figf2 ( a ) Intracellular spermidine of five - day - oldΔspe1 cells , compared with wild - type cells . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) . ( b ) Chronological ageing of wild - type ( ▪ , o ) and polyamine - depleted Δspe1 ( ▴ , ▿ ) yeast cells with ( open symbols ) and without ( closed symbols ) supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ) . Cells were tested for cell death markers at day 3 . ( c ) Fluorescence microscopy of DHE → Eth conversion in wild - type and Δspe1 cells indicating ROS production . Scale bars , 10 μm . ( d ) Quantification ( FACS analysis ) of ROS accumulation using DHE → Eth conversion of wild - type ( WT ) and Δspe1 cells with and without supplementation of low doses of spermidine . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) . ( e ) Quantification ( FACS analysis ) of phosphatidylserine externalization and loss of membrane integrity using annexin V / PI co - staining ( at day 5 ) and of DNA fragmentation using TUNEL staining ( at day 3 ) of chronologically ageing wild - type ( WT ) and Δspe1 cells with or without supplementation of 0 . 1 mM spermidine as indicated . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(a) Intracellular spermidine of five-day-oldΔspe1 cells, compared with wild-type cells. Data represent means ± s.e.m. (n = 3; *P 0.001).(b) Chronological ageing of wild-type (▪, o) and polyamine-depleted Δspe1 (▴, ▿) yeast cells with (open symbols) and without (closed symbols) supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4). Cells were tested for cell death markers at day 3.(c) Fluorescence microscopy of DHE→Eth conversion in wild-type and Δspe1 cells indicating ROS production. Scale bars, 10 μm.(d) Quantification (FACS analysis) of ROS accumulation using DHE→Eth conversion of wild-type (WT) and Δspe1 cells with and without supplementation of low doses of spermidine. Data represent means ± s.e.m. (n = 4; *P 0.001).(e) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity using annexin V/PI co-staining (at day 5) and of DNA fragmentation using TUNEL staining (at day 3) of chronologically ageing wild-type (WT) and Δspe1 cells with or without supplementation of 0.1 mM spermidine as indicated. Data represent means ± s.e.m. (n = 3; *P 0.001).


figf3 ( a ) Fluorescence microscopy of DHE → Eth conversion and annexin V ( green ) / PI ( red ) co - staining of wild - type cells at day 18 of the chronological ageing experiment shown in Fig . 1b . Scale bars , 10 μm . ( b , c ) Quantification of DHE → Eth conversion ( b ) and annexin V / PI co - staining ( c ) using FACS analysis performed at indicated time - points of the chronological ageing experiment shown in Fig . 1b . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 001 ) . ( d ) Electron microscopy of young log - phase cells ( day 0 ) and of 20 - day - old wild - type cells aged without ( control ) or with spermidine ( 4 mM ) . Representative cells are shown ( for an overview see Supplementary Information , Fig . S3c ) . ( e ) Fluorescence microscopy of chronologically aged wild - type cells ( day 3 and 14 ) expressing an EGFP - tagged version of the yeast HMGB1 homologue ( Nhp6A - EGFP ) with or without ( control ) addition of 4 mM spermidine . Scale bars , 5 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) Fluorescence microscopy of DHE→Eth conversion and annexin V (green)/PI (red) co-staining of wild-type cells at day 18 of the chronological ageing experiment shown in Fig. 1b. Scale bars, 10 μm. (b, c) Quantification of DHE→Eth conversion (b) and annexin V/PI co-staining (c) using FACS analysis performed at indicated time-points of the chronological ageing experiment shown in Fig. 1b. Data represent means ± s.e.m. (n = 3; *P 0.001).(d) Electron microscopy of young log-phase cells (day 0) and of 20-day-old wild-type cells aged without (control) or with spermidine (4 mM). Representative cells are shown (for an overview see Supplementary Information, Fig. S3c).(e) Fluorescence microscopy of chronologically aged wild-type cells (day 3 and 14) expressing an EGFP-tagged version of the yeast HMGB1 homologue (Nhp6A-EGFP) with or without (control) addition of 4 mM spermidine. Scale bars, 5 μm.


