( 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 .

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

(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 .

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

(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 ) .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-SMALL_MOLECULE O O O O O

(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 .

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

(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 .

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

(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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-ORGANISM B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O

(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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

(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 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-EXP_ASSAY O B-GENEPROD O

(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 .

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

(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 .

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

(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 .

O O O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O

(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 .

O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O

(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 .

O O O O O B-CELL_LINE O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

(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 .

O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

(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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-ORGANISM B-GENEPROD I-GENEPROD O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O

(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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM B-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

(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 .

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

(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 .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-ORGANISM B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

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

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 .

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

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 .

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

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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 .

O O O O O B-GENEPROD B-ORGANISM O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-TISSUE O O O B-ORGANISM O O O O O O O O O O O O O

(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 .

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

(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 .

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

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

O O O O O B-GENEPROD B-ORGANISM O O O O O O O O O O O B-ORGANISM O O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY 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). (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 .

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

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 .

B-GENEPROD B-ORGANISM O O O O O O O O O O B-CELL_TYPE O O O B-TISSUE O O B-GENEPROD B-ORGANISM O B-GENEPROD O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O

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 .

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

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 .

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

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 .

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

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 .

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


Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( A ) Box - plot of the enrichment of the RNAs identified by Mmi1 RNA - IPs coupled to high throughput sequencing . The log of the average enrichments obtained from two independent RNA - IPs is plotted . The enrichment is relative to the no antibody RNA - IPs conducted in parallel of Mmi1 RNA - IPs . The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA - IPs are shown in blue and green , respectively . Crosses represent newly identified Mmi1 targets and dots known targets .

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

Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (A) Box-plot of the enrichment of the RNAs identified by Mmi1 RNA-IPs coupled to high throughput sequencing. The log of the average enrichments obtained from two independent RNA-IPs is plotted. The enrichment is relative to the no antibody RNA-IPs conducted in parallel of Mmi1 RNA-IPs. The mRNAs and lncRNAs enriched at least two folds in both Mmi1 RNA-IPs are shown in blue and green, respectively. Crosses represent newly identified Mmi1 targets and dots known targets.


Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( D ) RNA - IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1 ' s binding to ssm4 mRNA . The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA - IPs . Loading was monitored using an anti - Tub1 ( tubulin ) antibody .

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Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (D) RNA-IPs showing the impact of Mmi1 YTH domain point mutations on Mmi1's binding to ssm4 mRNA. The lower part shows a Western blot monitoring the protein level of WT and mutant Mmi1 proteins in the cells used for the RNA-IPs. Loading was monitored using an anti-Tub1 (tubulin) antibody.


Figure 1 . Extensive identification of Mmi1 RNA targets by combining RNA - IP sequencing and computational approaches . ( E , F ) RNA - IPs showing the YTH - dependent association of Mmi1 to the lncRNAs identified in ( A ) and ( B ) . Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for ( D , E and F ) .

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Figure 1. Extensive identification of Mmi1 RNA targets by combining RNA-IP sequencing and computational approaches. (E, F) RNA-IPs showing the YTH-dependent association of Mmi1 to the lncRNAs identified in (A) and (B). Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for (D, E and F).


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( A ) Upper part , scheme of S . pombe sexual differentiation . Lower part , microscopy images showing WT and mmi1 ∆ cells , and mmi1 ∆ cells expressing Mmi1 - R351E or Mmi1 - R381E mutant proteins . Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium . The percentage of cells that underwent differentiation and iodine vapor assays , conducted on the corresponding patches , are shown at the bottom of each image .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (A) Upper part, scheme of S. pombe sexual differentiation. Lower part, microscopy images showing WT and mmi1∆ cells, and mmi1∆ cells expressing Mmi1-R351E or Mmi1-R381E mutant proteins. Images were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium. The percentage of cells that underwent differentiation and iodine vapor assays, conducted on the corresponding patches, are shown at the bottom of each image.


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( B ) Mating assay showing the percentage of zygotes forming over time in the same cells as in ( A ) .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (B) Mating assay showing the percentage of zygotes forming over time in the same cells as in (A).


