentage of ciliated cells (n=191 CTRL and n=197 siMeCP2 cells) (G) and the cilium length (n=50 CTRL and n=50 siMeCP2 cells) (H). The graphs show the mean ± S.E of three independent experiments (***p<0.001, by Mann-Whitney test (G) or Student's t-test (H)).


Representative immunofluorescence of primary cilium in WT and Mecp2 null primary cortical neurons at DIV7 and DIV14 . Scale bar 10 µm , and 5 µm in the enlarged image . Arrows indicate not ciliated cells .

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Representative immunofluorescence of primary cilium in WT and Mecp2 null primary cortical neurons at DIV7 and DIV14. Scale bar 10 µm, and 5 µm in the enlarged image. Arrows indicate not ciliated cells.


Neurons were fixed at DIV3 , DIV7 and DIV14 and primary cilium detected by immunostaining with anti - AC3 antibody . Percentage of ciliated cells ( J ) and cilium length ( K ) were analysed . Histograms represent the average of three independent experiments , analysing neurons derived from 3 different experiments ( n = 156 WT DIV3 ; n = 49 KO DIV3 ; n = 177 WT DIV7 ; n = 82 KO DIV7 ; n = 59 WT DIV14 ; n = 37 KO DIV14 ) ( mean ± S . E . , * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post hoc test ) .

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Neurons were fixed at DIV3, DIV7 and DIV14 and primary cilium detected by immunostaining with anti-AC3 antibody. Percentage of ciliated cells (J) and cilium length (K) were analysed. Histograms represent the average of three independent experiments, analysing neurons derived from 3 different experiments (n=156 WT DIV3; n=49 KO DIV3; n=177 WT DIV7; n=82 KO DIV7; n=59 WT DIV14; n=37 KO DIV14) (mean ± S.E., **p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post hoc test).


Primary cortical astrocytes were fixed at DIV15 and stained with anti - acetylated α - tubulin ( red ) and anti - ɣ - tubulin ( green ) antibodies . Scale bar 20 µm , and 10 µm in the enlarged image . Arrow indicates not ciliated cell .

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Primary cortical astrocytes were fixed at DIV15 and stained with anti-acetylated α-tubulin (red) and anti-ɣ-tubulin (green) antibodies. Scale bar 20 µm, and 10 µm in the enlarged image. Arrow indicates not ciliated cell.


Percentage of ciliated cells ( M ) and cilium length ( N ) were analysed ( n = 26 WT and n = 34 Mecp2 null astrocytes ) . Histograms represent the average of three independent experiments ( mean ± S . E . , * p < 0 . 05 , Student ' s t test ) .

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Percentage of ciliated cells (M) and cilium length (N) were analysed (n=26 WT and n=34 Mecp2 null astrocytes). Histograms represent the average of three independent experiments (mean ± S.E., *p<0.05, Student's t test).


Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP . Neurons were fixed at DIV7 and immunostained with anti - AC3 antibody to detect the primary cilium . GFP - positive cells represent infected cells . Scale bar 20 µm , and 10 µm in the enlarged image .

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Representative immunofluorescence of Mecp2 null cortical neurons infected at DIV0 with lentiviruses expressing GFP or MeCP2iresGFP. Neurons were fixed at DIV7 and immunostained with anti-AC3 antibody to detect the primary cilium. GFP-positive cells represent infected cells. Scale bar 20 µm, and 10 µm in the enlarged image.


Western blot comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons . Data are normalized to total protein content , visualized by a TGX stain - free technology .

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Western blot comparing MeCP2 levels in Mecp2 null neurons infected with lentiviruses expressing GFP or MeCP2iresGFP with respect to WT neurons. Data are normalized to total protein content, visualized by a TGX stain-free technology.


MECP2 expression in null neurons induced a significant recovery of ciliogenesis . ( C ) The graph represents the percentage of ciliated cells , counting n = 50 WT and n = 100 null cells of two different preparations ( mean ± S . E . , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post - hoc test ) . Statistical analysis reported a significant interaction ( F ( 1 , 135 ) = 8 . 264 ) , a significant genotype effect ( F ( 1 , 135 ) = 37 . 59 ) and a significant infection effect ( F ( 1 , 135 ) = 26 . 89 ) . ( D ) The graph represents the mean ± S . E . of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses , counting n = 28 cells of two different preparations ( * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post - hoc test ) . Two - way ANOVA reported a significant interaction ( F ( 1 , 103 ) = 6 . 405 ) , a significant genotype effect ( F ( 1 , 103 ) = 35 ) and a significant infection effect ( F ( 1 . 103 ) = 4 . 039 ) .

