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

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


( d - i ) CftrF508del mice treated with cystamine or PBS in 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 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 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 . Densitometric of blots is shown in Supplementary Information , Fig . S9 .

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


CftrF508del mice ( a - d ) ( a ) Confocal microscopy of LC3 ( top ) p62 ( bottom ) in lung tissues from CftrF508del mice . Nuclei counterstained with DAPI ( blue ) . Scale bar , 10 μm . Representative of ten PBS - treated ten NAC - treated mice . ( b ) 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 in 15 - 20 different randomly taken sections for each mouse lung for each condition ) ; means ± s . d . for three separate experiments . Asterisk , P 0 . 05 versus PBS - treated mice ; ANOVA .

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


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

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


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

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


Scnn1b - Tg mice ( e - k ) treated with NAC or PBS ( n = 10 per group ) . ( g ) FRET of TG2 - Alexa 546 after SUMO - 1 Cy5 photobleaching in lung tissues from PBS - treated WT mice 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 phospho - p42 / 44 in lung tissues from PBS - treated WT mice 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 NAC - treated Scnn1b - Tg mice . Haematoxylin . 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 ( left ) in PBS - treated NAC - treated Scnn1b - Tg mice . Nuclei counterstained with DAPI ( blue ) . Scale bar , 150 μm . Right ; number of COX - 2 - positive per mm2 of lung tissue in NAC - treated Scnn1b - treated mice - Tg PBS . Means ± s . d . Asterisk , P 0 . 05 versus mice - treated mice ; ANOVA . 1 versus PBS - treated mice ; ANOVA .

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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 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 of blot is shown in Supplementary Information , Fig . S9 .

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

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 targets by combining - IP sequencing computational approaches . ( A ) Box - plot of enrichment of RNAs identified by Mmi1 - IPs coupled to high throughput sequencing . log of average enrichments obtained from two independent - IPs is plotted . enrichment is relative to no antibody - IPs conducted in parallel of Mmi1 - IPs . mRNAs lncRNAs enriched at least two folds in both Mmi1 - IPs are shown in blue green , respectively . Crosses represent newly identified Mmi1 targets 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 targets by combining - IP sequencing computational approaches . ( D ) - IPs showing impact of Mmi1 YTH domain point mutations on Mmi1 ' s binding to ssm4 mRNA . lower part shows a Western blot monitoring level of WT mutant Mmi1 in used for - 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 targets by combining - IP sequencing computational approaches . ( E , F ) - IPs showing YTH - dependent association of Mmi1 to lncRNAs identified in ( A ) ( B ) . Average fold enrichment is shown with error bars that indicate mean average deviations for three independent experiments for ( D , E 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 showing WT mmi1 ∆ , mmi1 ∆ expressing Mmi1 - R351E or Mmi1 - R381E mutant . were taken after 24 hours of induction of sexual differentiation by growth on SPAS medium . of that underwent differentiation iodine vapor assays , conducted on corresponding patches , are shown at 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 showing of zygotes forming over time in same 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 - IPs showing 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 showing of zygotes formed over time in WT nam1 - 1 . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) ( B , E 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 level of Flag - Byr2 over first 4 hours of sexual differentiation in WT nam1 - 1 . 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 of WT ( h90 ) transformed with an empty plasmid ( Control ) nam1 - 1 transformed with either an empty plasmid ( Control ) or a plasmid expressing Byr2 ( 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 prevents nam1 read - through transcription from repressing downstream MAPKKK gene byr2 . ( A ) Northern blots showing nam1 byr2 during 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 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 prevents nam1 read - through transcription from repressing downstream MAPKKK gene byr2 . ( B ) ChIPs showing occupancy of elongating RNAPII ( RNAPII - S2P ) over nam1 - byr2 locus , in WT nam1 - 1 . RNAPII - S2P was immunoprecipitated with an antibody recognizing heptameric repeats ( present in C - terminal domain of 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 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 prevents nam1 read - through transcription from repressing downstream MAPKKK gene byr2 . ( C ) RT - qPCRs showing accumulation of nam1 read - through transcripts ( RT1 - qPCR ) nam1 lncRNAs ( RT2 - qPCR ) in with or without transcription terminator Ttef inserted at 3 ' end of nam1 ( scheme ) . Black arrow , primer used for strand - specific reverse transcription ( RT ) ; Black , location of region amplified by PCR . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E 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 prevents nam1 read - through transcription from repressing downstream MAPKKK gene byr2 . ( D ) Microscopy of , respectively , WT ( h90 ) , nam1 - 1 nam1 - 1 - Ttef , after induction of sexual differentiation for 24 hours . of sporulation iodine vapor assays are shown at bottom of . 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 prevents nam1 read - through transcription from repressing downstream MAPKKK gene byr2 . ( E ) RT - qPCRs showing accumulation of nam1 read - through transcripts in mmi1 ∆ rrp6 ∆ . Black arrow as in ( C ) . . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E 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 prevents nam1 read - through transcription from repressing downstream MAPKKK gene byr2 . ( F ) ChIPs monitoring enrichment of H3K9me2 over nam1 - byr2 locus mei4 gene in WT rrp6 ∆ . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( B , C , E 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 level of nam5 / 6 / 7 lncRNA population in mmi1 - ts3 , dcr1 ∆ , clr4 ∆ single mutant in mmi1 - ts3 dcr1 ∆ mmi1 - ts3 clr4 ∆ double mutant , at permissive ( 25ºC ) 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 accumulation of nam7 lncRNAs nam7 - L read - through transcripts , in same conditions as in ( B ) . Red arrow heads point to expected PCR products while other bands correspond to non - specific PCR products . From scheme agarose gels : black arrows , primers used for three different reverse transcriptions ; black lines red numbers , expected PCR products of three different RT - PCRs ; M , 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 of nam5 / 6 / 7 lncRNA population in double mutant rrp6 ∆ dcr1 ∆ , relative to single mutant rrp6 ∆ dcr1 ∆ . 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 - Student ' s t - .