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( E ) Mmi1 RNA - IPs showing the specific loss of binding of Mmi1 to nam1 - 1 lncRNA but not to mei4 mRNA , another target of Mmi1 .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (E) Mmi1 RNA-IPs showing the specific loss of binding of Mmi1 to nam1-1 lncRNA but not to mei4 mRNA, another target of Mmi1.


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( F ) Mating assay showing the percentage of zygotes formed over time in WT and nam1 - 1 cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) ( B , E and F ) .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (F) Mating assay showing the percentage of zygotes formed over time in WT and nam1-1 cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) (B, E and F).


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( G ) Western blots showing the level of Flag - Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1 - 1 cells . Tubulin ( Tub1 ) level was used as a loading control .

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Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (G) Western blots showing the level of Flag-Byr2 protein over the first 4 hours of sexual differentiation in WT and nam1-1 cells. Tubulin (Tub1) level was used as a loading control.


Figure 2 . Mmi1 binding to nam1 lncRNA controls MAPK - mediated entry into sexual differentiation . ( H ) Microscopy images of WT ( h90 ) cells transformed with an empty plasmid ( Control ) and nam1 - 1 cells transformed with either an empty plasmid ( Control ) or a plasmid expressing Byr2 protein ( Byr2 ) , after 24 hours of induction of sexual differentiation . Scale bar , 10 µm .

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

Figure 2. Mmi1 binding to nam1 lncRNA controls MAPK-mediated entry into sexual differentiation. (H) Microscopy images of WT (h90) cells transformed with an empty plasmid (Control) and nam1-1 cells transformed with either an empty plasmid (Control) or a plasmid expressing Byr2 protein (Byr2), after 24 hours of induction of sexual differentiation. Scale bar, 10 µm.


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( A ) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation . Ribosomal RNAs ( rRNAs ) stained with ethidium bromide were used as loading controls . Black lines indicate probes used to detect nam1 and byr2 RNAs .

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

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (A) Northern blots showing nam1 and byr2 RNA levels during the first 4 hours of sexual differentiation. Ribosomal RNAs (rRNAs) stained with ethidium bromide were used as loading controls. Black lines indicate probes used to detect nam1 and byr2 RNAs.


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( B ) ChIPs showing the occupancy of the elongating RNAPII ( RNAPII - S2P ) over nam1 - byr2 locus , in WT and nam1 - 1 cells . RNAPII - S2P was immunoprecipitated with an antibody recognizing the heptameric repeats ( present in the C - terminal domain of the polymerase ) when it is phosphorylated on its serine 2 . Black lines , genomic regions investigated . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (B) ChIPs showing the occupancy of the elongating RNAPII (RNAPII-S2P) over nam1-byr2 locus, in WT and nam1-1 cells. RNAPII-S2P was immunoprecipitated with an antibody recognizing the heptameric repeats (present in the C-terminal domain of the polymerase) when it is phosphorylated on its serine 2. Black lines, genomic regions investigated. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( C ) RT - qPCRs showing the accumulation of nam1 read - through transcripts ( RT1 - qPCR ) and nam1 lncRNAs ( RT2 - qPCR ) in cells with or without the transcription terminator Ttef inserted at the 3 ' end of nam1 ( scheme ) . Black arrow , primer used for the strand - specific reverse transcription ( RT ) ; Black line , location of the region amplified by PCR . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (C) RT-qPCRs showing the accumulation of nam1 read-through transcripts (RT1-qPCR) and nam1 lncRNAs (RT2-qPCR) in cells with or without the transcription terminator Ttef inserted at the 3' end of nam1 (scheme). Black arrow, primer used for the strand-specific reverse transcription (RT); Black line, location of the region amplified by PCR. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( D ) Microscopy images of , respectively , WT ( h90 ) , nam1 - 1 and nam1 - 1 - Ttef cells , after induction of sexual differentiation for 24 hours . The percentage of sporulation and iodine vapor assays are shown at the bottom of the images . Scale bar , 10 µm .

