D NIE - 115 cells stably expressing mSt - INPP5E ( WT , D477N , or ΔCAAX ) were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . The number showing to the right side indicates the colocalization rate of LAMP1 with mSt - INPP5E . Bar , 10 µm .

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D NIE-115 cells stably expressing mSt-INPP5E (WT, D477N, or ΔCAAX) were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. The number showing to the right side indicates the colocalization rate of LAMP1 with mSt-INPP5E. Bar, 10 µm.


A N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg / ml DQ - BSA for 12 h and chased for 2 h . Cells were fixed and stained with anti - LAMP1 antibodies and analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . B Quantitation of signal intensities in A showing colocalization of DQ - BSA with LAMP1 ( means ± s . d . ; n > 50 cells from three independent experiments ) . Baf . A1 was used as a control for inhibition of delivery and degradation of DQ - BSA via the endocytic pathway . * * , P < 0 . 01 ; n . s . , non significant .

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A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium with 20 µg/ml DQ-BSA for 12 h and chased for 2 h. Cells were fixed and stained with anti-LAMP1 antibodies and analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.B Quantitation of signal intensities in A showing colocalization of DQ-BSA with LAMP1 (means ± s.d.; n > 50 cells from three independent experiments). Baf.A1 was used as a control for inhibition of delivery and degradation of DQ-BSA via the endocytic pathway. **, P < 0.01; n.s., non significant.


C N1E - 115 cells treated with siControl , siINPP5Es , or siCHMP5 were stimulated with 100 ng / ml EGF for the indicated time periods , and then analyzed by immunoblot using anti - EGFR and anti - α - tubulin antibodies . Levels of CHMP5 mRNA72 h after transfection of N1E - 115 cells with siControl or siCHMP5 , as analyzed by RT - PCR . D Quantitation of EGFR degradation ratio in C . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 .

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C N1E-115 cells treated with siControl, siINPP5Es, or siCHMP5 were stimulated with 100 ng/ml EGF for the indicated time periods, and then analyzed by immunoblot using anti-EGFR and anti-α-tubulin antibodies. Levels of CHMP5 mRNA72 h after transfection of N1E-115 cells with siControl or siCHMP5, as analyzed by RT-PCR.D Quantitation of EGFR degradation ratio in C. Results represent means ± s.d. of three independent experiments. **, P < 0.01.


A NIE - 115 cells stably expressing mSt - 2xML1N or - 2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1 . Cells were fixed and stained with anti - LAMP1 or anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm .

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A NIE-115 cells stably expressing mSt-2xML1N or -2xPLCδ1PH were cultured in growth medium with or without 200 nM Torin1. Cells were fixed and stained with anti-LAMP1 or anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.


B NIE - 115 cells stably expressing mSt - 2xML1N treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm .

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B NIE-115 cells stably expressing mSt-2xML1N treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.


C Quantitation of signal intensities in B showing mSt - 2xML1N colocalizing with LAMP1 ( means ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 ; * , P < 0 . 05 .

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C Quantitation of signal intensities in B showing mSt-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01; *, P < 0.05.


D N1E - 115 cells stably expressing GFP - 2xML1N treated with siControl or siINPP5E were transiently transfected with mSt - INPP5E ( WT , D477N ) . Quantitation of signal intensities showing GFP - 2xML1N colocalizing with LAMP1 ( means ± s . d . ; n > 20 cells from three independent experiments ) . * * , P < 0 . 01 ; * , P < 0 . 05 .

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D N1E-115 cells stably expressing GFP-2xML1N treated with siControl or siINPP5E were transiently transfected with mSt-INPP5E (WT, D477N). Quantitation of signal intensities showing GFP-2xML1N colocalizing with LAMP1 (means ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01; *, P < 0.05.


E NIE - 115 cells stably expressing mCherry - 2xFYVE treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies , and then analyzed by immunofluorescence microscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . F Quantitation of signal intensities in E showing mCherry - 2xFYVE colocalizing with LAMP1 ( means ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 .

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E NIE-115 cells stably expressing mCherry-2xFYVE treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies, and then analyzed by immunofluorescence microscopy. Insets show the boxed areas at high magnification. Bar, 10 µm.F Quantitation of signal intensities in E showing mCherry-2xFYVE colocalizing with LAMP1 (means ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.