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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 ) - 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 - Student ' s t - .

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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 - RNAi - mediated silencing of pericentromeric transcription alternate during progression of cycle . ( A ) ChIPs assessing localization of Mmi1 to pericentromeric in WT clr4 ∆ .

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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 - RNAi - mediated silencing of pericentromeric transcription alternate during progression of cycle . ( B ) ChIPs showing localization of Mmi1 ( dashed ) H3K9me2 ( black ) to pericentromeric nam5 / 6 / 7 regions during progression of cycle . synchronization was achieved by using cdc25 - ts ( see experimental procedures for more details ) . synchronization was monitored by measuring of 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 - RNAi - mediated silencing of pericentromeric transcription alternate during progression of cycle . ( C ) RT - qPCRs monitoring of nam5 / 6 / 7 ( left part ) dg ( middle part ) lncRNA populations during cycle . Average fold enrichment is shown with error bars that indicate mean average deviations ( n = 3 ) for ( A , B 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 - RNAi - mediated silencing of pericentromeric transcription alternate during progression of cycle . ( D ) RT - PCRs monitoring accumulation of nam7 lncRNAs nam7 - L read - through transcripts in G2 / M G1 / S phases from synchronized as in ( B ) . Red arrow heads point to expected PCR products . other bands are non - specific PCR products . From scheme : black arrows , primers used for reverse transcriptions ; black lines 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 association of each of p - tau biomarkers with in Aβ - negative ( A - ; blue ; n = 250 ) Aβ - positive ( A + ; red ; n = 131 ) groups . solid lines indicate regression 95 % confidence intervals for each of groups . For each group , standardized regression coefficients ( β ) P - values were computed using a linear model adjusting for sex . Additionally , we computed ' 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 box - plot comparing each of p - tau biomarker between Aβ - negative ( A - ; blue ; n = 250 ) Aβ - positive ( A + ; red ; n = 131 ) groups . Aβ positivity was defined as CSF Aβ42 / 40 < 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 changes between each p - tau biomarker as a function of CSF Aβ42 / 40 . horizontal - axes directions were inverted ; lower CSF Aβ42 / 40 reflects higher Aβ pathology . For each Aβ status group , we computed standardized regression coefficients ( β ) P - values , adjusted for sex . solid lines indicate regression 95 % confidence intervals for each of Aβ status groups . dashed green lines indicate CSF Aβ42 / 40 cutoff . Participants were also colour - coded on 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 box - plot comparing each of p - tau biomarker between Aβ negative ( A - ; blue ; n = 287 ) 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 changes between each p - tau biomarker as a function of Aβ PET Centiloids ( CL ) . standardized regression coefficients ( β ) P - values were computed using a linear model adjusting for sex . solid lines indicate regression 95 % confidence intervals . dashed green lines indicate CL12 CL30 cutoffs . Participants were also colour - coded on CSF Aβ42 / 40 ( 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 box - plots depicting comparison between each of 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 ) . box - plots depict median ( horizontal bar ) , interquartile range ( IQR , hinges ) 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 was performed to 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 ; 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.


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

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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.


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

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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 with CAOP syndrome ( 14 - year - old 1 5 - year - old 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 eosin ( H & E ) of skin biopsies from 1 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 ( ) showing keratinocyte mitochondria ( red arrow ) in 1 ( A ) in 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 of mitochondrial number in 1 ( C ) Mbtps1 - cKO mouse model ( D ) . ( C ) Left panel : n = 13 biological replicates of normal individuals , n = 10 biological replicates of 1 . Middle right panel : n = 121 biological samples of normal individuals 1 . ( D ) Left panel : n = 23 biological replicates of Mbtps1 - loxP mice ; n = 15 biological replicates of Mbtps1 - cKO mice . Middle panel right panel : n = 113 biological replicates of Mbtps1 - loxP mice 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 p . Ter1053Arg ) was diffusely localized in cytosol showed lower mitochondrial localization compared with wild - type S1P , which suggested that these variants disrupt its mitochondrial import . arrowheads indicate colocation ( yellow ) of S1P ( green ) 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 component separation showing that S1P was enriched in mitochondria . Biochemical fractionation of whole lysate ( WCL ) , cytosol ( Cyto , tubulin ) , mitochondria ( Mito , COX IV ) , lysosomes ( Lyso , LAMP1 ) , Golgi apparatus ( Golgi , GM130 ) , endoplasmic reticulum ( ER , ERp72 ) , nucleus ( Nue , Lamin B ) from HaCaT .

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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 revealed a lower expression of mutant S1P ( p . Val355Gly 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 impaired binding of mutant S1P ( p . Val355Gly p . Ter1053Arg ) to translocase of outer ( TOM ) 70 translocase of inner ( TIM ) 23 was detected by a coimmunoprecipitation ( Co - IP ) .