O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (D) Microscopy images of, respectively, WT (h90), nam1-1 and nam1-1-Ttef cells, after induction of sexual differentiation for 24 hours. The percentage of sporulation and iodine vapor assays are shown at the bottom of the images. Scale bar, 10 µm.


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( E ) RT - qPCRs showing the accumulation of nam1 read - through transcripts in mmi1 ∆ and rrp6 ∆ cells . Black arrow and line as in ( C ) . . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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

Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (E) RT-qPCRs showing the accumulation of nam1 read-through transcripts in mmi1∆ and rrp6∆ cells. Black arrow and line as in (C). . Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 3 . Mmi1 promotes transcription termination of nam1 non - coding gene and prevents nam1 read - through transcription from repressing the downstream MAPKKK gene byr2 . ( F ) ChIPs monitoring the enrichment of H3K9me2 over nam1 - byr2 locus and mei4 gene in WT and rrp6 ∆ cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E and F ) .

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Figure 3. Mmi1 promotes transcription termination of nam1 non-coding gene and prevents nam1 read-through transcription from repressing the downstream MAPKKK gene byr2. (F) ChIPs monitoring the enrichment of H3K9me2 over nam1-byr2 locus and mei4 gene in WT and rrp6∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (B, C, E and F).


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( B ) Northern blot showing the level of nam5 / 6 / 7 lncRNA population in mmi1 - ts3 , dcr1 ∆ , clr4 ∆ single mutant cells and in mmi1 - ts3 dcr1 ∆ and mmi1 - ts3 clr4 ∆ double mutant cells , at the permissive ( 25ºC ) and restrictive ( 36ºC ) temperatures .

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Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (B) Northern blot showing the level of nam5/6/7 lncRNA population in mmi1-ts3, dcr1∆, clr4∆ single mutant cells and in mmi1-ts3 dcr1∆ and mmi1-ts3 clr4∆ double mutant cells, at the permissive (25ºC) and restrictive (36ºC) temperatures.


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( C ) RT - PCRs monitoring the accumulation of nam7 lncRNAs and nam7 - L read - through transcripts , in the same cells and conditions as in ( B ) . Red arrow heads point to the expected PCR products while the other bands correspond to non - specific PCR products . From the scheme and the agarose gels : black arrows , primers used for the three different reverse transcriptions ; black lines and red numbers , expected PCR products of the three different RT - PCRs ; M , DNA ladder Markers ; tub1 , tubulin control .

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

Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (C) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts, in the same cells and conditions as in (B). Red arrow heads point to the expected PCR products while the other bands correspond to non-specific PCR products. From the scheme and the agarose gels: black arrows, primers used for the three different reverse transcriptions; black lines and red numbers, expected PCR products of the three different RT-PCRs; M, DNA ladder Markers; tub1, tubulin control.


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( D ) RT - qPCRs showing the levels of nam5 / 6 / 7 lncRNA population in the double mutant rrp6 ∆ dcr1 ∆ cells , relative to the single mutant rrp6 ∆ and dcr1 ∆ cells . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( D , E ) . P value was calculated using a two - tailed Student ' s t - test .

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Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (D) RT-qPCRs showing the levels of nam5/6/7 lncRNA population in the double mutant rrp6∆ dcr1∆ cells, relative to the single mutant rrp6∆ and dcr1∆ cells. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.


Figure 4 . Mmi1 drives Rrp6 - dependent heterochromatin gene silencing at pericentromeric regions . ( E ) RNA - IPs showing that Rrp6 - Myc13 binds to nam5 / 6 / 7 lncRNAs in a Mmi1 - dependent manner . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( D , E ) . P value was calculated using a two - tailed Student ' s t - test .

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Figure 4. Mmi1 drives Rrp6-dependent heterochromatin gene silencing at pericentromeric regions. (E) RNA-IPs showing that Rrp6-Myc13 binds to nam5/6/7 lncRNAs in a Mmi1-dependent manner. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (D, E). P value was calculated using a two-tailed Student's t-test.


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( A ) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4 ∆ cells .

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Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (A) ChIPs assessing the localization of Mmi1 to pericentromeric DNA in WT and clr4∆ cells.