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

MECP2 expression in null neurons induced a significant recovery of ciliogenesis. (C) The graph represents the percentage of ciliated cells, counting n=50 WT and n=100 null cells of two different preparations (mean ± S.E., ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Statistical analysis reported a significant interaction (F (1,135) = 8.264), a significant genotype effect (F (1,135) = 37.59) and a significant infection effect (F (1,135) = 26.89). (D) The graph represents the mean ± S.E. of cilium length in WT and Mecp2 null neurons infected with GFP or MeCP2iresGFP expressing lentiviruses, counting n=28 cells of two different preparations (**p<0.01, ***p<0.001, by two-way ANOVA followed by Bonferroni post-hoc test). Two-way ANOVA reported a significant interaction (F (1,103) = 6.405), a significant genotype effect (F (1,103) = 35) and a significant infection effect (F (1.103) = 4.039).


MECP2 - silenced or control hTERT - RPE - 1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h . Cells were stained with anti - Smo and anti - acetylated α - tubulin antibodies . The percentages of Smo - positive cilia were calculated . The graph shows the average of three independent experiments ( mean ± S . E . , * * * p < 0 . 001 , by two - way ANOVA followed by Bonferroni post hoc test ) . Two - way ANOVA demonstrated a significant silencing effect ( F ( 1 , 12 ) = 160 ; p < 0 . 0001 ) , a significant SAG treatment ( F ( 2 , 12 ) = 259 . 4 ; p < 0 . 0001 ) and a significant interaction ( F ( 2 , 12 ) = 36 . 85 ; p < 0 . 0001 ) .

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

MECP2-silenced or control hTERT-RPE-1 cells were starved for 48 h before treatment with 200 nM and 500 nM SAG for 24 h. Cells were stained with anti-Smo and anti-acetylated α-tubulin antibodies. The percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *** p<0.001, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA demonstrated a significant silencing effect (F (1, 12) = 160; p<0.0001), a significant SAG treatment (F (2, 12) = 259.4; p<0.0001) and a significant interaction (F (2, 12) = 36.85; p<0.0001).


WT or Mecp2 null MEF cells were treated with SAG ( 200 and 500 nM ) for 24 h and the percentages of Smo - positive cilia were calculated . The graph shows the average of three independent experiments ( mean ± S . E . , * p < 0 . 05 , by two - way ANOVA followed by Bonferroni post hoc test ) . Two - way ANOVA indicated a significant genotype effect ( F ( 1 , 12 ) = 11 . 22 ; p < 0 . 01 ) and a significant SAG treatment ( F ( 2 , 12 ) = 11 . 7 ; p < 0 . 0001 ) .

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

WT or Mecp2 null MEF cells were treated with SAG (200 and 500 nM) for 24 h and the percentages of Smo-positive cilia were calculated. The graph shows the average of three independent experiments (mean ± S.E., *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant genotype effect (F (1, 12) = 11.22; p<0.01) and a significant SAG treatment (F (2, 12) = 11.7; p<0.0001).


Gli1 protein levels were measured by Western blot in untreated ( UT ) and 100 nM SAG - treated WT and Mecp2 null MEF cells ( n = 5 / 6 ) . Representative bands of Gli1 , Mecp2 and α - tubulin are depicted above the histogram , which shows the mean ± S . E of the percentage of Gli1 expression levels , compared to the untreated WT samples ( * * p < 0 . 01 , * * * p < 0 . 001 , by two - way ANOVA , followed by Bonferroni post hoc test ) . Two - way ANOVA reported a significant interaction ( F ( 1 , 18 ) = 6 . 652 ; p < 0 . 05 ) , a significant genotype effect ( F ( 1 , 18 ) = 15 . 95 ; p < 0 . 001 ) and a significant treatment ( F ( 1 , 18 ) = 20 . 15 ; p < 0 . 001 ) .

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 O O O O O O O

Gli1 protein levels were measured by Western blot in untreated (UT) and 100 nM SAG-treated WT and Mecp2 null MEF cells (n=5/6). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (**p<0.01, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA reported a significant interaction (F (1, 18) = 6.652; p<0.05), a significant genotype effect (F (1,18) = 15.95; p<0.001) and a significant treatment (F (1, 18) = 20.15; p<0.001).


Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons , following SAG treatment ( 200 and 400 nM ) . Representative bands of Gli1 , Mecp2 and α - tubulin are depicted above the histogram , which shows the mean ± S . E of the percentage of Gli1 expression levels , compared to the untreated WT samples ( * p < 0 . 05 , * * * p < 0 . 001 , by two - way ANOVA , followed by Bonferroni post hoc test ) . Two - way ANOVA indicated a significant interaction ( F ( 2 , 20 ) = 3 . 788 ; p < 0 . 05 ) , a significant genotype effect ( F ( 1 , 20 ) = 4 . 362 ; p < 0 . 05 ) and a significant treatment ( F ( 2 , 20 ) = 13 . 5 ; p < 0 . 001 ) .

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

Gli1 protein levels were measured by Western blot in WT and Mecp2 null neurons, following SAG treatment (200 and 400 nM). Representative bands of Gli1, Mecp2 and α-tubulin are depicted above the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to the untreated WT samples (*p<0.05, ***p<0.001, by two-way ANOVA, followed by Bonferroni post hoc test). Two-way ANOVA indicated a significant interaction (F (2, 20) = 3.788; p<0.05), a significant genotype effect (F (1, 20) = 4.362; p<0.05) and a significant treatment (F (2, 20) = 13.5; p<0.001).