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 of S1P - binding . Total lysate was subjected to coimmunoprecipitation ( Co - IP ) with normal IgG or S1P antibody . purified was separated by SDS - PAGE then subjected to silver . arrows indicate bands containing S1P , ETFA ETFB . 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 ETFB in HaCaT . Total lysate was subjected to Co - IP with normal IgG , ETFA ( left panel ) or ETFB ( right panel ) antibody . interaction between S1P 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 direct interaction between S1P ETFA - ETFB was validated by a GST pull - down . Left panel : in vitro - translated S1P was pulled down by purified GST - ETFA fusion . Right panel : in vitro - translated S1P was pulled down by purified GST - ETFB fusion .

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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 of S1P , ETFA ETFB in normal controls 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 decreased of ETFA ETFB were observed in skin biopsy of 1 . Quantitative RT - PCR ( upper panel , n = 6 biological replicates of normal controls ; n = 6 technical replicates of 1 ) immunohistochemistry ( lower panel , n = 6 biological replicates ; n = 3 technical replicates ) of S1P , ETFA ETFB .

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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 of S1P , ETFA ETFB in both Mbtps1 - conditional knockout ( cKO ) 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 ETFA ETFB in vivo . Quantitative RT - PCR ( upper panel , n = 6 biological replicates ) immunohistochemistry ( lower panel , n = 6 biological replicates ) of S1P , ETFA ETFB in Mbtps1 - cKO mice 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 showed that MBTPS1 knockout induced rapid degradation of ETFA ETFB in HaCaT . Left panel : Representative western blotting of ETFA ETFB during CHX chase . Right panel : of immunoblotting results corresponding to 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 p . Ter1053Arg ) only weakly interacted with ETFA ETFB . We constructed Flag - tagged wild - type mutant S1P HaCaT lines then performed a Co - IP with Flag antibody , interaction between S1P 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 p . Ter1053Arg ) , decreased association between ETFA ETFB . We performed a Co - IP with an ETFA antibody , interaction between ETFA 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.


H Wild - type S1P , but not mutant S1P ( p . Val355Gly p . Ter1053Arg ) , enhanced incorporation of FAD into ETF . visible spectra of flavin show two shoulder peaks at 420 nm 460 nm , indicating incorporation of FAD in ETF . In presence of wild - type S1P , two peaks were shifted by 0 . 02 OD units , whereas additional mutant S1P ( p . Val355Gly p . Ter1053Arg ) weakly shifted two peaks by 0 . 005 - 0 . 01 OD units .

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H Wild-type S1P, but not mutant S1P (p.Val355Gly and p.Ter1053Arg), enhanced the incorporation of FAD into the ETF complex. The visible spectra of flavin show two shoulder peaks at 420 nm and 460 nm, indicating the incorporation of FAD in the ETF complex. In the presence of the wild-type S1P protein, the two peaks were shifted by 0.02 OD units, whereas the additional mutant S1P (p.Val355Gly and p.Ter1053Arg) weakly shifted the two peaks by 0.005-0.01 OD units.


Mitochondrial respiration ( OCR ) in control ( Ctrl ) MBTPS1 - knockout ( KO ) HaCaT was quantified in real time using a Seahorse extracellular flux analyser . Right subpanels : Quantiﬁcation of basal respiration , maximal respiration , OCR - coupled ATP production in mitochondrial respiration ( n = 5 biological replicates ) .

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Mitochondrial respiration (OCR) in control (Ctrl) and MBTPS1-knockout (KO) HaCaT cells was quantified in real time using a Seahorse extracellular flux analyser. Right subpanels: Quantiﬁcation of basal respiration, maximal respiration, and OCR-coupled ATP production in mitochondrial respiration (n = 5 biological replicates).


B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation ( OXPHOS ) glycolysis in Ctrl MBTPS1 - KO HaCaT ( n = 4 biological replicates ) .

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B Quantiﬁcation of relevant metabolites in mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).


C Nonmitochondrial respiration ( ECAR ) was quantified in real time using a Seahorse extracellular flux analyser . of non - glycolytic acidification , glycolysis , glycolytic capacity , glycolytic reserve in Ctrl MBTPS1 - KO HaCaT ( n = 4 biological replicates ) .

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C Nonmitochondrial respiration (ECAR) was quantified in real time using a Seahorse extracellular flux analyser. Quantification of non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve in Ctrl and MBTPS1-KO HaCaT cells (n = 4 biological replicates).


D MBTPS1 knockout led to a decrease in global ATP production in HaCaT . Control MBTPS1 - KO HaCaT were cultured in 6 - well dishes . A standard curve was generated to calculate sample ATP concentrations using an ATP Lite Luminescence Kit ( n = 4 biological replicates ) .

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D MBTPS1 knockout led to a decrease in global ATP production in HaCaT cells. Control and MBTPS1-KO HaCaT cells were cultured in 6-well dishes. A standard curve was generated to calculate the sample ATP concentrations using an ATP Lite Luminescence Assay Kit (n = 4 biological replicates).


E MBTPS1 knockout significantly increased generation of mitochondrial reactive oxygen species ( Mito SOX ) in HaCaT . Left panel : Representative immunofluorescence of Mito SOX in HaCaT . Scale bars : 100 µm . Right panel : immunofluorescence was quantified to calculate relative Mito ROS level in HaCaT ( n = 4 biological replicates ) .

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E MBTPS1 knockout significantly increased the generation of mitochondrial reactive oxygen species (Mito SOX) in HaCaT cells. Left panel: Representative immunofluorescence images of Mito SOX in HaCaT cells. Scale bars: 100 µm. Right panel: The immunofluorescence intensity was quantified to calculate the relative Mito ROS level in HaCaT cells (n = 4 biological replicates).