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( B ) ChIPs showing the localization of Mmi1 ( dashed line ) and H3K9me2 ( black line ) to the pericentromeric nam5 / 6 / 7 DNA regions during the progression of the cell cycle . Cell synchronization was achieved by using cdc25 - ts cells ( see experimental procedures for more details ) . Cell synchronization was monitored by measuring the percentage of cells with a septum ( right part ) .

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Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (B) ChIPs showing the localization of Mmi1 (dashed line) and H3K9me2 (black line) to the pericentromeric nam5/6/7 DNA regions during the progression of the cell cycle. Cell synchronization was achieved by using cdc25-ts cells (see experimental procedures for more details). Cell synchronization was monitored by measuring the percentage of cells with a septum (right part).


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( C ) RT - qPCRs monitoring the levels of nam5 / 6 / 7 ( left part ) and dg ( middle part ) lncRNA populations during the cell cycle . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( A , B and C ) .

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Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (C) RT-qPCRs monitoring the levels of nam5/6/7 (left part) and dg (middle part) lncRNA populations during the cell cycle. Average fold enrichment is shown with error bars that indicate mean average deviations (n=3) for (A, B and C).


Figure 5 . Mmi1 - and RNAi - mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle . ( D ) RT - PCRs monitoring the accumulation of nam7 lncRNAs and nam7 - L read - through transcripts in G2 / M and G1 / S phases from synchronized cells as in ( B ) . Red arrow heads point to the expected PCR products . The other bands are non - specific PCR products . From the scheme : black arrows , primers used for the reverse transcriptions ; black lines and red numbers , regions amplified by PCR .

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

Figure 5. Mmi1- and RNAi-mediated silencing of pericentromeric DNA transcription alternate during the progression of the cell cycle. (D) RT-PCRs monitoring the accumulation of nam7 lncRNAs and nam7-L read-through transcripts in G2/M and G1/S phases from synchronized cells as in (B). Red arrow heads point to the expected PCR products. The other bands are non-specific PCR products. From the scheme: black arrows, primers used for the reverse transcriptions; black lines and red numbers, regions amplified by PCR.


A - E Scatter plots showing the association of each of the p - tau biomarkers with age in the Aβ - negative ( A - ; blue ; n = 250 ) and the Aβ - positive ( A + ; red ; n = 131 ) groups . The solid lines indicate the regression line and the 95 % confidence intervals for each of the groups . For each group , the standardized regression coefficients ( β ) and the P - values were computed using a linear model adjusting for sex . Additionally , we computed the ' Age x Aβ status ' interaction term . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

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

A-E Scatter plots showing the association of each of the p-tau biomarkers with age in the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n = 131) groups. The solid lines indicate the regression line and the 95% confidence intervals for each of the groups. For each group, the standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for sex. Additionally, we computed the 'Age x Aβ status' interaction term. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


( A , B , C , D , E ) Dot and box - plot comparing each of the p - tau biomarker between the Aβ - negative ( A - ; blue ; n = 250 ) and the Aβ - positive ( A + ; red ; n = 131 ) groups . Aβ positivity was defined as CSF Aβ42 / 40 ratio < 0 . 071 .

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(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ-negative (A-; blue; n = 250) and the Aβ-positive (A+; red; n =131) groups. Aβ positivity was defined as CSF Aβ42/40 ratio < 0.071.


( F , G , H , I , J ) Scatter plots depicting the changes between each p - tau biomarker as a function of CSF Aβ42 / 40 . The horizontal - axes directions were inverted ; lower CSF Aβ42 / 40 ratio reflects higher Aβ pathology . For each Aβ status group , we computed the standardized regression coefficients ( β ) and the P - values , adjusted for age and sex . The solid lines indicate the regression line and the 95 % confidence intervals for each of the Aβ status groups . The dashed green lines indicate the CSF Aβ42 / 40 cutoff . Participants were also colour - coded based on the Aβ PET CL scale ( ≤ 12CL , black ; > 12CL , turquoise ; Aβ PET non - available , grey ) .

