( A , B ) Comparative solvent side views of the small subunit with the bacterial SD - chamber on the 30S from the 70S POST state ( PDB 2HGR ) ( Yusupova et al , 2006 ) and the corresponding region of the eukaryotic 40S in the classical 80S * HCV IRES complex . Analogous proteins and rRNA helices are highlighted in the same colours , respectively . ( A ) Crystal structure of the 30S subunit with mRNA . The SD - helix is established between the mRNA ( orange ) and the 3 ' end of the 16S rRNA ( blue ) . ( B ) Atomic model of the 40S subunit with the HCV IRES model ( pink ) . The mRNA - part and S2 of the IRES are shown in orange and cyan , the 3 ' - end of the 18S rRNA is shown in blue .

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

(A, B) Comparative solvent side views of the small subunit with the bacterial SD-chamber on the 30S from the 70S POST state (PDB 2HGR) (Yusupova et al, 2006) and the corresponding region of the eukaryotic 40S in the classical 80S*HCV IRES complex. Analogous proteins and rRNA helices are highlighted in the same colours, respectively. (A) Crystal structure of the 30S subunit with mRNA. The SD-helix is established between the mRNA (orange) and the 3' end of the 16S rRNA (blue).(B) Atomic model of the 40S subunit with the HCV IRES model (pink). The mRNA-part and S2 of the IRES are shown in orange and cyan, the 3'-end of the 18S rRNA is shown in blue.


( C ) Overview and zoomed view of Classical 80S * HCVIRES . The cryo - EM map of the Classical 80S * HCVIRES was filtered according to resolution . ( D and E ) mRNA exit channel from in Classical 80S * HCVIRES and POST state ( PDB 5AJ0 ) ( Behrmann et al , 2015 ) . The two structures are aligned at the 40S body . We note eS28 as part of the 40S head is differently located because of the head tilt . The surface representation was created using the pdb models . Shown are the 18S rRNA ( yellow ) , ribosomal proteins ( gray ) , eS26 ( cyan ) eS28 ( purple ) , HCVIRES ( pink ) .

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

(C) Overview and zoomed view of Classical 80S*HCVIRES. The cryo-EM map of the Classical 80S*HCVIRES was filtered according to resolution.(D and E) mRNA exit channel from in Classical 80S*HCVIRES and POST state (PDB 5AJ0) (Behrmann et al, 2015). The two structures are aligned at the 40S body. We note eS28 as part of the 40S head is differently located because of the head tilt. The surface representation was created using the pdb models. Shown are the 18S rRNA (yellow), ribosomal proteins (gray), eS26 (cyan) eS28 (purple), HCVIRES (pink).


( A ) Comparison of HCV IRES from 80S * HCV IRES Classical ( pink ) and tRNAs from POST state ( gray ) ( PDB 5AJ0 ) ( Behrmann et al , 2015 ) in a common 40S body alignment of the cryo - EM maps . ( B ) Same as above but in a common 40S head alignment .

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

(A) Comparison of HCV IRES from 80S*HCV IRES Classical (pink) and tRNAs from POST state (gray)(PDB 5AJ0) (Behrmann et al, 2015) in a common 40S body alignment of the cryo-EM maps.(B) Same as above but in a common 40S head alignment.


( C ) Comparison of the 40S subunit and bound ligands of the classical 80S * HCVIRES complex ( orange ) and 80S * HCVIRES * Met - tRNA * eIF5B * GMPPNP complex ( POST - like state ) ( yellow ) ( EMD 2683 ) ( Yamamoto et al , 2014 ) in common 60S alignment . ( D ) The models of the 80S * HCVIRES Classical complex ( gray ) , 80S * HCVIRES * Met - tRNA * eIF5B * GMPPNP complex ( POST - like state ) ( green ) ( PDB 4UPZ ) ( Yamamoto et al , 2014 ) and 80S * HCVIRES Classical / no head tilt complex ( pink ) are rigid body fitted into the corresponding densities in a common 40S body alignment . The Helix 68 from the 28S rRNA is depicted as well ( blue ) .

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

(C) Comparison of the 40S subunit and bound ligands of the classical 80S*HCVIRES complex (orange) and 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (yellow)(EMD 2683) (Yamamoto et al, 2014) in common 60S alignment.(D) The models of the 80S*HCVIRES Classical complex (gray), 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (green)(PDB 4UPZ) (Yamamoto et al, 2014) and 80S*HCVIRES Classical/no head tilt complex (pink) are rigid body fitted into the corresponding densities in a common 40S body alignment. The Helix 68 from the 28S rRNA is depicted as well (blue).


( A and B ) Comparison of the 80S * HCV IRES Classical complex and Classical / no head tilt complex ( 40S yellow , 60S blue , IRES pink , P - tRNA green ) at intersubunit view . Inter subunit bridge 1b / c and 1a are indicated as B1b / c and B1a , respectively . The cryo - EM map of classical 80S * HCV IRES classical complex was filtered to 6 Å . ( C and D ) Close up of the mRNA tunnel . The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å

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

(A and B) Comparison of the 80S*HCV IRES Classical complex and Classical/no head tilt complex (40S yellow, 60S blue, IRES pink, P-tRNA green) at intersubunit view. Inter subunit bridge 1b/c and 1a are indicated as B1b/c and B1a, respectively. The cryo-EM map of classical 80S*HCV IRES classical complex was filtered to 6 Å.(C and D) Close up of the mRNA tunnel. The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å units


, Total protein levels of Munc18 - 1 . HEK293T cells transfected with WT or mutant Munc18 - 1 variants and either GFP , syntaxin - 11 - 264 or syntaxin - 11 - 180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins , normalized to β - actin ( Synt - 1A = syntaxin - 1A ) . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test , followed by Dunn ' s multiple comparison test ; n = 11 independent experiments ; exact p values are shown in Appendix Table S1 ) .

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

, Total protein levels of Munc18-1. HEK293T cells transfected with WT or mutant Munc18-1 variants and either GFP, syntaxin-11-264 or syntaxin-11-180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins, normalized to β-actin (Synt-1A = syntaxin-1A). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 by Kruskal-Wallis test, followed by Dunn's multiple comparison test; n = 11 independent experiments; exact p values are shown in Appendix Table S1).


Solubility of Munc18 - 1 . HEK293T cells transfected as in ( C ) were solubilized in 0 . 1 % Triton X - 100 ( TX ) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting . TX - soluble Munc18 - 1 was measured as percent of total Munc18 - 1 by quantitative immunoblotting . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test followed by Dunn ' s multiple comparison test , or by one - way ANOVA followed by Bonferroni post - hoc test ; n = 5 - 9 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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

Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).


Same as in ( A , B ) except that compounds 9 , 10 , 11 , 13 , 14 and 16 were added at 0 . 25 , 1 , 5 , 20 , 100 , or 250 µM . Note , that the data for 250 µM were not plotted in ( D ) because of causing neuronal death . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 by Kruskal - Wallis test with Dunn ' s multiple comparison test , or one - way ANOVA with Dunnett ' s post - hoc test ; n = 6 - 15 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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

Same as in (A, B) except that compounds 9, 10, 11, 13, 14 and 16 were added at 0.25, 1, 5, 20, 100, or 250 µM. Note, that the data for 250 µM were not plotted in (D) because of causing neuronal death. Data are means ± SEM (*p < 0.05, **p < 0.01 by Kruskal-Wallis test with Dunn's multiple comparison test, or one-way ANOVA with Dunnett's post-hoc test; n = 6-15 independent experiments; exact n and p values are shown in Appendix Table S1).


Limited proteolysis . Recombinant purified proteins were incubated with increasing concentrations of trypsin . Remaining protein levels were analyzed by quantitative immunoblotting . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , by 2 - way ANOVA and Dunnett ' s multiple - comparison test ; n = 4 independent experiments ; exact p values are shown in Appendix Table S1 ) .

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

Limited proteolysis. Recombinant purified proteins were incubated with increasing concentrations of trypsin. Remaining protein levels were analyzed by quantitative immunoblotting. Data are means ± SEM (* p < 0.05, **p < 0.01, ****p < 0.0001, by 2-way ANOVA and Dunnett's multiple-comparison test; n = 4 independent experiments; exact p values are shown in Appendix Table S1).