figf4 ( a ) Relative acetylation ( normalized to controls at each day ) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis . A representative blot using site specific antibodies is shown in b . Data represent means ± s . e . m . ( n = 3 ) . Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification ( examples are shown in Supplementary Information , Fig . S4 ) . For calculation details see section on Methods . P values indicate the result of a two - factor ANOVA corrected by Bonferroni post hoc test . ( b ) Immunoblot of whole - cell extracts of wild - type cells chronologically aged to designated time - points with ( + ) or without ( − ) spermidine application . Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues . Full scans of blots are available in Supplementary Information , Fig . S5 . ( c ) Relative acetylation ( normalized to wild - type controls at each day ) of indicated histone H3 Lys residues of Δspe1 cells ( open bars ) , compared with wild - type cells ( closed bars ) during chronological ageing . Data represent means ± s . e . m . ( n = 3 ) . P values indicate the result of a two - factor ANOVA corrected by Bonferroni post hoc test . ( d ) Relative acetylation ( normalized to day 1 ) of histone H3 Lys 18 residue of chronologically ageing wild - type cells . Data represent means ± s . e . m . ( n = 3 ) . ( e ) Relative acetylation ( normalized to controls ) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine . Data represent quantification of two independent experiments performed with cells obtained from two different donors . ( f ) Immunoblot analysis of liver cell extracts ( applied in serial dilutions ) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age . Blots were probed with antibodies recognizing total histone H3 ( C - terminal epitope ) or specific for acetylated lysine 18 residue ( N - terminal epitope ) . Full scans of blots are available in Supplementary Information , Fig . S5 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Relative acetylation (normalized to controls at each day) of indicated histone H3 Lys residues determined by quantification of immunoblot analysis. A representative blot using site specific antibodies is shown in b. Data represent means ± s.e.m. (n = 3). Serial dilutions of protein extracts were applied in western blots to verify linearity before quantification (examples are shown in Supplementary Information, Fig. S4). For calculation details see section on Methods. P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test. (b) Immunoblot of whole-cell extracts of wild-type cells chronologically aged to designated time-points with (+) or without (−) spermidine application. Blots were probed with antibodies against total histone H3 or H3 acetylation sites at the indicated Lys residues. Full scans of blots are available in Supplementary Information, Fig. S5.(c) Relative acetylation (normalized to wild-type controls at each day) of indicated histone H3 Lys residues of Δspe1 cells (open bars), compared with wild-type cells (closed bars) during chronological ageing. Data represent means ± s.e.m. (n = 3). P values indicate the result of a two-factor ANOVA corrected by Bonferroni post hoc test.(d) Relative acetylation (normalized to day 1) of histone H3 Lys 18 residue of chronologically ageing wild-type cells. Data represent means ± s.e.m. (n = 3).(e) Relative acetylation (normalized to controls) of histone H3 Lys 14 and 18 residues of cultured human PBMC after 6 days of incubation with or without 20 nM spermidine. Data represent quantification of two independent experiments performed with cells obtained from two different donors.(f) Immunoblot analysis of liver cell extracts (applied in serial dilutions) obtained from mice fed with 3 mM spermidine for 200 days and respective control mice of the same age. Blots were probed with antibodies recognizing total histone H3 (C-terminal epitope) or specific for acetylated lysine 18 residue (N-terminal epitope). Full scans of blots are available in Supplementary Information, Fig. S5.