A S1P deficiency - induced decrease in ETFA ETFB was significantly reversed by riboflavin ( Rib ) in a - dependent manner in MBTPS1 - KO HaCaT . GAPDH was used as a loading control . MBTPS1 - KO were initially cultured in DMEM for 24 h supplemented with 0 , 2 . 5 , 5 , or 10 µM riboflavin for 3 days . expression of ETFA ETFB was then detected by immunoblotting .

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A The S1P deficiency-induced decrease in ETFA and ETFB was significantly reversed by riboflavin (Rib) in a concentration-dependent manner in MBTPS1-KO HaCaT cells. GAPDH was used as a loading control. MBTPS1-KO cells were initially cultured in DMEM for 24 h and supplemented with 0, 2.5, 5, or 10 µM riboflavin for 3 days. The expression of ETFA and ETFB was then detected by immunoblotting.


B S1P deficiency - induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation ETFA / B overexpression in HaCaT . Left panel : Mitochondrial respiration ( OCR ) was quantified in real time using a Seahorse extracellular flux analyser .

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B The S1P deficiency-induced abnormalities in mitochondrial respiration can be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells. Left panel: Mitochondrial respiration (OCR) was quantified in real time using a Seahorse extracellular flux analyser.


C decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation ETFA / B overexpression in HaCaT ( n = 4 biological replicates ) .

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C The decreased global ATP production caused by S1P deficiency could be significantly reversed by riboflavin supplementation and ETFA/B overexpression in HaCaT cells (n = 4 biological replicates).


D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation . representative head image of 1 before therapy is shown in Figure 1A ( panel 1 ) .

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D Inflammatory lesions in CAOP syndrome were significantly improved by riboflavin supplementation. The representative head image of patient 1 before therapy is shown in Figure 1A (panel 1).


( a ) Activation of gB - specific CD8 + T hybridoma ( which expresses β - galactosidase as an indicator of T activation ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , then incubated for 12 h at 37 ° C with hybridoma . A595 , absorbance at 595 nm .

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(a) Activation of the gB-specific CD8+ T cell hybridoma (which expresses β-galactosidase as an indicator of T cell activation) by macrophages infected for various times (horizontal axis) with HSV-1, then incubated for 12 h at 37 °C with the hybridoma. A595, absorbance at 595 nm.


b ) Activation of hybridoma as described in a , with addition of dimethyl sulfoxide ( DMSO ; negative control ) , bafilomycin A ( Baf ) , brefeldin A ( BFA ) or MG - 132 at 2 h after macrophage infection .

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b) Activation of the hybridoma as described in a, with the addition of dimethyl sulfoxide (DMSO; negative control), bafilomycin A (Baf), brefeldin A (BFA) or MG-132 at 2 h after macrophage infection.


( c ) Activation of hybridoma as described in a , with addition of bafilomycin A at 2 h after macrophage infection .

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(c) Activation of the hybridoma as described in a, with the addition of bafilomycin A at 2 h after macrophage infection.


( d ) Immunofluorescence microscopy of gB ( blue ) LAMP - 1 ( red ) in HSV - 1 - infected macrophages ; pink indicates colocalization . Original magnification , × 40 .

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(d) Immunofluorescence microscopy of gB (blue) and LAMP-1 (red) in HSV-1-infected macrophages; pink indicates colocalization. Original magnification, × 40.


( e ) CD8 + T - stimulatory capacity ( as described in a ) of uninfected macrophages ( Mock ) , of BMA ( BMA - HSV ) or J774 ( J774 - HSV ) macrophages infected for 8 h with HSV - 1 , of cocultures of J774 macrophages ( H - 2d ) infected for 1 h with HSV - 1 , then mixed with uninfected BMA ( H - 2b ) macrophages at a of 1 : 1 cultured together for 8 h ( J774 - HSV + BMA ) .

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(e) CD8+ T cell-stimulatory capacity (as described in a) of uninfected macrophages (Mock), of BMA (BMA-HSV) or J774 (J774-HSV) macrophages infected for 8 h with HSV-1, and of cocultures of J774 macrophages (H-2d) infected for 1 h with HSV-1, then mixed with uninfected BMA (H-2b) macrophages at a ratio of 1:1 and cultured together for 8 h (J774-HSV + BMA).


( a ) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2 - 8 h ( left margin ) with HSV - 1 . Original magnification , × 20 .

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(a) Immunofluorescence microscopy of LC3 expression in macrophages infected for 2-8 h (left margin) with HSV-1. Original magnification, × 20.


( b ) Activation of gB - specific CD8 + T hybridoma ( described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with HSV - 1 , with ( DMEM + 3 - MA ) or without ( DMEM ) addition of 3 - methyladenine 2 h after infection , then incubated for 12 h at 37 ° C with hybridoma .

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(b) Activation of the gB-specific CD8+ T cell hybridoma (described in Fig. 1a) by macrophages infected for various times (horizontal axis) with HSV-1, with (DMEM + 3-MA) or without (DMEM) the addition of 3-methyladenine 2 h after infection, then incubated for 12 h at 37 °C with the hybridoma.


( c ) Activation of hybridoma ( as described in b ) by macrophages transfected for 60 h with control ( Ctrl ) siRNA or Atg5 - specific siRNA , then infected for 8 h or 12 h with HSV - 1 .