Munc18 - 1 knockout neurons expressing G544D Munc18 - 1 were subjected to analysis of mean firing rate before addition of compounds ( 0 h ) or 48 hours after vehicle ( DMSO ) or compound addition . 16 electrodes per well were analyzed for neuronal firing ( A ; purple boxes indicate network activity ) . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA and Dunnett ' s multiple comparison test , or Kruskal - Wallis test followed by Dunn ' s post - hoc test ; n = 11 - 16 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

Munc18-1 knockout neurons expressing G544D Munc18-1 were subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).


Uptake of synaptotagmin - 1 antibody during high K + stimulation . Neurons expressing cre recombinase and / or WT , R406H or G544D Munc18 - 1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay . Endocytosed synaptotagmin - 1 antibody was quantified by immunostaining ( A - C ; scale bar = 30 µm ; 4 - PBA = 4 - phenylbutyrate ) , via counting the number of pixels > intensity of 15 ( D ) . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s multiple comparison test ; n = 3 - 10 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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

Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).


Plot of change in fluorescence from baseline to peak fluorescence for Munc18 - 1 G544D ( H ) Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way one - way ANOVA and Dunnett ' s multiple comparison test ; n = 7 - 23 ; exact n and p values are shown in Appendix Table S1 ) . Note , that compound 10 is fluorescent at a similar wavelength than pHluorin , so experiments at concentrations higher than 1 µM were not possible .

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

Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.


Heat - induced paralysis . Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 ° C over a period of 120 min , and paralysis was scored at indicated time points . Data are means ± SEM ( n = 3 - 4 independent experiments on ten worms per experiment ; exact n and p values are shown in Appendix Table S1 ) . ( B , D , F , H , J , L ) Quantification of paralysis at 20 minutes . Data are means ± SEM ( * , # p < 0 . 05 , * * , # # p < 0 . 01 , * * * , # # # p < 0 . 001 , # as compared to WT , * as compared to mutant by two - way ANOVA and Dunnett ' s multiple comparison test ; n = 3 - 4 independent experiments on ten worms per experiment ; exact n and p values are shown in Appendix Table S1 ) .

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

Heat-induced paralysis. Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 °C over a period of 120 min, and paralysis was scored at indicated time points. Data are means ± SEM (n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1). (B, D, F, H, J, L) Quantification of paralysis at 20 minutes. Data are means ± SEM (*,#p < 0.05, **,##p < 0.01, ***,###p < 0.001, # as compared to WT, * as compared to mutant by two-way ANOVA and Dunnett's multiple comparison test; n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1).


Rescue of the subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP were immobilized , and the ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger puncta , broken arrowheads to single , smaller puncta ( N , O ) . Scale bar in ( N ) and ( O ) = 10 µm . 4 - PBA = 4 - phenylbutyrate .

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

Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta (N, O). Scale bar in (N) and (O) = 10 µm. 4-PBA = 4-phenylbutyrate.


Rescue of the subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP at 1 , 5 , 20 or 100 µM compound were immobilized , and the ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger puncta , broken arrowheads to single , smaller

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

Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP at 1, 5, 20 or 100 µM compound were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta


Quantification of soluble and aggregated Munc18 - 1 . Data are means ± SEM ( * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA and Dunnett ' s multiple comparison test ; n = 3 - 4 worms ; exact n and p values are shown in Appendix Table S1 ) .

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

Quantification of soluble and aggregated Munc18-1. Data are means ± SEM (**p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-4 worms; exact n and p values are shown in Appendix Table S1).


( A ) The hair coats of Sirt7 − / − and Sirt7 + / + mice were clipped on P60 in the mid second telogen phase . Images were captured at P60 , P120 and P150 .

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

(A) The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid second telogen phase. Images were captured at P60, P120 and P150.


( B ) The postnatal day when the hair coat recovered by 25 % , 50 % , 75 % and 100 % in shaved Sirt7 − / − and Sirt7 + / + mice . n = 6 mice / genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7−/− and Sirt7+/+ mice. n = 6 mice/genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


( C ) H & E staining of the skin in unshaved Sirt7 − / − and Sirt7 + / + mice on P90 and P130 .

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

(C) H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130.


( D ) Representative immunofluorescence images showing Ki67 and p - cadherin ( Pcad , labeling HG cells ) expression in Sirt7 − / − and Sirt7 + / + HFs on P74 and P90 . The HF boundary and dermal papilla ( DP ) is denoted by the dotted and dashed lines , respectively .

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

(D) Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) is denoted by the dotted and dashed lines, respectively.


( E ) Percent Ki67 positive cells in each hair germ in D ; n = 3 mice and 10 HGs were counted in each mouse .

(E) Percent Ki67 positive cells in each hair germ in D; n = 3 mice and 10 HGs were counted in each mouse.


( F ) Telogen duration in Sirt7 − / − and Sirt7 + / + mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

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

(F) Telogen duration in Sirt7−/− and Sirt7+/+ mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


( G ) The hair coats of Sirt7 - TG and WT mice were clipped on P45 and images were captured on P56 and P90 .

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

(G) The hair coats of Sirt7-TG and WT mice were clipped on P45 and images were captured on P56 and P90.


( H ) The postnatal day when the hair coat recovered by 25 % , 50 % , 75 % and 100 % in shaved Sirt7 - TG and WT mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

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

(H) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


( I ) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7 - TG and WT HFs at P60 and P65 . The HF boundary and DP is noted by the dotted and dashed lines , respectively .

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

(I) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7-TG and WT HFs at P60 and P65. The HF boundary and DP is noted by the dotted and dashed lines, respectively.


( J ) The percentage of Ki67 positive cells in each HG ; n = 3 mice and 10 HGs were counted in each mouse .

(J) The percentage of Ki67 positive cells in each HG; n = 3 mice and 10 HGs were counted in each mouse.


( K ) H & E staining of skin tissues in unshaved Sirt7 - TG and WT mice on P65 .

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

(K) H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65.


( L ) Telogen duration in Sirt7 - TG and WT mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

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

(L) Telogen duration in Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


( A - B ) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation - induced hair cycle in WT mice . P56 , P69 , P74 and P84 indicate the postnatal day in the second hair cycle . Pd , post depilation . Dashed line indicates bulge ( Bu ) of a hair follicle and solid line indicates the Ki - 67 positive staining region .

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(A-B) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation-induced hair cycle in WT mice. P56, P69, P74 and P84 indicate the postnatal day in the second hair cycle. Pd, post depilation. Dashed line indicates bulge (Bu) of a hair follicle and solid line indicates the Ki-67 positive staining region.


( C ) The hair coats of Sirt7f / f and Sirt7f / f ; K15 - Cre mice were clipped at P60 and images were captured on P60 , P125 and P150 .

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(C) The hair coats of Sirt7f/f and Sirt7f/f;K15-Cre mice were clipped at P60 and images were captured on P60, P125 and P150.


( D ) The postnatal day of hair coat recovery by 25 % , 50 % 75 % and 100 % in shaved Sirt7f / f and Sirt7f / f ; K15 - Cre mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

(D) The postnatal day of hair coat recovery by 25%, 50% 75% and 100% in shaved Sirt7f/f and Sirt7f/f;K15-Cre mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


( E ) H & E staining of the skin in Sirt7f / f and Sirt7f / f ; K15 - Cre mice at P100 .

(E) H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100.


( F ) Representative immunofluorescence images showing Ki67 / Pcad expression ( upper ) and Cd34 / Ki67 expression ( lower ) in Sirt7fl / fl and Sirt7f / f ; K15 - Cre HFs on P56 , P74 and P85 . The white dashed line indicates bulge of hair follicle .

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(F) Representative immunofluorescence images showing Ki67/Pcad expression (upper) and Cd34/Ki67 expression (lower) in Sirt7fl/fl and Sirt7f/f;K15-Cre HFs on P56, P74 and P85. The white dashed line indicates bulge of hair follicle.


( G ) The percentage of Ki67 - positive cells per HG ; n = 3 mice and 10 HGs were counted in each mouse .

(G) The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse.


( H ) Ki67 - positive cells per bulge ; n = 3 mice and 10 bulges were counted in each mouse .

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(H) Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse.


( A ) Representative colonies formed by Sirt7 + / + , Sirt7 - / - and Sirt7 - TG bulge SCs that were puriﬁed from telogen - phase HFs and allowed to grow for 2 weeks . ( B - C ) Quantiﬁcations of colony number and size in ( A ) ; n = 3 mice per genotype .

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(A) Representative colonies formed by Sirt7+/+, Sirt7-/- and Sirt7-TG bulge SCs that were puriﬁed from telogen-phase HFs and allowed to grow for 2 weeks. (B-C) Quantiﬁcations of colony number and size in (A); n = 3 mice per genotype.


Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice ( D )

Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D)


proteins in HFSC colonies from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice Nfatc1 protein levels were quantified using Image J Plus 6 . 0 ( E , n = 3 mice for each group ) .

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proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice Nfatc1 protein levels were quantified using Image J Plus 6.0 (E, n = 3 mice for each group).


( F ) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs .

(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.


( G ) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells .

(G) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells.


( H ) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments ( n = 3 ) . The relative luciferase activity was determined by dual luciferase reporter assay .

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(H) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments (n=3). The relative luciferase activity was determined by dual luciferase reporter assay.


( I - J ) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f / f , Sirt7f / f ; K15 - Cre and Sirt7 - TG mice at the indicted time points . The black dashed line indicates the bulge ( Bu ) of hair follicle .

(I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle.


( K - L ) qPCR analysis of Nfatc1 - targeted genes in HFSCs colonies cultured from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice , n = 3 for each genotypes .

(K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.


( A - B ) Immunoblot analysis of the interactions between ectopic HA - SIRT7 and FLAG - NFATc1in HEK293T cells .

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(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells.


( C ) Co - immunoprecipitation ( CoIP ) between endogenous NFATc1 and SIRT7 in HEK293T cells .

(C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells.


( D ) Immunoblots showing endogenous NFATc1 pull - down by GST - SIRT7 in HEK293T cells .

(D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells.


( E ) Immunoblot analysis of HA - SIRT7 WT and various mutants in the anti His - NFATc1 immunoprecipitates . ∆ N indicates N - terminal deletion ; ∆ C indicates C - terminal deletion ; AC indicates N - terminal and C - terminal deletion .

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(E) Immunoblot analysis of HA-SIRT7 WT and various mutants in the anti His-NFATc1 immunoprecipitates. ∆N indicates N-terminal deletion; ∆C indicates C-terminal deletion; AC indicates N-terminal and C-terminal deletion.


( F ) Immunoblot analysis of HA - SIRT7 levels in truncated FLAG - NFATc1 immunoprecipitates . N indicates the N - terminal domain ; C indicates C - terminal domain .

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(F) Immunoblot analysis of HA-SIRT7 levels in truncated FLAG-NFATc1 immunoprecipitates. N indicates the N-terminal domain; C indicates C-terminal domain.


( G - H ) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells .

(G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells.


( I - J ) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments .

(I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments.


Immunoblot analysis of NFATc1 - WT / K612R acetylation based on SIRT7 deacetylation assays in vitro ( K )

Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K)


Immunoblot analysis of NFATc1 - WT / K612R acetylation based on SIRT7 deacetylation assays in vivo ( L ) .

Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vivo (L).


Immunoblots showing NFATc1 pan phosphorylation ( probing to anti S / T antibody ) and acetylation levels under the indicated treatments .

Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.


( O ) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants , noting that K612R represents deacetylation while K612Q hyperacetylation .

(O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.


( A ) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide ( CHX ) , with or without SIRT7 overexpression . ( B ) Quantification of NFATc1 levels in ( A ) from 3 independent experiments .

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(A) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide (CHX), with or without SIRT7 overexpression. (B) Quantification of NFATc1 levels in (A) from 3 independent experiments.


( C ) Immunoblots of Nfatc1 protein levels in Sirt7 + / + and Sirt7 - / - keratinocytes . ( D ) Quantification of Nfatc1 levels in ( C ) from 3 independent experiments .

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(C) Immunoblots of Nfatc1 protein levels in Sirt7+/+ and Sirt7-/- keratinocytes. (D) Quantification of Nfatc1 levels in (C) from 3 independent experiments.


( E - F ) Immunoblots ( E ) and related quantitative data ( F , n = 3 ) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA - SIRT7 or a SIRT7 enzyme - dead mutant ( H187Y ) .

(E-F) Immunoblots (E) and related quantitative data (F, n=3) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA-SIRT7 or a SIRT7 enzyme-dead mutant (H187Y).


( G - H ) Immunoblots ( G ) and quantitative data ( H , n = 3 ) showing NFATc1 levels in HEK293T cells transfected with NFATc1 - CA , with or without ectopic SIRT7 , treated with MG132 .

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(G-H) Immunoblots (G) and quantitative data (H, n=3) showing NFATc1 levels in HEK293T cells transfected with NFATc1-CA, with or without ectopic SIRT7, treated with MG132.


( I - J ) Immunoblots ( I ) and related quantitative data ( J , n = 3 ) showing NFATc1 - WT and NFATc1 - K612R protein turnover in HEK293T cells .

(I-J) Immunoblots (I) and related quantitative data (J, n=3) showing NFATc1-WT and NFATc1-K612R protein turnover in HEK293T cells.


Immunoblots showing the interaction ( by co - immunoprecipitation ) between ectopic NFATc1 / PA28γ in HEK293T cells ( K ) ( L ) after manipulating SIRT7 levels .

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Immunoblots showing the interaction (by co-immunoprecipitation) between ectopic NFATc1/PA28γ in HEK293T cells (K) (L) after manipulating SIRT7 levels.


Immunoblots showing the interaction ( by co - immunoprecipitation ) between endogenous NFATc1 / PA28γ in HaCaT cells ( L ) after manipulating SIRT7 levels .

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Immunoblots showing the interaction (by co-immunoprecipitation) between endogenous NFATc1/PA28γ in HaCaT cells (L) after manipulating SIRT7 levels.


( M - N ) Immunoblot ( M ) and quantitative data ( N , n = 3 ) analysis of NFATc1 levels upon overexpression of HA - SIRT7 with or without PA28γ oligo siRNAs treatment .

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(M-N) Immunoblot (M) and quantitative data (N, n=3) analysis of NFATc1 levels upon overexpression of HA-SIRT7 with or without PA28γ oligo siRNAs treatment.


( O ) Co - immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA - SIRT7 or an enzyme - dead mutant ( H187Y ) .

(O) Co-immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA-SIRT7 or an enzyme-dead mutant (H187Y).


( A ) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7 + / + and Sirt7 - / - mice with or without Cyclosporin A ( CsA ) treatment . The black dashed line indicates bulge ( Bu ) of hair follicle . ( B ) Quantitative analysis of Nfatc1 levels in ( A , n = 10 hair follicles ) using ImageJ plus 6 . 0 .

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(A) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7+/+ and Sirt7-/- mice with or without Cyclosporin A (CsA) treatment. The black dashed line indicates bulge (Bu) of hair follicle. (B) Quantitative analysis of Nfatc1 levels in (A, n=10 hair follicles) using ImageJ plus 6.0.


( C ) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7 + / + and Sirt7 - / - HFs with or without CsA treatment .

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(C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.


( D ) The percentage of Ki67 - positive cells per HG in ( C , n = 10 hair follicles ) .

(D) The percentage of Ki67-positive cells per HG in (C, n=10 hair follicles).


( E ) H & E staining of the skin in the indicated treatment groups at P100 .

(E) H&E staining of the skin in the indicated treatment groups at P100.


( F ) Representative images showing haircoat recovery after the indicated treatments . The dashed circle indicates the shaved area ; the dotted line indicates the hair growth boundary .

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(F) Representative images showing haircoat recovery after the indicated treatments. The dashed circle indicates the shaved area; the dotted line indicates the hair growth boundary.


( A ) Immunoblot analysis of Sirt7 expression in young ( 3M ) and aged ( 18M ) HFSCs . # 1 , # 2 , # 3 represented individual mice . Below , quantification of Sirt7 protein levels ; n = 3 mice for each group ; the Y - axis represents the relative band intensity ( measured with Image J ® ) normalized to the signal in young mice .

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(A) Immunoblot analysis of Sirt7 expression in young (3M) and aged (18M) HFSCs. #1, #2, #3 represented individual mice. Below, quantification of Sirt7 protein levels; n=3 mice for each group; the Y-axis represents the relative band intensity (measured with Image J®) normalized to the signal in young mice.


( B - D ) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young ( 3 months ) and old ( 18 months ) mice ( B ) , and the relative levels of Sirt7 and Nfatc1 - positive cells in the bulge ( C - D ) . The white / black dashed line indicates bulge ( Bu ) of hair follicle .

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(B-D) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young (3 months) and old (18 months) mice (B), and the relative levels of Sirt7 and Nfatc1-positive cells in the bulge (C-D). The white/black dashed line indicates bulge (Bu) of hair follicle.