figf5 ( a ) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 ) . ( b ) Chronological ageing of wild - type ( ▪ , o ) and Δiki3Δsas3 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 4 ) . ( c ) Quantification ( FACS analysis ) of phosphatidylserine externalization and loss of membrane integrity ( annexinV / PI costaining ) as well as ROS production ( DHE - > Eth conversion ) performed at day 20 of the experiment shown in b . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 001 ) . ( d ) Relative inhibition of histone acetyltransferase activity ( HAT - activity ) by spermidine determined by an in vitro HAT - activity assay of yeast nuclear extracts of wild - type cells . Data represent means ± s . e . m . of three independent experiments ( * P = 0 . 024 ) . ( g ) Relative change of ATG7 , ATG11 and ATG15 mRNA levels by spermidine supplementation ( normalised to controls ) after ten days of chronological ageing as determined by reverse transcriptase real - time PCR . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 05 and * * P 0 . 01 ) . ( h ) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter ( pATG7 ) determined by chromatin immunoprecipitation with H3 - K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR . Signals specific for indicated promoter regions were initially compared with non - immunoprecipitated samples and afterwards normalized to the pATG7 signals . Data represent means ± s . e . m . ( n = 3 ) . The P value was calculated using a two - factor ANOVA with promoter and condition ( treated and non - treated ) as independent factors .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Relative acetylation of histone H3 Lys 9 and 14 residues determined by quantification of immunoblot analysis performed at day 20 of the ageing experiment shown in panel b. Data represent means ± s.e.m. (n = 3; *P 0.05). (b) Chronological ageing of wild-type (▪, o) and Δiki3Δsas3 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4). (c) Quantification (FACS analysis) of phosphatidylserine externalization and loss of membrane integrity (annexinV/PI costaining) as well as ROS production (DHE->Eth conversion) performed at day 20 of the experiment shown in b. Data represent means ± s.e.m. (n = 4; *P 0.001).(d) Relative inhibition of histone acetyltransferase activity (HAT-activity) by spermidine determined by an in vitro HAT-activity assay of yeast nuclear extracts of wild-type cells. Data represent means ± s.e.m. of three independent experiments (*P = 0.024).(g) Relative change of ATG7, ATG11 and ATG15 mRNA levels by spermidine supplementation (normalised to controls) after ten days of chronological ageing as determined by reverse transcriptase real-time PCR. Data represent means ± s.e.m. (n = 3; *P 0.05 and **P 0.01).(h) Relative histone H3 Lys 18 acetylation of indicated promoter regions normalized to the acetylation of ATG7 promoter (pATG7) determined by chromatin immunoprecipitation with H3-K18Ac specific antibody and subsequent quantification of precipitated promoter DNA using quantitative real time PCR. Signals specific for indicated promoter regions were initially compared with non-immunoprecipitated samples and afterwards normalized to the pATG7 signals. Data represent means ± s.e.m. (n = 3). The P value was calculated using a two-factor ANOVA with promoter and condition (treated and non-treated) as independent factors.


figf6 ( a ) Fluorescence microscopy of wild - type yeast cells expressing an EGFP - Atg8p fusion protein with or without ( control ) treatment of 4 mM spermidine for 48 h . White arrows indicate vacuolar localization of EGFP - Atg8p indicative of autophagy . Scale bars , 5 μm . ( b ) Relative alkaline phosphatase activity ( ALP activity ) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with ( open bars ) or without ( closed bars ) application of spermidine ( 4 mM ) . Data represent means ± s . e . m . ( n = 3 * ; P 0 . 01 and * * P 0 . 001 ) . ( c ) Relative alkaline phosphatase activity ( ALP activity ) indicative of autophagy during chronological ageing of wild - type ( closed bars ) and Δiki3Δsas3 ( open bars ) cells . Data represent means ± s . e . m . ( n = 3 ; * P 0 . 01 and * * P 0 . 