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(c) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control (Ctrl) siRNA or Atg5-specific siRNA, then infected for 8 h or 12 h with HSV-1.


( d ) Immunoblot of Atg - 5 in siRNA - treated macrophages .

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(d) Immunoblot analysis of Atg-5 in siRNA-treated macrophages.


( e ) Activation of hybridoma ( as described in b ) by macrophages infected with HSV - 1 incubated at 37 ° C ( Basal ) , incubated for 12 h at 39 ° C before being infected with HSV - 1 ( heat shock ( HS ) ) , or treated with rapamycin during HSV - 1 infection ( Rapa ) , with ( + Baf ) or without addition of bafilmycin A at 2 h after infection .

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(e) Activation of the hybridoma (as described in b) by macrophages infected with HSV-1 and incubated at 37 °C (Basal), incubated for 12 h at 39 °C before being infected with HSV-1 (heat shock (HS)), or treated with rapamycin during HSV-1 infection (Rapa), with (+ Baf) or without the addition of bafilmycin A at 2 h after infection.


( f ) Activation of hybridoma ( as described in b ) by macrophages transfected for 60 h with control siRNA or Atg5 - specific siRNA , then infected for 8 h with HSV - 1 , with ( + ) or without ( − ) addition of rapamycin at 2 h after infection . Results in b , c , e , f are normalized to results obtained for CD8 + T stimulated with macrophages infected for 8 h at 37 ° C without further treatment ( b , e ) or infected macrophages treated with control siRNA ( c , f ) are presented in arbitrary units . Data are representative of three independent experiments ( a , d ) or are from three independent experiments ( mean s . e . m . of triplicate samples ; b , c , e , f ) .

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(f) Activation of the hybridoma (as described in b) by macrophages transfected for 60 h with control siRNA or Atg5-specific siRNA, then infected for 8 h with HSV-1, with (+) or without (−) the addition of rapamycin at 2 h after infection. Results in b,c,e,f are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 8 h at 37 °C without further treatment (b,e) or infected macrophages treated with control siRNA (c,f) and are presented in arbitrary units. Data are representative of three independent experiments (a,d) or are from three independent experiments (mean and s.e.m. of triplicate samples; b,c,e,f).


( a - c ) Immunofluorescence microscopy of uninfected macrophages incubated at 37 ° C ( control ; a ) , subjected to mild heat shock ( b ) or treated with rapamycin ( c ) , then stained with anti - LC3 .

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(a-c) Immunofluorescence microscopy of uninfected macrophages incubated at 37 °C (control; a), subjected to mild heat shock (b) or treated with rapamycin (c), then stained with anti-LC3.


d ) Immunofluorescence microscopy of expression of LC3 , gB GFP ( VP26 ) by macrophages infected for various times ( left margin ) with HSV - 1 . indicates colocalization .

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d) Immunofluorescence microscopy of the expression of LC3, gB and GFP (VP26) by macrophages infected for various times (left margin) with HSV-1. White indicates colocalization.


( e ) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h ( left margin ) with wild - type HSV - 1 ( WT ) or HSV - 1 lacking ICP34 . 5 ( Δ34 . 5 ) . Blue , of nuclei with DAPI ( 4 , 6 - diamidino - 2 - phenylindole ) . Original magnification , × 100 ( a - c ) or × 63 ( d , e ) . Results in a - e are representative of three independent experiments .

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(e) Immunofluorescence microscopy of macrophages infected for 6 h or 8 h (left margin) with wild-type HSV-1 (WT) or HSV-1 lacking ICP34.5 (Δ34.5). Blue, staining of nuclei with DAPI (4,6-diamidino-2-phenylindole). Original magnification, × 100 (a-c) or × 63 (d,e). Results in a-e are representative of three independent experiments.


( f ) Activation of gB - specific CD8 + T hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( horizontal axis ) with wild - type HSV - 1 17 + ( WT 17 + ) or Δ34 . 5 HSV - 1 . Data are from three independent experiments ( mean s . e . m . of triplicate samples ) .

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(f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (horizontal axis) with wild-type HSV-1 strain 17+ (WT 17+) or Δ34.5 HSV-1. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


( g , h ) Immunoblot ( IB ; g ) flow cytometry ( h ) of expression of HSV - 1 ( g ) gB ( h ) in macrophages infected for various times ( above lanes ( g ) or plots ( h ) ) with wild - type or Δ34 . 5 HSV - 1 . Ctrl , control ( uninfected BMA macrophages ; h ) . Data are representative of two ( g ) or three ( h ) independent experiments .

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(g,h) Immunoblot analysis (IB; g) and flow cytometry (h) of the expression of HSV-1 proteins (g) and gB (h) in macrophages infected for various times (above lanes (g) or plots (h)) with wild-type or Δ34.5 HSV-1. Ctrl, control (uninfected BMA macrophages; h). Data are representative of two (g) or three (h) independent experiments.


( i ) Activation of gB - specific CD8 + T hybridoma ( as described in Fig . 1a ) by macrophages infected for various times ( below graph ) with wild - type or Δ34 . 5 HSV - 1 , with ( + ) or without ( - ) addition of bafilomycin A at 2 h after infection . Results in f , i are normalized to results obtained for CD8 + T stimulated with macrophages infected for 6 h with wild - type ( f ) or with infected macrophages incubated without bafilomycin ( i ) are presented in arbitrary units . Data are from three independent experiments ( mean s . e . m . of triplicate samples ) .