( E ) Images were captured of the hair coats of Sirt7 + / + and Sirt7 - / - mice at 15 months . The yellow dashed lines indicate hair thinning or hair loss .

(E) Images were captured of the hair coats of Sirt7+/+ and Sirt7-/- mice at 15 months. The yellow dashed lines indicate hair thinning or hair loss.


( F ) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15 - month - old Sirt7 + / + and Sirt7 - / - mice . The white / black dashed line indicates bulge ( Bu ) of hair follicle .

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(F) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15-month-old Sirt7+/+ and Sirt7-/- mice. The white/black dashed line indicates bulge (Bu) of hair follicle.


( G ) Quantification of Nfatc1 - positive cells per bulge in ( F , n = 10 hair follicles ) .

(G) Quantification of Nfatc1-positive cells per bulge in (F, n=10 hair follicles).


( H ) Images captured of the skin in the plucked regions ( blue dotted lines ) of WT ( 3 months ) , aged WT ( 20 months ) and aged Sirt7 - TG mice ( 20 months ) , 14 days after depilation . Pigmentation of the skin indicates hair regrowth . n = 3 mice per genotype .

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(H) Images captured of the skin in the plucked regions (blue dotted lines) of WT (3 months), aged WT (20 months) and aged Sirt7-TG mice (20 months), 14 days after depilation. Pigmentation of the skin indicates hair regrowth. n = 3 mice per genotype.


( I ) Hair re - growth scores were analyzed for young WT , old WT and old Sirt7 - TG mice in ( H ) , n = 3 mice for each group .

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(I) Hair re-growth scores were analyzed for young WT, old WT and old Sirt7-TG mice in (H), n=3 mice for each group.


( J ) The hair coat recovery score ( described in methods ) after the back hair of old WT and old Sirt7 - TG mice was shaved . n = 3 mice per genotype .

(J) The hair coat recovery score (described in methods) after the back hair of old WT and old Sirt7-TG mice was shaved. n = 3 mice per genotype.


( K ) Images captured of the hair coats of WT and Sirt7 - TG mice at 28 months - of - age . The yellow dashed lines indicate the hair thinning or hair loss regions .

(K) Images captured of the hair coats of WT and Sirt7-TG mice at 28 months-of-age. The yellow dashed lines indicate the hair thinning or hair loss regions.


Speed distribution profile of PMN treated with 0 . 5 µM E4 or DMSO for 30 min . Graph shows an increase in the rate of axonal transport upon E4 treatment ( DMSO , 153 endosomes , 13 axons ; E4 , 165 endosomes , 13 axons ; N = 4 independent experiments , data shown are mean ± SEM ) .

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Speed distribution profile of PMN treated with 0.5 µM E4 or DMSO for 30 min. Graph shows an increase in the rate of axonal transport upon E4 treatment (DMSO, 153 endosomes, 13 axons; E4, 165 endosomes, 13 axons; N=4 independent experiments, data shown are mean ± SEM).


PMN treated with 0 . 5 µM E4 for 30 min showed a significant decrease in phospho - IGF1R levels compared to controls . Protein levels were normalised to β - actin ( * * * P < 0 . 001 , Students t - test , N = 3 independent experiments , data shown are mean ± SEM ) .

PMN treated with 0.5 µM E4 for 30 min showed a significant decrease in phospho-IGF1R levels compared to controls. Protein levels were normalised to β-actin (*** P< 0.001, Students t-test, N=3 independent experiments, data shown are mean ± SEM).


PMN were treated with 1 µM PPP ( blue ) or 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Example images of DIV6 PMN treated with 1 µM PPP or 50 ng / ml IGF1 . PMN were stained with 30 nM AlexaFluor555 - HcT . The scale bar is 50 µm .

PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example images of DIV6 PMN treated with 1 µM PPP or 50 ng/ml IGF1. PMN were stained with 30 nM AlexaFluor555-HcT. The scale bar is 50 µm.


PMN were treated with 1 µM PPP ( blue ) or 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Example of HcT - containing organelle tracking in the axon of PMN . Each colour denotes a single endosome tracked over time . The scale bar is 10 µm .

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PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example of HcT-containing organelle tracking in the axon of PMN. Each colour denotes a single endosome tracked over time. The scale bar is 10 µm.


PMN were treated with 1 µM PPP ( blue ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Graph shows the average velocity of HcT - containing organelles , after PPP treatment compared to controls ( DMSO : 138 endosomes , 24 axons , 6 , 433 movements ; PPP : 130 endosomes , 23 axons , 5 , 222 movements ) ( * * P = 0 . 0061 , Student ' s t - test , N = 4 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Percent of time HcT - containing organelles pausing per axon . There was no difference in pausing between PPP - treated neurons and controls ( P = 0 . 45 , Student ' s T - test , N = 4 independent experiments data shown are mean ± SEM ) .

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PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles, after PPP treatment compared to controls (DMSO: 138 endosomes, 24 axons, 6,433 movements; PPP: 130 endosomes, 23 axons, 5,222 movements) (** P= 0.0061, Student's t-test, N=4 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. There was no difference in pausing between PPP-treated neurons and controls (P=0.45, Student's T-test, N=4 independent experiments data shown are mean ± SEM).


PMN were treated with 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Graph shows the average velocity of HcT - containing organelles after IGF1 treatment compared to control ( water : 138 endosomes , 22 axons , 5 , 119 movements ; IGF1 : 106 endosomes , 20 axons , 5 , 202 movements ; * * * P = 2 . 83x10 - 5 , Student ' s T - test , N = 3 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Percent of time HcT - containing organelles spent pausing per axon . IGF1 significantly increased the pausing time of HcT - containing organelles ( * P = 0 . 011 , Student ' s T - test , N = 3 independent experiments , data shown are mean ± SEM ) .

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PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles after IGF1 treatment compared to control (water: 138 endosomes, 22 axons, 5,119 movements; IGF1: 106 endosomes, 20 axons, 5,202 movements; *** P=2.83x10-5, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles spent pausing per axon. IGF1 significantly increased the pausing time of HcT-containing organelles (* P=0.011, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).


PMN were treated with 1 µM PPP ( blue ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Speed distribution profile of PPP - treated neurons and controls . PPP caused an increase in instantaneous velocities of HcT - containing organelles ( N = 3 independent experiments , data shown are mean ± SEM ) .

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PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of PPP-treated neurons and controls. PPP caused an increase in instantaneous velocities of HcT-containing organelles (N=3 independent experiments, data shown are mean ± SEM).


PMN were treated with 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Speed distribution profile of IGF1 - treated neurons and controls . IGF1 caused a decrease in instantaneous velocities of HcT - containing organelles compared to controls ( N = 3 independent experiments , data shown are mean ± SEM ) .

PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of IGF1-treated neurons and controls. IGF1 caused a decrease in instantaneous velocities of HcT-containing organelles compared to controls (N=3 independent experiments , data shown are mean ± SEM).


Western blot showing the levels of pAKT ( Ser473 ) , total Akt , pErk1 / 2 ( T202 / Y204 ) and total Erk1 / 2 after treatment with 1 µM PPP or 50 ng / ml IGF1 for 60 min . Protein was normalised to GAPDH . Western blot quantification . PMN treated with PPP showed a significant decrease in Akt activation ( * P = 0 . 049 , N = 5 independent experiments , Student ' s T - test ) , but no significant difference was seen on Erk1 / 2 activation ( P = 0 . 07 , Student ' s T - test , N = 5 independent experiments ) . 50 ng / ml IGF1 treated neurons showed a significant increase in Akt activation ( * * P = 0 . 008 , Student ' s T - test , N = 3 independent experiments ) . In contrast , no change in Erk1 / 2 activation ( P = 0 . 88 , Student ' s T - test , N = 3 independent experiments ) was observed ( data shown are mean ± SEM ) .

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

Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Protein was normalised to GAPDH. Western blot quantification. PMN treated with PPP showed a significant decrease in Akt activation (* P=0.049, N=5 independent experiments, Student's T-test), but no significant difference was seen on Erk1/2 activation (P=0.07, Student's T-test, N=5 independent experiments). 50 ng/ml IGF1 treated neurons showed a significant increase in Akt activation (** P=0.008, Student's T-test, N=3 independent experiments). In contrast, no change in Erk1/2 activation (P=0.88, Student's T-test, N=3 independent experiments) was observed (data shown are mean ± SEM).