O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O

(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of CSF Aβ42/40. The horizontal-axes directions were inverted; lower CSF Aβ42/40 ratio reflects higher Aβ pathology. For each Aβ status group, we computed the standardized regression coefficients (β) and the P-values, adjusted for age and sex. The solid lines indicate the regression line and the 95% confidence intervals for each of the Aβ status groups. The dashed green lines indicate the CSF Aβ42/40 cutoff. Participants were also colour-coded based on the Aβ PET CL scale (≤12CL, black; >12CL, turquoise; Aβ PET non-available, grey).


( A , B , C , D , E ) Dot and box - plot comparing each of the p - tau biomarker between the Aβ negative ( A - ; blue ; n = 287 ) and the Aβ positive ( A + ; red ; n = 42 ) groups . Aβ positivity was defined with Aβ PET visual read .

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(A, B, C, D, E) Dot and box-plot comparing each of the p-tau biomarker between the Aβ negative (A-; blue; n = 287) and the Aβ positive (A+; red; n = 42) groups. Aβ positivity was defined with Aβ PET visual read.


( F , G , H , I , J ) Scatter plots depicting the changes between each p - tau biomarker as a function of Aβ PET Centiloids ( CL ) . The standardized regression coefficients ( β ) and the P - values were computed using a linear model adjusting for age and sex . The solid lines indicate the regression line and the 95 % confidence intervals . The dashed green lines indicate the CL12 and CL30 cutoffs . Participants were also colour - coded based on the CSF Aβ42 / 40 ratio ( A - , black ; A + , turquoise ) .

O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(F, G, H, I, J) Scatter plots depicting the changes between each p-tau biomarker as a function of Aβ PET Centiloids (CL). The standardized regression coefficients (β) and the P-values were computed using a linear model adjusting for age and sex. The solid lines indicate the regression line and the 95% confidence intervals. The dashed green lines indicate the CL12 and CL30 cutoffs. Participants were also colour-coded based on the CSF Aβ42/40 ratio (A-, black; A+, turquoise).


( K , L , M , N , O ) Dot and box - plots depicting comparison between each of the p - tau biomarker between Centiloid scale groups : ( i ) ≤ 12CL ( blue ; n = 278 ) , ( ii ) 12 - 30CL ( subthreshold Aβ pathology group ; grey ; n = 28 ) , ( iii ) > 30CL ( red ; n = 25 ) . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) .

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(K, L, M, N, O) Dot and box-plots depicting comparison between each of the p-tau biomarker between Centiloid scale groups: (i) ≤12CL (blue; n = 278), (ii) 12 - 30CL (subthreshold Aβ pathology group; grey; n = 28), (iii) >30CL (red; n = 25). The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers).


A - C ROC analysis was performed to test the accuracy to discriminate between Aβ - positive ( A + ) from Aβ - negative ( A - ) individuals . Aβ positivity was defined as CSF Aβ42 / 40 < 0 . 071 ( A ) , Aβ PET positive visual read ( B ) or Aβ PET Centiloid ( CL ) > 12 ( C ) . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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

A-C ROC analysis was performed to test the accuracy to discriminate between Aβ-positive (A+) from Aβ-negative (A-) individuals. Aβ positivity was defined as CSF Aβ42/40 < 0.071 (A), Aβ PET positive visual read (B) or Aβ PET Centiloid (CL) > 12 (C). Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


A - D Dot and box - plot showing the levels of each p - tau biomarker in each of the AT groups . Aβ - positive ( A + ) was defined by a CSF Aβ42 / 40 < 0 . 071 and Tau - positive ( T + ) by an Elecsys CSF Mid - p - tau181 > 24 pg / ml . The box - plots depict the median ( horizontal bar ) , interquartile range ( IQR , hinges ) and 1 . 5 x IQR ( whiskers ) . The horizontal dashed line indicates the median of the p - tau biomarker in the A - T - group . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; N , N - terminal ; p - tau , phosphorylated tau .