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(i) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages infected for various times (below graph) with wild-type or Δ34.5 HSV-1, with (+) or without (-) the addition of bafilomycin A at 2 h after infection. Results in f,i are normalized to results obtained for CD8+ T cells stimulated with macrophages infected for 6 h with wild-type virus (f) or with infected macrophages incubated without bafilomycin (i) and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Electron microscopy of macrophages 10 h after infection with HSV - 1 . ( a ) Arrows indicate - coiled structures emerging from nucleus of an infected . ( b - d ) Four - layered structures formed by coiling of . ( e , f ) Glucose - 6 - phosphatase ( black deposits ) on autophagosome - like structures emerging from envelope or free in cytoplasm , viral capsids in cytoplasm engulfed in lumen of an autophagosome - like compartment . N , nucleus ; VP , viral particles . Scale bars , 1 μm ( a ) , 0 . 25 μm ( b - d , f ) or 0 . 4 μm ( e ) . are representative of three independent experiments with at least 100 profiles in each .

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Electron microscopy of macrophages 10 h after infection with HSV-1. (a) Arrows indicate membrane-coiled structures emerging from the nucleus of an infected cell. (b-d) Four-layered membrane structures formed by coiling of the nuclear membrane. (e,f) Glucose-6-phosphatase (black deposits) on autophagosome-like structures emerging from the nuclear envelope or free in the cytoplasm, and viral capsids in the cytoplasm engulfed in the lumen of an autophagosome-like compartment. N, nucleus; VP, viral particles. Scale bars, 1 μm (a), 0.25 μm (b-d,f) or 0.4 μm (e). Images are representative of three independent experiments with at least 100 cell profiles in each.


Immunoelectron microscopy of macrophages 10 h after infection with HSV - 1 . ( a - c ) Accumulation of LC3 ( a , b ) gB ( c ) . ( d ) Fusion of four - layered structures lysosomes preloaded with bovine serum albumin - gold ( BSA - gold ; black dots ) . Original magnification , × 54 , 800 ( a , b ) , × 69 , 000 ( c ) or × 38 , 000 ( d ) . are representative of three ( a - c ) or two ( d ) independent experiments .

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Immunoelectron microscopy of macrophages 10 h after infection with HSV-1. (a-c) Accumulation of LC3 (a,b) and gB (c). (d) Fusion of four-layered membrane structures and lysosomes preloaded with bovine serum albumin-gold (BSA-gold; black dots). Original magnification, × 54,800 (a,b), × 69,000 (c) or × 38,000 (d). Images are representative of three (a-c) or two (d) independent experiments.


( a ) Activation of gB - specific CD8 + T hybridoma ( as described in Fig . 1a ) by macrophages exposed to DMSO ( negative control ) , mild heat shock , IL - 1β or IFN - γ .

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(a) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages exposed to DMSO (negative control), mild heat shock, IL-1β or IFN-γ.


( b ) Dansylcadaverin of untreated macrophages ( Basal ) or macrophages exposed to mild heat shock , IFN - γ or IL - 1β infected for 8 h with wild - type HSV - 1 , normalized to signal obtained in basal conditions presented in arbitrary units .

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(b) Dansylcadaverin staining of untreated macrophages (Basal) or macrophages exposed to mild heat shock, IFN-γ or IL-1β and infected for 8 h with wild-type HSV-1, normalized to the signal obtained in basal conditions and presented in arbitrary units.


( c - f ) Activation of gB - specific CD8 + T hybridoma ( as described in Fig . 1a ) by macrophages incubated at 37 ° C ( c ) or exposed to mild heat shock ( d ) , IL - 1β ( e ) or IFN - γ ( f ) infected for 8 h with wild - type HSV - 1 with addition of DMSO ( negative control ) , 3 - methyladenine , bafilomycin , brefeldin A or MG - 132 at 2 h after infection . Results in a , c - f are normalized to results obtained for CD8 + T stimulated with macrophages incubated with DMSO in each condition are presented in arbitrary units . Data are from three independent experiments ( mean s . e . m . of triplicate samples ) .

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(c-f) Activation of the gB-specific CD8+ T cell hybridoma (as described in Fig. 1a) by macrophages incubated at 37 °C (c) or exposed to mild heat shock (d), IL-1β (e) or IFN-γ (f) and infected for 8 h with wild-type HSV-1 with the addition of DMSO (negative control), 3-methyladenine, bafilomycin, brefeldin A or MG-132 at 2 h after infection. Results in a,c-f are normalized to results obtained for CD8+ T cells stimulated with macrophages incubated with DMSO in each condition and are presented in arbitrary units. Data are from three independent experiments (mean and s.e.m. of triplicate samples).


Confirmation of peptide entry into neurons . Cultures were incubated with Bio - TMyc ( 25 µM , 1 h ) ( panel b ) or left untreated ( panel a ) . Arrowheads highlight peptide permeability , detected by fluorescein Avidin D ( green ) , into neurons labeled with neuronal - specific antibody NeuN ( red ) . Peptide is not detected in some neurons ( arrow ) non - neuronal ( asterisk ) . Confocal microscopy correspond to single sections are representative of five independent experiments . Scale bar , 10 μm .