Graph shows the average velocity of HcT - containing organelles treated with Capivasertib or Ipatasertib ( 10 nM ) for 30 min compared to controls ( DMSO : 483 endosomes , 34 axons , 41 , 897 movements ; Capivasertib : 658 endosomes , 43 axons , 49 , 841 movements ; Ipatasertib : 605 endosomes , 32 axons , 41 , 485 movements ) ( * * P = 2 x10 - 16 , One - way ANOVA , Tukey ' s post - hoc test : DMSO - Capivasertib and DMSO - Ipatasertib * * * P < 0 . 0001 ; N = 6 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Percent of time HcT - containing organelles pausing per axon . Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing ( * P = 1 . 14 x10 - 7 , One - way ANOVA , Tukey ' s post - hoc test : DMSO - Capivasertib P = 9 . 37x10 - 5 , DMSO - Ipatasertib P = 2 x10 - 7 ; data shown are mean ± SEM , N = 6 independent experiments ) .

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

Graph shows the average velocity of HcT-containing organelles treated with Capivasertib or Ipatasertib (10 nM) for 30 min compared to controls (DMSO: 483 endosomes, 34 axons, 41,897 movements; Capivasertib: 658 endosomes, 43 axons, 49,841 movements; Ipatasertib: 605 endosomes, 32 axons, 41,485 movements) (** P=2 x10-16, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib and DMSO-Ipatasertib *** P <0.0001; N=6 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing (* P=1.14 x10-7, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib P= 9.37x10-5, DMSO-Ipatasertib P= 2 x10-7; data shown are mean ± SEM, N=6 independent experiments).


PMN were treated with either an shRNA targeting IGF1R ( 1 µl ) or a scrambled control 6 h after plating . At DIV 6 - 7 , retrograde axonal transport was analysed using 30 nM AlexaFluor555 - HcT . Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls ( IGF1R shRNA : 108 endosomes , 21 axons , 801 movements ; scrambled shRNA : 94 endosomes , 20 axons , 777 movements , * P = 0 . 011 , Student ' s T - test , N = 3 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control . IGF1R knockdown caused an increase in instantaneous velocities of HcT - containing organelles and decreased the amount of pausing ( N = 3 independent experiments , data shown are mean ± SEM ) .

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

PMN were treated with either an shRNA targeting IGF1R (1 µl) or a scrambled control 6 h after plating. At DIV 6-7, retrograde axonal transport was analysed using 30 nM AlexaFluor555-HcT. Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls (IGF1R shRNA: 108 endosomes, 21 axons, 801 movements; scrambled shRNA: 94 endosomes, 20 axons, 777 movements, * P=0.011, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control. IGF1R knockdown caused an increase in instantaneous velocities of HcT-containing organelles and decreased the amount of pausing (N=3 independent experiments, data shown are mean ± SEM).


Western blot of PMN treated with 1 µM PPP or 50 ng / ml IGF1 for 60 min . Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins , all proteins were assessed from the same blot . Hook3 expression didn ' t change after treatment with PPP or IGF1 compared to controls ( DMSO , 100 % ; PPP , 102 . 16 ± 4 . 3 % , P = 0 . 67 , Student ' s T - test , N = 3 independent experiments ; water , 100 % ; IGF1 , 93 . 48 ± 4 . 2 % , P = 0 . 26 , N = 3 independent experiments , data shown are mean ± SEM ) . However , BICD1 expression was significantly increased in PMN treated with PPP compared to control ( DMSO , 100 % ; PPP , 137 . 1 ± 7 . 3 % , * * P = 0 . 002 , Student ' s T - test , N = 7 independent experiments ) . In contrast , IGF1R stimulation by IGF1 had no effect on BICD1 expression ( water , 100 % ; IGF1 , 88 . 1 ± 14 . 0 % , P = 0 . 49 , Student ' s T - test , N = 3 independent experiments , data shown are mean ± SEM ) . BICD2 expression didn ' t change after treatment with PPP or IGF1 compared to controls ( DMSO , 100 % ; PPP , 104 . 44 ± 6 . 5 % , P = 0 . 51 , N = 3 independent experiments : water , 100 % ; IGF1 , 106 ± 7 . 5 % , P = 0 . 51 , Student ' s T - test , N = 3 independent experiments , data shown are mean ± SEM ) .

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

Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins, all proteins were assessed from the same blot. Hook3 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 102.16 ± 4.3%, P=0.67, Student's T-test, N=3 independent experiments; water, 100%; IGF1, 93.48 ± 4.2%, P=0.26, N=3 independent experiments, data shown are mean ± SEM). However, BICD1 expression was significantly increased in PMN treated with PPP compared to control (DMSO, 100%; PPP, 137.1 ± 7.3%, **P=0.002, Student's T-test, N=7 independent experiments). In contrast, IGF1R stimulation by IGF1 had no effect on BICD1 expression (water, 100%; IGF1, 88.1 ± 14.0%, P=0.49, Student's T-test, N=3 independent experiments, data shown are mean ± SEM). BICD2 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 104.44 ± 6.5%, P=0.51, N=3 independent experiments: water, 100%; IGF1, 106 ± 7.5%, P=0.51, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).


Western blot of PMN pre - treatment with cycloheximide ( 50 µg / ml ) or MG - 132 ( 10 µM ) for 30 min before incubation with 1 µM PPP for 60 min . Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells . ( DMSO , grey : 100 % , N = 7 independent experiments ; PPP , blue : 137 . 1 ± 7 . 3 % , N = 7 independent experiments ; CHX , pink : 89 . 5 ± 12 . 7 % , N = 5 independent experiments ; MG - 132 , teal : 124 ± 28 . 7 , N = 3 independent experiments ; P = 0 . 009 , one - way ANOVA ; Tukey ' s post - hoc test : DMSO - PPP * P = 0 . 04 , PPP - PPP + CHX * P = 0 . 01 ; data shown are mean ± SEM ) .

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

Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min. Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells. (DMSO, grey: 100%, N=7 independent experiments; PPP, blue: 137.1 ± 7.3%, N=7 independent experiments; CHX, pink: 89.5 ± 12.7%, N=5 independent experiments; MG-132, teal: 124 ± 28.7, N=3 independent experiments; P=0.009, one-way ANOVA; Tukey's post-hoc test: DMSO-PPP * P=0.04, PPP-PPP+CHX * P=0.01; data shown are mean ± SEM).


Puromycin - PLA signal in PMN grown in microfluidics devices . Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment . The scale bars are 10 µm . White arrows point to puromycin - PLA puncta within the β3 - tubulin mask ; the empty arrow shows a PLA puncta not associated with neurons .

Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons.


Quantification of puromycin - PLA signal . PPP causes an increase in PLA puncta in the axon of PMN , which is abolished by treatment with 40 µM anisomycin ( DMSO , grey : 100 % ; PPP , blue : 139 . 3 ± 7 . 7 % ; PPP + anisomycin , purple : 86 . 3 ± 10 . 0 ; P = 0 . 005 , one - way ANOVA , Tukey ' s post - hoc test ; DMSO vs PPP , * P = 0 . 02 ; PPP vs PPP + anisomycin , * * 0 . 005 , N = 3 independent experiments , 27 - 57 images , data shown are mean ± SEM ) .

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

Quantification of puromycin-PLA signal. PPP causes an increase in PLA puncta in the axon of PMN, which is abolished by treatment with 40 µM anisomycin (DMSO, grey: 100%; PPP, blue: 139.3 ± 7.7%; PPP + anisomycin, purple: 86.3 ± 10.0; P=0.005, one-way ANOVA, Tukey's post-hoc test; DMSO vs PPP, * P=0.02; PPP vs PPP + anisomycin, ** 0.005, N=3 independent experiments, 27-57 images, data shown are mean ± SEM).


The graph shows the average velocity of HcT - containing organelles in vivo in D72 / 73 SOD1G93A mice and wild type littermate controls . PPP ( blue ) treatment increased the transport rate of signalling endosomes in wild type and SOD1G93A mice compared to animals treated with vehicle control ( grey ) ( WT control : cargo 371 , movements 15 , 272 , N = 4 independent experiments ; WT + PPP : cargo 388 , movements 14 , 512 , N = 4 independent experiments ; SOD1G93A control : cargo 322 , movements 15 , 425 , N = 4 independent experiments ; SOD1G93A + PPP : cargo 404 , movements 17 , 643 , N = 4 independent experiments ; P = 2 . 24x10 - 12 ( treatment ) , P = 2 . 22 x10 - 16 ( genotype ) , two - way ANOVA , Tukey ' s post - hoc test : all conditions * * * P < 0 . 005 , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . PPP treatment does not alter the pausing of signalling endosomes ( WT : 16 . 1 ± 1 . 5 % , WT + PPP : 13 . 1 ± 1 . 5 % ; SOD1G93A : 16 . 6 ± 1 . 9 % , SOD1G93A + PPP : 16 . 2 ± 2 . 1 % ; P = 0 . 36 ( treatment ) , P = 0 . 32 ( genotype ) , N = 4 independent experiments for each condition , data shown are mean ± SEM .