001 ) . ( d ) Chronological ageing of wild - type ( ▪ , o ) and Δatg7 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 4 mM ) at day 1 . Data represent means ± s . e . m . ( n = 4 ) . ( e ) Chronological ageing on water of wild - type ( ▪ , o ) and Δatg7 ( ▴ , ▿ ) with ( open symbols ) or without ( closed symbols ) addition of spermidine ( 8 mM ) at day 0 . Data represent means ± s . e . m . ( n = 4 ) . ( f ) Quantification ( fluorescence reader ) of ROS production ( DHE → Eth conversion ) by wild - type and Δatg7 cells with and without supplementation of spermidine ( 8 mM ) , obtained from the ageing experiment shown in panel e . Data represent means ± s . e . m . ( n = 4 ; * P 0 . 07 and * * P 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) Fluorescence microscopy of wild-type yeast cells expressing an EGFP-Atg8p fusion protein with or without (control) treatment of 4 mM spermidine for 48 h. White arrows indicate vacuolar localization of EGFP-Atg8p indicative of autophagy. Scale bars, 5 μm.(b) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of pho8ΔC60 yeast with (open bars) or without (closed bars) application of spermidine (4 mM). Data represent means ± s.e.m. (n = 3 *; P 0.01 and **P 0.001).(c) Relative alkaline phosphatase activity (ALP activity) indicative of autophagy during chronological ageing of wild-type (closed bars) and Δiki3Δsas3 (open bars) cells. Data represent means ± s.e.m. (n = 3; *P 0.01 and **P 0.001).(d) Chronological ageing of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (4 mM) at day 1. Data represent means ± s.e.m. (n = 4).(e) Chronological ageing on water of wild-type (▪, o) and Δatg7 (▴, ▿) with (open symbols) or without (closed symbols) addition of spermidine (8 mM) at day 0. Data represent means ± s.e.m. (n = 4).(f) Quantification (fluorescence reader) of ROS production (DHE→Eth conversion) by wild-type and Δatg7 cells with and without supplementation of spermidine (8 mM), obtained from the ageing experiment shown in panel e. Data represent means ± s.e.m. (n = 4; *P 0.07 and **P 0.01).


figf7 ( a ) Fluorescence microscopy of Hoechst - counterstained HeLa cells transiently transfected with LC3 - GFP subjected to 100 μM spermidine for 6 h . Representative pictures are shown . ( b ) Percentage of adherent cells exhibiting a clear LC3 - GFP relocalization into cytoplasmic vacuoles . Numbers were determined using micrographs of Hoechst - counterstained HeLa cells as representatively shown in a . Data represent means ± s . d . ( n = 3 ) . ( c ) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days , compared with controls ( without spermidine ) . Nuclei were visualized by Hoechst staining . Scale bars , 10 μm . ( d ) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10 % glucose ( starved ) for 48 h , compared with normal food ( control ) . Data represent means ± s . e . m . of at least 20 flies for each group ( * P 0 . 01 ) . ( e , f ) Survival of Drosophila during ageing without ( control ) and with supplementation of food at various concentrations of spermidine ( as indicated ) . Autophagy - deficient flies ( f ) homozygous mutant for Atg7 ( Δatg7 ) were compared with flies capable of autophagy ( e ) and heterozygous for Atg7 ( control ) . For details of strains and additional wild - type controls , see Supplementary Information Methods and Fig . S6h , i . ( g ) Fluorescence microscopy of C . elegans transgenic embryos expressing a full - length plgg - 1DsRED : : LGG - 1 fusion protein indicative of autophagic activity . Shown are two representative pictures of embryos untreated ( control ) or treated with spermidine ( 0 . 2 mM ) supplementation of food . ( h ) Quantification of autophagic activity through measurement of DsRED : : LGG - 1 pixel intensity from images of wild - type animals shown in g , and bec - 1 RNAi knockdown animals ( bec - 1 RNAi ) . Data represent means ± s . e . m . ( n = 3 ) with at least 25 images processed for each trial . ( i ) Survival of C . elegans during ageing with and without ( control ) supplementation of food ( UV - killed E . coli ) with spermidine ( 0 . 2 mM ) . Wild - type ( N2 ) animals were compared with bec - 1 RNAi animals deficient in autophagy induction . For mean life spans see Supplementary Information , Table S3 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Fluorescence microscopy of Hoechst-counterstained HeLa cells transiently transfected with LC3-GFP subjected to 100 μM spermidine for 6 h. Representative pictures are shown. (b) Percentage of adherent cells exhibiting a clear LC3-GFP relocalization into cytoplasmic vacuoles. Numbers were determined using micrographs of Hoechst-counterstained HeLa cells as representatively shown in a. Data represent means ± s.d. (n = 3).