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

A-D Dot and box-plot showing the levels of each p-tau biomarker in each of the AT groups. Aβ-positive (A+) was defined by a CSF Aβ42/40 < 0.071 and Tau-positive (T+) by an Elecsys CSF Mid-p-tau181 > 24 pg/ml. The box-plots depict the median (horizontal bar), interquartile range (IQR, hinges) and 1.5 x IQR (whiskers). The horizontal dashed line indicates the median of the p-tau biomarker in the A-T- group. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; N, N-terminal; p-tau, phosphorylated tau.


The graphs represent the z - scores changes of each CSF biomarker as a function of CSF Aβ42 / 40 ratio ( as proxy of disease progression ) using a robust local weighted regression method . The z - scores were calculated using the mean and the SD of each CSF biomarker in the A - T - group as a reference . The solid lines depict the trajectory of each CSF biomarker . The dashed lines depict the trajectories of the plasma biomarkers . The vertical black dashed line indicates the CSF Aβ42 / 40 cutoff for A + . Note that the CSF p - tau biomarkers reach the 2 z - scores ( depicted with an horizontal dashed line ) with the following sequence : Mid - p - tau231 , N - p - tau181 , N - p - tau217 , Mid - p - tau181 and t - tau . Abbreviations : CSF , cerebrospinal fluid ; Mid , mid - region ; NfL , Neurofilament light ; N , N - terminal ; p - tau , phosphorylated tau ; t - tau , total tau .

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

The graphs represent the z-scores changes of each CSF biomarker as a function of CSF Aβ42/40 ratio (as proxy of disease progression) using a robust local weighted regression method. The z-scores were calculated using the mean and the SD of each CSF biomarker in the A-T- group as a reference. The solid lines depict the trajectory of each CSF biomarker. The dashed lines depict the trajectories of the plasma biomarkers. The vertical black dashed line indicates the CSF Aβ42/40 cutoff for A+. Note that the CSF p-tau biomarkers reach the 2 z-scores (depicted with an horizontal dashed line) with the following sequence: Mid-p-tau231, N-p-tau181, N-p-tau217, Mid-p-tau181 and t-tau. Abbreviations: CSF, cerebrospinal fluid; Mid, mid-region; NfL, Neurofilament light; N, N-terminal; p-tau, phosphorylated tau; t-tau, total tau.


A Representative clinical picture of two patients with CAOP syndrome ( 14 - year - old patient 1 and 5 - year - old patient 2 ) .

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A Representative clinical picture of two patients with CAOP syndrome (14-year-old patient 1 and 5-year-old patient 2).


B Haematoxylin and eosin ( H & E ) staining of skin biopsies from patient 1 and healthy controls . Scale bars : 200 µm .

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B Haematoxylin and eosin (H&E) staining of skin biopsies from patient 1 and healthy controls. Scale bars: 200 µm.


A , B Representative transmission electron microscopy ( TEM ) images showing keratinocyte mitochondria ( red arrow ) in patient 1 ( A ) and in the Mbtps1 - conditional knockout ( cKO ) mouse model ( B ) . Scale bars : 2 µm .

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A, B Representative transmission electron microscopy (TEM) images showing keratinocyte mitochondria (red arrow) in patient 1 (A) and in the Mbtps1-conditional knockout (cKO) mouse model (B). Scale bars: 2 µm.


C , D Quantification of the mitochondrial number and morphology in patient 1 ( C ) and the Mbtps1 - cKO mouse model ( D ) . ( C ) Left panel : n = 13 biological replicates of normal individuals , n = 10 biological replicates of patient 1 . Middle and right panel : n = 121 biological samples of normal individuals and patient 1 . ( D ) Left panel : n = 23 biological replicates of Mbtps1 - loxP mice ; n = 15 biological replicates of Mbtps1 - cKO mice . Middle panel and right panel : n = 113 biological replicates of Mbtps1 - loxP mice and Mbtps1 - cKO .

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C, D Quantification of the mitochondrial number and morphology in patient 1 (C) and the Mbtps1-cKO mouse model (D). (C) Left panel: n = 13 biological replicates of normal individuals, n = 10 biological replicates of patient 1. Middle and right panel: n = 121 biological samples of normal individuals and patient 1. (D) Left panel: n = 23 biological replicates of Mbtps1-loxP mice; n = 15 biological replicates of Mbtps1-cKO mice. Middle panel and right panel: n =113 biological replicates of Mbtps1-loxP mice and Mbtps1-cKO.