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

The graph shows the average velocity of HcT-containing organelles in vivo in D72/73 SOD1G93A mice and wild type littermate controls. PPP (blue) treatment increased the transport rate of signalling endosomes in wild type and SOD1G93A mice compared to animals treated with vehicle control (grey) (WT control: cargo 371, movements 15,272, N=4 independent experiments; WT + PPP: cargo 388, movements 14,512, N=4 independent experiments; SOD1G93A control: cargo 322, movements 15,425, N=4 independent experiments; SOD1G93A + PPP: cargo 404, movements 17,643, N=4 independent experiments; P=2.24x10-12 (treatment), P=2.22 x10-16 (genotype), two-way ANOVA, Tukey's post-hoc test: all conditions *** P<0.005, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). PPP treatment does not alter the pausing of signalling endosomes (WT: 16.1 ± 1.5%, WT + PPP: 13.1 ± 1.5%; SOD1G93A: 16.6 ± 1.9%, SOD1G93A + PPP: 16.2 ± 2.1%; P=0.36 (treatment), P=0.32 (genotype), N=4 independent experiments for each condition, data shown are mean ± SEM.


Treatment with PPP had no effect on Akt levels in the sciatic nerve ( WT : Control - 100 ± 8 . 6 , PPP - 91 . 5 ± 14 . 6 % ; SOD1 : Control - 101 . 4 ± 13 . 5 % , PPP - 117 . 6 ± 32 . 9 % , P = 0 . 81 ( treatment ) , P = 0 . 53 ( genotype ) , two - way ANOVA , N = 5 independent experiments , data shown are mean ± SEM , Appendix Fig S7D ) . Erk1 / 2 activation was down - regulated after treatment with PPP in the sciatic nerve of wild type and SOD1G93A mice ( WT : control - 100 ± 15 . 4 % , PPP - 64 . 4 ± 3 . 2 % ; SOD1 : Control - 78 . 0 ± 11 . 9 % , PPP - 51 . 2 ± 8 . 2 % , * P = 0 . 013 ( treatment ) , P = 0 . 13 ( genotype ) , two - way ANOVA , N = 5 independent experiments , data shown are mean ± SEM , Appendix Fig S7D ) . Protein was normalised to GAPDH protein levels .

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

Treatment with PPP had no effect on Akt levels in the sciatic nerve (WT: Control-100 ± 8.6, PPP-91.5 ± 14.6%; SOD1: Control-101.4 ± 13.5%, PPP-117.6 ± 32.9%, P=0.81 (treatment), P=0.53 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Erk1/2 activation was down-regulated after treatment with PPP in the sciatic nerve of wild type and SOD1G93A mice (WT: control-100 ± 15.4%, PPP-64.4 ± 3.2%; SOD1: Control-78.0 ± 11.9 %, PPP-51.2 ± 8.2%, * P=0.013 (treatment), P=0.13 (genotype), two-way ANOVA, N=5 independent experiments, data shown are mean ± SEM, Appendix Fig S7D). Protein was normalised to GAPDH protein levels.


( B ) Density of HEAP - AGO2 peaks across cytosolic lncRNAs ( n = 62 , blue ) and the 3 ' UTR regions of mRNAs ( n = 8798 , red ) with experimental evidence for AGO2 binding in mESCs ( > 0 AGO2 peaks ) . Central band of boxplot represents median , box depicts 25 - 75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Density of HEAP-AGO2 peaks across cytosolic lncRNAs (n=62, blue) and the 3'UTR regions of mRNAs (n=8798, red) with experimental evidence for AGO2 binding in mESCs (>0 AGO2 peaks). Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.


( D ) Distribution of the fold - change ( FC ) in degradation rate of mRNAs ( n = 13296 , red ) , micropeptide encoding transcripts ( n = 43 , pink ) , bona fide cytosolic ( n = 759 , blue ) and nuclear ( n = 4299 , grey ) lncRNAs in 4 - OHT treated ( KO ) relative to ethanol treated ( WT ) cells after 8 days treatment , horizontal dashed line represents a KO / WT FC in degradation rate of 1 . Statistics : NS - p > 0 . 05 , * - p - value < 0 . 05 . Two - tailed Mann - Whitney U test : p - value = 0 . 044 . Central band of boxplot represents median , box depicts 25 - 75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution .

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

(D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.


( F ) Distribution of the fold - change ( FC ) in degradation rates of mRNAs ( n = 29900 , red ) , cytosolic ( n = 474 , blue ) and nuclear ( n = 2348 , grey ) lncRNAs , in 4 - OHT treated ( KO ) relative to ethanol treated ( WT ) cells after 8 days of treatment ( estimated based on the 10 minutes 4sU pulse ) , horizontal dashed line represents a KO / WT FC in degradation rate of 1 Statistics : NS - p - value > 0 . 05 anband of boxplot represents median box depicts quantiles distribution and whiskers represent the > th and 95th quantiles of the distribution . ( two - tailed Mann - Whitney U test ) .

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

(F) Distribution of the fold-change (FC) in degradation rates of mRNAs (n=29900, red), cytosolic (n=474, blue) and nuclear (n=2348, grey) lncRNAs, in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days of treatment (estimated based on the 10 minutes 4sU pulse), horizontal dashed line represents a KO/WT FC in degradation rate of 1 Statistics: NS-p-value>0.05 anband of boxplot represents median box depicts quantiles distribution and whiskers represent the>th and 95th quantiles of the distribution. (two-tailed Mann-Whitney U test).


( B ) Fraction of cytosolic lncRNAs with experimental evidence for ribosomal binding with ( red ) or without ( blue ) an overlapping conserved short open reading frame .

O O O O O O 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) Fraction of cytosolic lncRNAs with experimental evidence for ribosomal binding with (red) or without (blue) an overlapping conserved short open reading frame.


( B ) Expression of GFP , lncRNA - c1 , GFP - lncRNA - c1 , lncRNA - c2 and GFP - lncRNA - c2 ( x - axis ) in 8 day 4 - OHT treated , miRNA depleted cells ( KO ) relative to ethanol treated ( WT ) mESC ( y - axis ) 24h hours post - transfection . Four independent biological replicates were treated , transfected and analyzed by RT - qPCR . Statistical significance represented on the figure based on comparison of KO / WT fold change in expression of GFP , with GFP - lncRNA - c1 and with GFP - lncRNA - c2 , ( paired two - tailed t - test p - value = 0 . 034 and 0 . 032 respectively ) and based on comparison of KO / WT fold change in expression of GFP - lncRNAc1 with lncRNA - c1 and GFP - lncRNA - c2 with lncRNA - c2 ( paired two - tailed t - test p - value = 0 . 012 and 0 . 018 respectively ) . Data is represented as mean ± SD and Each point corresponds to the results of one independent biological replicate . Data information : For all RTqPCR analyses , transcript expression was first normalized by the amount of Act - β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression . Each point corresponds to the results of one independent biological replicate . Statistics : NS - p - value > 0 . 05 , * - p - value < 0 . 05 , * * - p - value < 0 . 01 and * * * - p - value < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Expression of GFP, lncRNA-c1, GFP-lncRNA-c1, lncRNA-c2 and GFP-lncRNA-c2 (x-axis) in 8 day 4-OHT treated, miRNA depleted cells (KO) relative to ethanol treated (WT) mESC (y-axis) 24h hours post-transfection. Four independent biological replicates were treated, transfected and analyzed by RT-qPCR. Statistical significance represented on the figure based on comparison of KO/WT fold change in expression of GFP, with GFP-lncRNA-c1 and with GFP-lncRNA-c2, (paired two-tailed t-test p-value= 0.034 and 0.032 respectively) and based on comparison of KO/WT fold change in expression of GFP-lncRNAc1 with lncRNA-c1 and GFP-lncRNA-c2 with lncRNA-c2 (paired two-tailed t-test p-value= 0.012 and 0.018 respectively). Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.