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Confirmation of peptide entry into neurons. Cultures were incubated with Bio-TMyc (25 µM, 1 h) (panel b) or left untreated (panel a). Arrowheads highlight peptide permeability, detected by fluorescein Avidin D (green), into neurons labeled with neuronal-specific antibody NeuN (red). Peptide is not detected in some neurons (arrow) and non-neuronal cells (asterisk). Confocal microscopy images correspond to single sections and are representative of five independent experiments. Scale bar, 10 μm.


Effect of TMyc on neuronal survival . cultures were incubated with TMyc ( 5 , 15 or 25 μM , 30 min ) subjected to treatment with NMDA ( 100 µM ) glycine ( 10 µM ) for 4 h . Specific neuronal viability was established expressed relative to values in cultures with no treatment . Means ± are represented ( n = 8 ) statistical was performed by ANOVA followed by post - hoc Tukey HSD .

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Effect of TMyc on neuronal survival. Primary cultures were incubated with TMyc (5, 15 or 25 μM, 30 min) and subjected to treatment with NMDA (100 µM) and glycine (10 µM) for 4 h. Specific neuronal viability was established and expressed relative to values in cultures with no treatment. Means ± SEM are represented (n = 8) and statistical analysis was performed by ANOVA test followed by post-hoc Tukey HSD test.


Effect of TFL457 , TFL482 TFL541 on TrkB - FL . Cultures preincubated with TrkB - FL peptides or TMyc ( 25 µM , 30 min ) , followed by chronic NMDA treatment ( 2 h ) , were compared to without peptide . of full - ( FL ) TrkB were established with panTrkB , an antibody for extracellular domain that also detects truncated forms ( tTrkB ) . Quantitation of peptide interference of TrkB - FL downregulation . Receptor were normalized to NSE expressed relative to values obtained in cultures without peptide or NMDA . Means ± are represented was performed by ANOVA followed by post - hoc Tukey HSD ( * p = 0 . 02 ; n = 4 ) .

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Effect of TFL457, TFL482 and TFL541 on TrkB-FL levels. Cultures preincubated with TrkB-FL peptides or TMyc (25 µM, 30 min), followed by chronic NMDA treatment (2 h), were compared to cells without peptide. Levels of full-length (FL) TrkB were established with panTrkB, an antibody for the extracellular domain that also detects the truncated forms (tTrkB). Quantitation of peptide interference of TrkB-FL downregulation. Receptor levels were normalized to NSE and expressed relative to values obtained in cultures without peptide or NMDA. Means ± SEM are represented and analysis was performed by ANOVA test followed by post-hoc Tukey HSD test (*p = 0.02; n = 4).


Effect of TFL457 , TFL482 TFL541 on neuronal viability after chronic excitotoxicity . Cultures were preincubated with peptides treated with NMDA for 0 - 6 h as before . Values for each time point were represented relative to those of neurons incubated with same peptide but no NMDA . Mean ± of eleven ( TFL457 ) , three ( TFL482 ) seven ( TFL541 ) independent experiments is given .

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Effect of TFL457, TFL482 and TFL541 on neuronal viability after chronic excitotoxicity. Cultures were preincubated with peptides and treated with NMDA for 0-6 h as before. Values for each time point were represented relative to those of neurons incubated with the same peptide but no NMDA. Mean ± SEM of eleven (TFL457), three (TFL482) and seven (TFL541) independent experiments is given.


Effect of TFL518 on TrkB - FL . Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457 .

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Effect of TFL518 on TrkB-FL levels. Cultures incubated with TFL518 as before were compared to those treated with TMyc or TFL457.


TFL518 effect on neuronal viability . Cultures were treated analyzed as above . No significant differences were found for TFL518 while neuroprotection by TFL457 was

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TFL518 effect on neuronal viability. Cultures were treated and analyzed as above. No significant differences were found for TFL518 while neuroprotection by TFL457 was confirmed


Effect of TFL457 on neuronal viability after acute excitotoxicity . were induced with NMDA ( 50 μM ) glycine ( 10 μM ) for 1 h , after agonists removal , culture proceeded for 20 h with DL - AP5 ( 200 μM ) TMyc or TFL457 ( 15 μM ) . Mean ± relative to cultures incubated with TMyc but no NMDA is represented .

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Effect of TFL457 on neuronal viability after acute excitotoxicity. Cells were induced with NMDA (50 μM) and glycine (10 μM) for 1 h and, after agonists removal, culture proceeded for 20 h with DL-AP5 (200 μM) and TMyc or TFL457 (15 μM). Mean ± SEM relative to cultures incubated with TMyc but no NMDA is represented.


Effect of TFL457 on Y515 Y816 phosphorylation . Cultures preincubated with TMyc or TFL457 ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) analyzed with panTrkB or phosphospecific antibodies . Quantitation of pY515 pY816 . Mean ± of normalized pY515 ( n = 7 ) or pY816 ( n = 4 ) obtained after NMDA - treatment ( 2 h ) are represented relative to those found in with same peptide but no NMDA . Statistical was performed by unpaired Student t ( * p = 0 . 046 ; n . s . = non - significant ) .

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Effect of TFL457 on Y515 and Y816 phosphorylation. Cultures preincubated with TMyc or TFL457 (25 μM, 30 min) were treated with NMDA (0-6 h) and analyzed with panTrkB or phosphospecific antibodies. Quantitation of pY515 and pY816. Mean ± SEM of normalized pY515 (n = 7) or pY816 (n = 4) levels obtained after NMDA-treatment (2 h) are represented relative to those found in cells with the same peptide but no NMDA. Statistical analysis was performed by unpaired Student t test (*p = 0.046; n.s. = non-significant).


by immunoprecipitation of TFL457 effect on pY816 . Cultures preincubated treated as before with NMDA ( 2 h ) , were immunoprecipitated with antibodies specific for phosphorylated tyrosine ( pY ) . pY816 was analyzed by WB in immunoprecipates .