E Immunofluorescence experiments showed that mutant S1P ( p . Val355Gly and p . Ter1053Arg ) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild - type S1P , which suggested that these variants disrupt its mitochondrial import . The white arrowheads indicate the colocation ( yellow ) of S1P ( green ) and the mitochondrial tracer ( red ) . Scale bars : 20 μm ( panels 1 - 4 ) ; scale bars : 2 μm ( panel 5 ) .

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E Immunofluorescence experiments showed that mutant S1P (p.Val355Gly and p.Ter1053Arg) was diffusely localized in the cytosol and showed lower mitochondrial localization compared with wild-type S1P, which suggested that these variants disrupt its mitochondrial import. The white arrowheads indicate the colocation (yellow) of S1P (green) and the mitochondrial tracer (red). Scale bars: 20 μm (panels 1-4); scale bars: 2 μm (panel 5).


F Cellular component separation assay showing that S1P was enriched in mitochondria . Biochemical fractionation of the whole cell lysate ( WCL ) , cytosol ( Cyto , tubulin ) , mitochondria ( Mito , COX IV ) , lysosomes ( Lyso , LAMP1 ) , Golgi apparatus ( Golgi , GM130 ) , endoplasmic reticulum ( ER , ERp72 ) , and nucleus ( Nue , Lamin B ) from HaCaT cells .

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F Cellular component separation assay showing that S1P was enriched in mitochondria. Biochemical fractionation of the whole cell lysate (WCL), cytosol (Cyto, tubulin), mitochondria (Mito, COX IV), lysosomes (Lyso, LAMP1), Golgi apparatus (Golgi, GM130), endoplasmic reticulum (ER, ERp72), and nucleus (Nue, Lamin B) from HaCaT cells.


G A component separation assay revealed a lower expression of mutant S1P ( p . Val355Gly and p . Ter1053Arg ) than wild - type S1P in mitochondria .

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G A component separation assay revealed a lower expression of mutant S1P (p.Val355Gly and p.Ter1053Arg) than wild-type S1P in mitochondria.


H The impaired binding of mutant S1P ( p . Val355Gly and p . Ter1053Arg ) to translocase of the outer membrane ( TOM ) 70 and translocase of the inner membrane ( TIM ) 23 was detected by a coimmunoprecipitation ( Co - IP ) assay .

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H The impaired binding of mutant S1P (p.Val355Gly and p.Ter1053Arg) to translocase of the outer membrane (TOM) 70 and translocase of the inner membrane (TIM) 23 was detected by a coimmunoprecipitation (Co-IP) assay.


A LC - MS / MS analysis of S1P - binding proteins . Total protein lysate was subjected to coimmunoprecipitation ( Co - IP ) with normal IgG or S1P antibody . The purified protein complex was separated by SDS - PAGE and then subjected to silver staining . The arrows indicate the bands containing S1P , ETFA and ETFB . The differential bands were then analysed by liquid chromatography - tandem mass spectrometry ( LC - MS / MS ) .

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A LC-MS/MS analysis of S1P-binding proteins. Total protein lysate was subjected to coimmunoprecipitation (Co-IP) with normal IgG or S1P antibody. The purified protein complex was separated by SDS-PAGE and then subjected to silver staining. The arrows indicate the bands containing S1P, ETFA and ETFB. The differential bands were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).


C S1P interacts with endogenous ETFA and ETFB in HaCaT cells . Total protein lysate was subjected to Co - IP with normal IgG , ETFA ( left panel ) or ETFB ( right panel ) antibody . The interaction between S1P and ETFA / ETFB was then detected by immunoblotting .

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C S1P interacts with endogenous ETFA and ETFB in HaCaT cells. Total protein lysate was subjected to Co-IP with normal IgG, ETFA (left panel) or ETFB (right panel) antibody. The interaction between S1P and ETFA/ETFB was then detected by immunoblotting.