( C ) Expression of GFP - lncRNA - c1 ΔMRE relative to GFP - lncRNA - c1 ( y - axis ) in ethanol treated ( WT , circles ) or 4 - OHT treated , miRNA - depleted cells ( KO , triangles , x - axis ) . Four independent biological replicates were treated , transfected and analyzed by RTqPCR . Data is represented as mean ± SD and Each point corresponds to the results of one independent biological replicate . Paired two - tailed t - test p - value = 0 . 0071 Data information : For all RTqPCR analyses , transcript expression was first normalized by the amount of Act - β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression . Each point corresponds to the results of one independent biological replicate . Statistics : NS - p - value > 0 . 05 , * - p - value < 0 . 05 , * * - p - value < 0 . 01 and * * * - p - value < 0 . 001 .

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

(C) Expression of GFP-lncRNA-c1 ΔMRE relative to GFP-lncRNA-c1 (y-axis) in ethanol treated (WT, circles) or 4-OHT treated, miRNA-depleted cells (KO, triangles, x-axis). Four independent biological replicates were treated, transfected and analyzed by RTqPCR. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0071 Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.


( F ) Immunoblot analysis of GFP ( GFP ) in protein extracts from mESCs transfected with mock , BoxB ( - 30 ) - GFP and GFP expressing vectors . ACTIN - β ( ACT - β ) was used as an internal control . One representative blot is depicted ( G ) Fold - change ( FC ) in normalized expression of GFP - lncRNA - c1 , GFP - lncRNA - c2 ( noBoxB , circles ) and BoxB ( - 30 ) - GFP - lncRNA - c1 , BoxB ( - 30 ) - GFP - lncRNA - c2 ( BoxB ( - 30 ) , triangles ) ( x - axis ) 4 - OHT treated , miRNA depleted mESCs ( KO ) relative to ethanol treated mESCs ( WT ) ( y - axis ) . Four independent biological replicates were analyzed . Data is represented as mean ± SD and Each point corresponds to the results of one independent biological replicate . Paired two - tailed t - test p - value = 0 . 0494 for GFP - lncRNA - c1 and p - value = 0 . 0355 for GPF - lncRNA - c2Data information : Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data .

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

(F) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(-30)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted (G) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(-30)-GFP-lncRNA-c1, BoxB (-30)-GFP-lncRNA-c2 (BoxB(-30), triangles) (x-axis) 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates were analyzed. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0494 for GFP-lncRNA-c1 and p-value=0.0355 for GPF-lncRNA-c2Data information: Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data.


( H ) Immunoblot analysis of GFP ( GFP ) in protein extracts from mESCs transfected with mock , BoxB ( + 339 ) - GFP and GFP expressing vectors . ACTIN - β ( ACT - β ) was used as an internal control . One representative blot is depicted . ( I ) Fold - change ( FC ) in normalized expression of GFP - lncRNA - c1 , GFP - lncRNA - c2 ( noBoxB , circles ) and BoxB ( + 339 ) - GFP - lncRNA - c1 , BoxB ( + 339 ) - GFP - lncRNA - c2 ( BoxB ( + 339 ) , triangles ) ( x - axis ) in 8 day 4 - OHT treated , miRNA depleted mESCs ( KO ) relative to ethanol treated mESCs ( WT ) ( y - axis ) . Four independent biological replicates analyzed . Data information : Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data .

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

(H) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(+339)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted. (I) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(+339)-GFP-lncRNA-c1, BoxB (+339)-GFP-lncRNA-c2 (BoxB(+339), triangles) (x-axis) in 8 day 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates analyzed.Data information: Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data.


( a - f ) Atg8 was co - incubated with Atg19 or Atg34 ( wild type and mutants ) at concentrations of 40 μM and 800 μM , respectively , and run on a size exclusion column . Aliquots of individual fractions were run on SDS - polyacrylamide gel electrophoresis ( SDS - PAGE ) gels and Coomassie stained . The white box indicates the N - terminal domain , the light grey box the coiled - coil domain , the dark grey box the Ams1 - binding domain and the purple box the C - terminal domain .

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(a-f) Atg8 was co-incubated with Atg19 or Atg34 (wild type and mutants) at concentrations of 40 μM and 800 μM, respectively, and run on a size exclusion column. Aliquots of individual fractions were run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and Coomassie stained. The white box indicates the N-terminal domain, the light grey box the coiled-coil domain, the dark grey box the Ams1-binding domain and the purple box the C-terminal domain.


( g - i ) Atg8 was co - incubated with the indicated Atg19 domains at 40 μM and run on a size exclusion column . Individual elution profiles are shown above ; Coomassie stained SDS - PAGE gels below . Columns : ( a - f , i ) S200 3 . 2 / 30 ; ( g , h ) S75 3 . 2 / 30 . M : size marker . All experiments shown have been carried out twice .

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(g-i) Atg8 was co-incubated with the indicated Atg19 domains at 40 μM and run on a size exclusion column. Individual elution profiles are shown above; Coomassie stained SDS-PAGE gels below. Columns: (a-f,i) S200 3.2/30; (g,h) S75 3.2/30. M: size marker. All experiments shown have been carried out twice.


a ) Anti - Atg8 western blot of the input and pellet fractions of a pulldown experiment using GST - Atg19 as bait and Atg8 as prey . The black vertical line indicates that unrelated samples were run on the same gel but in between the two samples on the left and the one on the right ( Supplementary Fig . 7 ) .

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a) Anti-Atg8 western blot of the input and pellet fractions of a pulldown experiment using GST-Atg19 as bait and Atg8 as prey. The black vertical line indicates that unrelated samples were run on the same gel but in between the two samples on the left and the one on the right (Supplementary Fig. 7).


( b ) Coomassie stained gels of pulldown experiments using the GST - prApe1 propeptide as bait to pull down Atg19 , the Atg19 - Atg34 chimaera and Atg34 , respectively . The Atg19 - Atg34 chimaera contained residues 1 - 361 of Atg19 and residues 348 - 412 of Atg34 .

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(b) Coomassie stained gels of pulldown experiments using the GST-prApe1 propeptide as bait to pull down Atg19, the Atg19-Atg34 chimaera and Atg34, respectively. The Atg19-Atg34 chimaera contained residues 1-361 of Atg19 and residues 348-412 of Atg34.


( c ) Yeast two - hybrid ( Y2H ) assay testing for the interaction of Atg11 with Atg19 , the Atg19 - Atg34 chimaera and Atg34 , respectively .

(c) Yeast two-hybrid (Y2H) assay testing for the interaction of Atg11 with Atg19, the Atg19-Atg34 chimaera and Atg34, respectively.


( d ) Anti - Atg8 western blot using the indicated GST fusion proteins as bait to pull down Atg8 . The pulldowns were conducted in the presence or absence of the prApe1 propeptide . See Supplementary Fig . 1 for input gel . All experiments shown have been carried out twice . Images of uncropped western blots and gels can be found in Supplementary Fig . 7 .

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

(d) Anti-Atg8 western blot using the indicated GST fusion proteins as bait to pull down Atg8. The pulldowns were conducted in the presence or absence of the prApe1 propeptide. See Supplementary Fig. 1 for input gel. All experiments shown have been carried out twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.


( a , b ) Anti - Atg8 western blots using the indicated GST fusion proteins shown on the left as bait to pull down Atg8 . See Supplementary Fig . 1 for input gels .

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(a,b) Anti-Atg8 western blots using the indicated GST fusion proteins shown on the left as bait to pull down Atg8. See Supplementary Fig. 1 for input gels.


( c ) Alignment of the Atg19 C - terminus lacking the LIR motif with the region around the LIR motif of human p62 ( NP _ 003891 . 1 ) . The amino acids shown in magenta and green indicate the residues mutated in the experiments shown in d - f .

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(c) Alignment of the Atg19 C-terminus lacking the LIR motif with the region around the LIR motif of human p62 (NP_003891.1). The amino acids shown in magenta and green indicate the residues mutated in the experiments shown in d-f.


( d ) Anti - Atg8 western blots with the indicated GST fusion proteins as bait to pull down Atg8 . Quantification of the amount of Atg8 pulled down by the indicated GST fusion protein . The graph is based on three independent experiments ( N = 3 ) , one of which is shown below the graph . Shown are the averages and s . d . WT , wild type .

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

(d) Anti-Atg8 western blots with the indicated GST fusion proteins as bait to pull down Atg8. Quantification of the amount of Atg8 pulled down by the indicated GST fusion protein. The graph is based on three independent experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. WT, wild type.