(c) LysoTracker Red staining of vacuoles indicative of autophagy in oesophagus tissue from flies fed with 1 mM spermidine for two days, compared with controls (without spermidine). Nuclei were visualized by Hoechst staining. Scale bars, 10 μm.(d) Quantification of autophagic vesicles per nucleus in LysoTracker Red stained muscle tissue of female flies fed with supplementation of 1 mM spermidine or with 10% glucose (starved) for 48 h, compared with normal food (control). Data represent means ± s.e.m. of at least 20 flies for each group (*P 0.01).(e, f) Survival of Drosophila during ageing without (control) and with supplementation of food at various concentrations of spermidine (as indicated). Autophagy-deficient flies (f) homozygous mutant for Atg7 (Δatg7) were compared with flies capable of autophagy (e) and heterozygous for Atg7 (control). For details of strains and additional wild-type controls, see Supplementary Information Methods and Fig. S6h, i.(g) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRED::LGG-1 fusion protein indicative of autophagic activity. Shown are two representative pictures of embryos untreated (control) or treated with spermidine (0.2 mM) supplementation of food. (h) Quantification of autophagic activity through measurement of DsRED::LGG-1 pixel intensity from images of wild-type animals shown in g, and bec-1 RNAi knockdown animals (bec-1 RNAi). Data represent means ± s.e.m. (n = 3) with at least 25 images processed for each trial.(i) Survival of C. elegans during ageing with and without (control) supplementation of food (UV-killed E. coli) with spermidine (0.2 mM). Wild-type (N2) animals were compared with bec-1 RNAi animals deficient in autophagy induction. For mean life spans see Supplementary Information, Table S3.


Figure 1A . Measurement of oxygen consumption rate ( OCR ) , extracellular acidification rate ( ECAR ) and oxidative burst of monocytes from COVID - 19 patients ( COVID ) and healthy controls ( CTR ) . OCR was measured in real time , under basal condition and in response to mitochondria inhibitors : oligomycin ( Oligo , 2 μM ) , cyanide - 4 - ( trifluoromethoxy ) phenylhydrazone ( FCCP , 0 . 5 μM ) , and antimycin A plus rotenone ( Rot / AA , 0 . 5 μM ) . Oxidative burst was measured in response to PMA / ionomycin ( PMA / Iono , 1 μg / mL ) . Scatter plots show the quantification of basal respiration ( indicated as Basal OCR ) , ATP - linked respiration ( ATP - linked ) , maximal respiration ( Max Resp ) , proton - leak , spare respiratory capacity ( Spare ) , oxidative burst immediate response and basal ECAR . The maximal respiration kinetic range and the oxidative burst kinetic range are also reported and were obtained by analyzing the area under the curve ( AUC ) from the sixth to the tenth measurement and from the tenth to the thirteenth measurement , respectively . Data represent individual values , mean and standard error of the mean ( CTR , n = 12 ; COVID , n = 13 ) . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure . Representative traces of OCR of monocytes from COVID - 19 patients ( COVID ) and healthy controls ( CTR ) are also reported . B . Representative electron micrographs of mitochondrial ultrastructural changes observed in COVID - 19 monocytes . Panels " a " and " c " are representative electron micrographs of monocytes from healthy controls , whereas panels " b " , " d " and " e " are representative electron micrographs of monocytes from COVID - 19 patients . In panel " f " the quantification of mitochondrial area , external perimeter , Feret ' s diameter , aspect ratio circularity and roundness are reported ( n = 225 mitochondria for each group ) . Black arrows in panel " d " indicate mitochondria ; N refers to nucleus . Exact p values are reported in panel " f " .