D The direct interaction between S1P and ETFA - ETFB was validated by a GST pull - down assay . Left panel : in vitro - translated S1P was pulled down by purified GST - ETFA fusion protein . Right panel : in vitro - translated S1P was pulled down by purified GST - ETFB fusion protein .

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D The direct interaction between S1P and ETFA-ETFB was validated by a GST pull-down assay. Left panel: in vitro-translated S1P was pulled down by purified GST-ETFA fusion protein. Right panel: in vitro-translated S1P was pulled down by purified GST-ETFB fusion protein.


A Representative immunohistochemical images of S1P , ETFA and ETFB in normal controls and patient 1 . Scale bars : 100 μm .

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A Representative immunohistochemical images of S1P, ETFA and ETFB in normal controls and patient 1. Scale bars: 100 μm.


B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1 . Quantitative RT - PCR analysis ( upper panel , n = 6 biological replicates of normal controls ; n = 6 technical replicates of patient 1 ) and immunohistochemistry ( lower panel , n = 6 biological replicates ; n = 3 technical replicates ) of the S1P , ETFA and ETFB levels .

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B Unchanged mRNA levels and decreased protein levels of ETFA and ETFB were observed in the skin biopsy of patient 1. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates of normal controls; n = 6 technical replicates of patient 1) and immunohistochemistry (lower panel, n = 6 biological replicates; n = 3 technical replicates) of the S1P, ETFA and ETFB levels.


C Representative immunohistochemistry images of S1P , ETFA and ETFB in both Mbtps1 - conditional knockout ( cKO ) and Mbtps1 - loxP mice . Scale bars : 100 μm .

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C Representative immunohistochemistry images of S1P, ETFA and ETFB in both Mbtps1-conditional knockout (cKO) and Mbtps1-loxP mice. Scale bars: 100 μm.


D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo . Quantitative RT - PCR analysis ( upper panel , n = 6 biological replicates ) and immunohistochemistry ( lower panel , n = 6 biological replicates ) of the S1P , ETFA and ETFB levels in Mbtps1 - cKO mice and Mbtps1 - loxP mice .

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D MBTPS1 gene knockout led to decreases in the ETFA and ETFB protein levels in vivo. Quantitative RT-PCR analysis (upper panel, n = 6 biological replicates) and immunohistochemistry (lower panel, n = 6 biological replicates) of the S1P, ETFA and ETFB levels in Mbtps1-cKO mice and Mbtps1-loxP mice.


E Cycloheximide ( CHX ) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells . Left panel : Representative western blotting images of the ETFA and ETFB protein levels during CHX chase . Right panel : Quantification of the immunoblotting results corresponding to the left panel ( n = 3 biological replicates ) .

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E Cycloheximide (CHX) chase analysis showed that MBTPS1 knockout induced rapid degradation of ETFA and ETFB proteins in HaCaT cells. Left panel: Representative western blotting images of the ETFA and ETFB protein levels during CHX chase. Right panel: Quantification of the immunoblotting results corresponding to the left panel (n = 3 biological replicates).


F Mutant S1P ( p . Val355Gly and p . Ter1053Arg ) only weakly interacted with ETFA and ETFB . We constructed Flag - tagged wild - type and mutant S1P HaCaT cell lines and then performed a Co - IP assay with Flag antibody , and the interaction between S1P and ETFA / ETFB was detected by immunoblotting .

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F Mutant S1P (p.Val355Gly and p.Ter1053Arg) only weakly interacted with ETFA and ETFB. We constructed Flag-tagged wild-type and mutant S1P HaCaT cell lines and then performed a Co-IP assay with Flag antibody, and the interaction between S1P and ETFA/ETFB was detected by immunoblotting.


G Wild - type S1P , but not mutant S1P ( p . Val355Gly and p . Ter1053Arg ) , decreased the association between ETFA and ETFB . We performed a Co - IP assay with an ETFA antibody , and the interaction between ETFA and ETFB was detected by immunoblotting .

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G Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), decreased the association between ETFA and ETFB. We performed a Co-IP assay with an ETFA antibody, and the interaction between ETFA and ETFB was detected by immunoblotting.