A N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - LAMP1 antibodies and Phalloidin , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 ( means ± s . d . ; n > 40 cells from three independent experiments ) . * * , P < 0 . 01 .

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A N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-LAMP1 antibodies and Phalloidin, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing Phalloidin colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.


B N1E - 115 cells treated with siControl or siINPP5Es were cultured in growth medium . Cells were fixed and stained with anti - phospho - Y421 cortactin and anti - LAMP1 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . Quantitation of signal intensities showing cortactin ( pY421 ) colocalizing with LAMP1 ( means ± s . d . ; n > 40 cells from three independent experiments ) . * * , P < 0 . 01 .

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B N1E-115 cells treated with siControl or siINPP5Es were cultured in growth medium. Cells were fixed and stained with anti-phospho-Y421 cortactin and anti-LAMP1 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Quantitation of signal intensities showing cortactin (pY421) colocalizing with LAMP1 (means ± s.d.; n > 40 cells from three independent experiments). **, P < 0.01.


C N1E - 115 cells were treated with 0 . 5 µM Latrunculin A ( Lat A ) in growth medium for 6 h . Cells were fixed and stained with anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Bar , 10 µm . Quantitation of the number of LC3puncta per cell . ( mean ± s . d . ; n > 100 cells from three independent experiments ) . * * , P < 0 . 01 .

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C N1E-115 cells were treated with 0.5 µM Latrunculin A (Lat A) in growth medium for 6 h. Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Bar, 10 µm. Quantitation of the number of LC3puncta per cell. (mean ± s.d.; n > 100 cells from three independent experiments). **, P < 0.01.


D NIE - 115 cells stably expressing LAMP1 - mCherry were treated with DMSO or 0 . 5 µM Lat A in growth medium for 4 h , subsequently cultured with 200 nM Torin1 and protease inhibitors ( 10 µg / ml Pepstatin A and 10 µg / ml E - 64 - d ) for 2 h ( Lat A treatment for total 6h ) . Cells were fixed and stained with anti - LC3 antibodies , and then analyzed by immunofluorescencemicroscopy . Insets show the boxed areas at high magnification . Bar , 10 µm . Percentages of colocalization of LC3 dots with LAMP1 dots ( mean ± s . d . ; n > 20 cells from three independent experiments ) . * * , P < 0 . 01 .

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D NIE-115 cells stably expressing LAMP1-mCherry were treated with DMSO or 0.5 µM Lat A in growth medium for 4 h, subsequently cultured with 200 nM Torin1 and protease inhibitors (10 µg/ml Pepstatin A and 10 µg/ml E-64-d) for 2 h (Lat A treatment for total 6h). Cells were fixed and stained with anti-LC3 antibodies, and then analyzed by immunofluorescencemicroscopy. Insets show the boxed areas at high magnification. Bar, 10 µm. Percentages of colocalization of LC3 dots with LAMP1 dots (mean ± s.d.; n > 20 cells from three independent experiments). **, P < 0.01.


B N1E - 115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf . A1 for 2 h , and analyzed by immunoblot using anti - LC3 and anti - GAPDH antibodies . The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence . C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3 - II levels in the presence and absence of Baf . A1 following normalization to the control proteinGAPDH . Results represent means ± s . d . of three independent experiments . * * , P < 0 . 01 ; * , P < 0 . 05 .

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B N1E-115 cells stably expressing the indicated mutants treated with siControl or siINPP5E were cultured in growth medium with or without 125 nM Baf.A1 for 2 h, and analyzed by immunoblot using anti-LC3 and anti-GAPDH antibodies. The mutants were resistant to mouse siINPP5E because target sequence of the siRNA is different from the human correspondent sequence.C Quantitation of protein signal intensities from immunoblots in B showing differences in LC3-II levels in the presence and absence of Baf.A1 following normalization to the control proteinGAPDH. Results represent means ± s.d. of three independent experiments. **, P < 0.01; *, P < 0.05.


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 .

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(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 .

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(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 ) .

(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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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). (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 .

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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). (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 B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). (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 .

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

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

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). (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 .

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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 .

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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 .

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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. (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 ) .

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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. (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 ) .

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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. (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 .

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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. (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 ) .

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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. (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 .

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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 .

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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 .

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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 ) .

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(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 ) .

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(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 .

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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.