( e ) Green fluorescent protein ( GFP ) - trap pulldown experiment using yeast cells with integrated GFP - Atg8 transformed with the indicated Myc - Atg19 constructs .

(e) Green fluorescent protein (GFP)-trap pulldown experiment using yeast cells with integrated GFP-Atg8 transformed with the indicated Myc-Atg19 constructs.


( f ) Pulldown experiment using the GST - prApe1 propeptide ( propep . ) to pull down Atg8 through the indicated Atg19 proteins . The graph is based on three experiments ( N = 3 ) , one of which is shown below the graph . Shown are the averages and s . d . See Supplementary Fig . 2 for input gel .

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

(f) Pulldown experiment using the GST-prApe1 propeptide (propep.) to pull down Atg8 through the indicated Atg19 proteins. The graph is based on three experiments (N = 3), one of which is shown below the graph. Shown are the averages and s.d. See Supplementary Fig. 2 for input gel.


g ) Representative profile of a size exclusion chromatography run using a 10 / 300 S200 column . Atg8 was run at 400 μM and the Atg19 C - terminus at 40 μM . For the complex run both proteins were preincubated at the concentrations mentioned . The average number of Atg8 molecules shifted into the complex peak per Atg19 C - terminus was 4 . 14 ( average of three experiments ) . The experiments shown in a , b , d , f , g have been conducted three times ; the experiment shown in e has been conducted twice . Images of uncropped western blots and gels can be found in Supplementary Fig . 7 .

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g) Representative profile of a size exclusion chromatography run using a 10/300 S200 column. Atg8 was run at 400 μM and the Atg19 C-terminus at 40 μM. For the complex run both proteins were preincubated at the concentrations mentioned. The average number of Atg8 molecules shifted into the complex peak per Atg19 C-terminus was 4.14 (average of three experiments). The experiments shown in a,b,d,f,g have been conducted three times; the experiment shown in e has been conducted twice. Images of uncropped western blots and gels can be found in Supplementary Fig. 7.


( a ) Giant unilamellar vesicles ( GUVs ) incubated with mCherry - Atg19 . The membrane was marked by incorporation of an Oregon Green labelled lipid . ( b ) GUV coated with GFP - Atg8 incubated with mCherry - Atg19 . ( c ) Schematic representation showing the proteins and reaction resulting in GFP - Atg8 conjugation to the GUV shown in b . ( d ) GUVs containing nickellipids incubated with GFP - Atg8 - 6 × His and wild - type mCherry - Atg19 .

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(a) Giant unilamellar vesicles (GUVs) incubated with mCherry-Atg19. The membrane was marked by incorporation of an Oregon Green labelled lipid. (b) GUV coated with GFP-Atg8 incubated with mCherry-Atg19. (c) Schematic representation showing the proteins and reaction resulting in GFP-Atg8 conjugation to the GUV shown in b. (d) GUVs containing nickellipids incubated with GFP-Atg8-6×His and wild-type mCherry-Atg19.


( f ) Quantification of membrane binding by the indicated mCherry - Atg19 proteins . The experimental set - up is shown in e . The graph is based on three experiments . Shown are the averages and s . d . of these three experiments ( N = 3 , on the basis of 318 GUVs for Atg19 , 171 GUVs for Atg19 W412A , 166 GUVs for Atg19 F376A , F379A , W412A , 188 GUVs for the enhanced GFP ( eGFP ) control ) . Scale bars , 5 μm . Abbreviations : 3 , Atg3 ; 5 , Atg5 ; 7 , Atg7 ; 8 , Atg8 ; 12 , Atg12 ; 16 , Atg16 ; 19 , Atg19 redundant . Experiments shown in a , b have been conducted twice and the experiment in d three times .

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(f) Quantification of membrane binding by the indicated mCherry-Atg19 proteins. The experimental set-up is shown in e. The graph is based on three experiments. Shown are the averages and s.d. of these three experiments (N = 3, on the basis of 318 GUVs for Atg19, 171 GUVs for Atg19 W412A, 166 GUVs for Atg19 F376A, F379A, W412A, 188 GUVs for the enhanced GFP (eGFP) control). Scale bars, 5 μm. Abbreviations: 3, Atg3; 5, Atg5; 7, Atg7; 8, Atg8; 12, Atg12; 16, Atg16; 19, Atg19 redundant. Experiments shown in a,b have been conducted twice and the experiment in d three times.


( a ) prApe1 propeptide recruitment to Atg8 - harbouring GUVs in the presence and absence of Atg19 . The experimental set - up is shown on the right .

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(a) prApe1 propeptide recruitment to Atg8-harbouring GUVs in the presence and absence of Atg19. The experimental set-up is shown on the right.


( b ) Atg19 - dependent envelopment of prApe1 - coated beads with Atg8 - positive membranes . The experimental set - up is shown on the right . The membrane was labelled by incorporation of a rhodamine - labelled lipid . (

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(b) Atg19-dependent envelopment of prApe1-coated beads with Atg8-positive membranes. The experimental set-up is shown on the right. The membrane was labelled by incorporation of a rhodamine-labelled lipid. (


( c ) Quantification of the percentage of GUV - associated eGFP - Ape1 - coated beads showing detectable membrane bending ( N = 3 ; 40 GUVs per condition ) . The experimental set - up is shown in b . The density of Atg8 - 6 × His on the membrane was titrated by different amounts of nickel lipids in the GUV membrane . The graph is based on at least three independent experiments . Shown are the averages and s . d . (

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

(c) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending (N = 3; 40 GUVs per condition). The experimental set-up is shown in b. The density of Atg8-6×His on the membrane was titrated by different amounts of nickel lipids in the GUV membrane. The graph is based on at least three independent experiments. Shown are the averages and s.d. (


( d ) Quantification of the percentage of GUV - associated eGFP - Ape1 - coated beads showing detectable membrane bending . The quantification is based on three independent experiments for the mutant Atg19 versions and 16 independent experiments for wild - type Atg19 and the no - Atg19 control ( Atg19 , N = 16 , total GUVs = 382 ; Atg19 W412A , N = 3 , total GUVs = 55 ; Atg19 F376A , F379A , N = 3 , total 66 GUVs ; Atg19 F376A , F379A , W412A , N = 3 , total GUVs = 84 ; Atg19 F376A , F379A , P385A , E386A , W412A , N = 3 , total GUVs = 60 ; Atg19 - 34 , N = 4 , total GUVs = 110 ; no Atg19 , N = 16 , total GUVs = 382 ) . Shown are the averages and s . d . The experimental set - up is as shown in b ; the p - values in c and d were calculated using a two - tailed Student t - test .

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(d) Quantification of the percentage of GUV-associated eGFP-Ape1-coated beads showing detectable membrane bending. The quantification is based on three independent experiments for the mutant Atg19 versions and 16 independent experiments for wild-type Atg19 and the no-Atg19 control (Atg19, N = 16, total GUVs = 382; Atg19 W412A, N = 3, total GUVs = 55; Atg19 F376A, F379A, N = 3, total 66 GUVs; Atg19 F376A, F379A, W412A, N = 3, total GUVs = 84; Atg19 F376A, F379A, P385A, E386A, W412A, N = 3, total GUVs = 60; Atg19-34, N = 4, total GUVs = 110; no Atg19, N = 16, total GUVs = 382). Shown are the averages and s.d. The experimental set-up is as shown in b; the p-values in c and d were calculated using a two-tailed Student t-test.


( e ) Visualization of Atg19 during the envelopment of prApe1 - coated beads with Atg8 - containing membranes . Note that Atg19 directly binds the prApe1 - coated beads ( arrows ) . The experimental set - up is shown on the right . Scale bars , 5 μM . Abbreviations : 8 , Atg8 ; 19 , Atg19 ; DIC , differential interference contrast . All experiments have been conducted at least three times .

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(e) Visualization of Atg19 during the envelopment of prApe1-coated beads with Atg8-containing membranes. Note that Atg19 directly binds the prApe1-coated beads (arrows). The experimental set-up is shown on the right. Scale bars, 5 μM. Abbreviations: 8, Atg8; 19, Atg19; DIC, differential interference contrast. All experiments have been conducted at least three times.


( a ) Anti - Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs . The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole . Rap . , rapamycin ; Log . ,

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

(a) Anti-Ape1 western blot of atg19Δ cells transformed with the indicated expression constructs. The lower Ape1 band indicates prApe1 processing and thus its delivery into the vacuole. Rap., rapamycin; Log.,