Gli1 mRNA expression levels upon SAG exposure in MEF cells ( n = 7 WT and n = 9 null samples ) ( F ; SAG 100 nM ) and cortical neurons ( n = 15 WT and n = 12 null samples ) ( G ; SAG 400 nM ) . The graphs show the mean ± S . E . of the fold change derived from at least three independent experiments ( mean ± S . E . , * * p < 0 . 01 , by two - way ANOVA , followed by Bonferroni post hoc test ) .

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Gli1 mRNA expression levels upon SAG exposure in MEF cells (n=7 WT and n=9 null samples) (F; SAG 100 nM) and cortical neurons (n= 15 WT and n=12 null samples) (G; SAG 400 nM). The graphs show the mean ± S.E. of the fold change derived from at least three independent experiments (mean ± S.E., **p<0.01, by two-way ANOVA, followed by Bonferroni post hoc test).


Representative immunostaining of Arl13b ( red ) and γ - tubulin ( green ) in the P14 mouse cortical layers . Nuclei were stained with DAPI ( blue ) . Scale bar 10 µm .

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Representative immunostaining of Arl13b (red) and γ-tubulin (green) in the P14 mouse cortical layers. Nuclei were stained with DAPI (blue). Scale bar 10 µm.


Primary cilium length was measured in the cortex of Mecp2 null mice Average cilium length was measured in the cortex ( mean ± S . E . , n = 4 WT and n = 3 Mecp2 null ) ( B ; * p < 0 . 05 , by Mann - Whitney test ) and for each cortical layer ( C ; * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 , by two - way ANOVA followed by Bonferroni post hoc test ) . In C , cilium length is analysed in separate cortical layers while in B , the values indicate the average of cilium lengths for each layer .

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Primary cilium length was measured in the cortex of Mecp2 null mice Average cilium length was measured in the cortex (mean ± S.E., n=4 WT and n=3 Mecp2 null) (B; *p<0.05, by Mann-Whitney test) and for each cortical layer (C; ***p<0.001, **p<0.01, *p<0.05, by two-way ANOVA followed by Bonferroni post hoc test). In C, cilium length is analysed in separate cortical layers while in B, the values indicate the average of cilium lengths for each layer.


Representative immunostaining of Arl13b ( green ) in the internal ( IGL ) and external ( EGL ) granular layers of Mecp2 null ( n = 3 ) and heterozygous ( het ) cerebella ( n = 3 ) at P7 , and the corresponding WT ( n = 3 males and n = 3 females ) . Nuclei were stained with DAPI ( blue ) . Scale bar 5 µm .

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Representative immunostaining of Arl13b (green) in the internal (IGL) and external (EGL) granular layers of Mecp2 null (n=3) and heterozygous (het) cerebella (n=3) at P7, and the corresponding WT (n=3 males and n=3 females). Nuclei were stained with DAPI (blue). Scale bar 5 µm.


Primary cilium length was measured in the cerebellum of Mecp2 null and heterozygous mice . Histograms show the mean ± S . E . of cilium length ( n = 3 males WT , n = 3 females WT , n = 3 Mecp2 null and Mecp2 het ; * * p < 0 . 01 , * * * p < 0 . 001 , by Mann - Whitney test ) in Mecp2 null ( E ) and heterozygous ( F ) mice , compared to the corresponding WT .

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Primary cilium length was measured in the cerebellum of Mecp2 null and heterozygous mice. Histograms show the mean ± S.E. of cilium length (n=3 males WT, n=3 females WT, n=3 Mecp2 null and Mecp2 het; **p<0.01, ***p<0.001, by Mann-Whitney test) in Mecp2 null (E) and heterozygous (F) mice, compared to the corresponding WT.


Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum ( n = 7 WT and n = 6 Mecp2 null ) . Representative bands of Gli1 and α - tubulin are depicted beside the histogram , which shows the mean ± S . E of the percentage of Gli1 expression levels , compared to WT animals ( * p < 0 . 05 , by Student ' s t test ) .

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

Gli1 protein levels were measured by Western blot in WT and Mecp2 null cerebellum (n=7 WT and n=6 Mecp2 null). Representative bands of Gli1 and α-tubulin are depicted beside the histogram, which shows the mean ± S.E of the percentage of Gli1 expression levels, compared to WT animals (*p<0.05, by Student's t test).


The mRNA expression levels of Gli1 and its downstream genes ( Cyclin D1 and D2 ) were analyzed in WT and Mecp2 null cerebella ( n = 8 WT and n = 7 Mecp2 null ) . The graph shows the fold change of transcripts compared to WT ( mean ± S . E . , * p < 0 . 05 , by Student ' s t test ) .

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The mRNA expression levels of Gli1 and its downstream genes (Cyclin D1 and D2) were analyzed in WT and Mecp2 null cerebella (n=8 WT and n=7 Mecp2 null). The graph shows the fold change of transcripts compared to WT (mean ± S.E., *p<0.05, by Student's t test).