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Analysis by immunoprecipitation of TFL457 effect on pY816 levels. Cultures preincubated and treated as before with NMDA (2 h), were immunoprecipitated with antibodies specific for phosphorylated tyrosine (pY). pY816 was analyzed by WB in immunoprecipates.


TFL457 effects on neuronal viability after inhibition of TrkB - FL signaling . Cultures were preincubated ( 30 min ) with inhibitors specific for PI3K ( Wortmannin , 100 nM ) , MAPK / ERK ( UO126 , 300 nM ) or PLCγ ( U - 73122 , 5 μM ) before incubation with TMyc or TFL457 ( 25 μM , 30 min ) . Viability was established 4 h after NMDA treatment . Means ± relative to untreated cultures are represented data were analyzed by ANOVA followed by post - hoc Tukey HSD ( * * * p = 0 . 0001 , * * p = 0 . 009 , * p = 0 . 02 , n . s . = non - significant , respectively for TMyc vs TFL457 in untreated or Wortmannin , UO126 or U - 73122 - treated ; * * p = 0 . 008 for untreated vs U - 73122 - treated preincubated with TFL457 ; n = 5 ) .

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TFL457 effects on neuronal viability after inhibition of TrkB-FL signaling. Cultures were preincubated (30 min) with inhibitors specific for PI3K (Wortmannin, 100 nM), MAPK/ERK (UO126, 300 nM) or PLCγ (U-73122, 5 μM) before incubation with TMyc or TFL457 (25 μM, 30 min). Viability was established 4 h after NMDA treatment. Means ± SEM relative to untreated cultures are represented and data were analyzed by ANOVA test followed by post-hoc Tukey HSD test (***p = 0.0001, **p = 0.009, *p = 0.02, n.s. = non-significant, respectively for TMyc vs TFL457 in untreated or Wortmannin, UO126 or U-73122-treated cells; **p = 0.008 for untreated vs U-73122-treated cells preincubated with TFL457; n = 5).


Dose - dependent TFL457 effects . Cultures incubated with TMyc o TFL457 ( 5 , 15 o 25 μM , 30 min ) were treated with NMDA ( 2 h ) compared to without peptide or NMDA .

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Dose-dependent TFL457 effects. Cultures incubated with TMyc o TFL457 (5, 15 o 25 μM, 30 min) were treated with NMDA (2 h) and compared to cells without peptide or NMDA.


Time - course TFL457 effects . preincubated with peptides ( 25 μM , 30 min ) were treated with NMDA ( 0 - 6 h ) .

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Time-course TFL457 effects. Cells preincubated with peptides (25 μM, 30 min) were treated with NMDA (0-6 h).


Quantitation of peptide effects . Normalized are presented relative to values obtained in incubated with same peptide but no NMDA . Means ± are given . Results were analyzed by a two - way ANOVA followed by post - hoc Bonferroni , comparing TMyc or TFL457 - treated for each time - point ( TrkB - FL , * * * p = 0 . 0009 * p = 0 . 02 ; CREB , * p = 0 . 0149 * p = 0 . 0148 ; pCREB , * * * p = 0 . 0007 * * p = 0 . 0012 ; MEF2D , * * p = 0 . 0023 * * p = 0 . 0047 . values are respectively for 2 or 4 h ; n = 6 ) .

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Quantitation of peptide effects. Normalized protein levels are presented relative to values obtained in cells incubated with the same peptide but no NMDA. Means ± SEM are given. Results were analyzed by a two-way ANOVA test followed by post-hoc Bonferroni test, comparing TMyc or TFL457-treated cells for each time-point (TrkB-FL, ***p = 0.0009 and *p = 0.02; CREB, *p = 0.0149 and *p = 0.0148; pCREB, ***p = 0.0007 and **p = 0.0012; MEF2D, **p = 0.0023 and **p = 0.0047. All values are respectively for 2 or 4 h; n = 6).


Effect of TFL457 on MEF2 - promoter activity . Cultures transfected with pMEF2 ( two minimal wild type MEF2 elements ) or pMEF2mut ( mutant ) were preincubated with CPPs ( 25 μM , 30 min ) treated with NMDA ( 100 µM , 2 h ) . Means ± of luciferase activities obtained in excitotoxicity , relative to values found in treated with same peptide no NMDA , are presented .

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Effect of TFL457 on MEF2-promoter activity. Cultures transfected with pMEF2 (two minimal wild type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 µM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented.


Effect on CRE - promoter activity . pCRE contains two minimal CREs . Peptide preincubation was as above with or without KG - 501 ( 10 µM ) . Mean ± luciferase activities are given relative to values in treated with same peptide but no NMDA or KG - 501 .

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Effect on CRE-promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG-501 (10 µM). Mean ± SEM luciferase activities are given relative to values in cells treated with the same peptide but no NMDA or KG-501.


Effect on NMDAR - subunit promoters . transfected with pGluN1 or pGluN2A were treated analyzed as in panel A ( pGluN1 , * p = 0 . 016 ; pGluN2A , * p = 0 . 029 ; n = 14 ) .

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Effect on NMDAR-subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel A (pGluN1, *p = 0.016; pGluN2A, *p = 0.029; n = 14).