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Measurement of oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and oxidative burst of monocytes from COVID-19 patients (COVID) and healthy controls (CTR). OCR was measured in real time, under basal condition and in response to mitochondria inhibitors: oligomycin (Oligo, 2 μM), cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP, 0.5 μM), and antimycin A plus rotenone (Rot/AA, 0.5 μM). Oxidative burst was measured in response to PMA/ionomycin (PMA/Iono, 1 μg/mL). Scatter plots show the quantification of basal respiration (indicated as Basal OCR), ATP-linked respiration (ATP-linked), maximal respiration (Max Resp), proton-leak, spare respiratory capacity (Spare), oxidative burst immediate response and basal ECAR. The maximal respiration kinetic range and the oxidative burst kinetic range are also reported and were obtained by analyzing the area under the curve (AUC) from the sixth to the tenth measurement and from the tenth to the thirteenth measurement, respectively. Data represent individual values, mean and standard error of the mean (CTR, n=12; COVID, n=13). Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure. Representative traces of OCR of monocytes from COVID-19 patients (COVID) and healthy controls (CTR) are also reported.B. Representative electron micrographs of mitochondrial ultrastructural changes observed in COVID-19 monocytes. Panels "a" and "c" are representative electron micrographs of monocytes from healthy controls, whereas panels "b", "d" and "e" are representative electron micrographs of monocytes from COVID-19 patients. In panel "f" the quantification of mitochondrial area, external perimeter, Feret's diameter, aspect ratio circularity and roundness are reported (n=225 mitochondria for each group). Black arrows in panel "d" indicate mitochondria; N refers to nucleus. Exact p values are reported in panel "f".


Figure 2B . Heatmaps representing different monocyte clusters identified by FlowSOM , with percentages in healthy donors ( CTR ) and COVID - 19 patients . The colors in the heat map represent the median of the arcsinh , 0 - 1 transformed marker expression calculated over cells from all the samples , varying from blue for lower expression to red for higher expression . Each cluster has a unique color assigned ( bar on the left which colors correspond to those used in panel A ) . C . Differential analysis between healthy donors ( CTR , n = 8 ) and COVID - 19 ( COVID , n = 7 ) . The heatmap represents arcsine - square - root transformed cell frequencies that were subsequently normalized per cluster ( rows ) to mean of zero and standard deviation of one . The color of the heat varies from blue , indicating relative under - representation , to red indicating relative over - representation . Bar and numbers at the right indicate significant differentially abundant clusters ( green ) and adjusted p values ( Differential analysis performed by diffcyt package present in the Catalyst package ) . The statistical analyses was done using GLMM ( according to the actual guidelines ) and all p - values were Bonferroni corrected . Clusters were sorted according to the adjusted p values , so that the cluster at the top shows the most significant abundance changes between the two conditions . D . Boxes and whiskers plots representing maximum , minimum , median value , 25 and 75 percentile of different subpopulations of monocytes in healthy donors ( CTR , n = 8 ) and COVID - 19 patients ( COVID , n = 7 ) . E . Scatter plots showing the frequency of PD - 1 + cells , PD - L1 + cells among total monocytes in healthy controls ( CTR , n = 8 ) and COVID - 19 patients ( COVID , n = 7 ) . A scatter plot reporting the median fluorescence intensity ( MFI ) of PD - L1 in total monocytes is also shown . Data represent individual values , mean ± standard error of the mean . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. Heatmaps representing different monocyte clusters identified by FlowSOM, with percentages in healthy donors (CTR) and COVID-19 patients. The colors in the heat map represent the median of the arcsinh, 0-1 transformed marker expression calculated over cells from all the samples, varying from blue for lower expression to red for higher expression. Each cluster has a unique color assigned (bar on the left which colors correspond to those used in panel A).C. Differential analysis between healthy donors (CTR, n=8) and COVID-19 (COVID, n=7). The heatmap represents arcsine-square-root transformed cell frequencies that were subsequently normalized per cluster (rows) to mean of zero and standard deviation of one. The color of the heat varies from blue, indicating relative under-representation, to red indicating relative over-representation. Bar and numbers at the right indicate significant differentially abundant clusters (green) and adjusted p values (Differential analysis performed by diffcyt package present in the Catalyst package). The statistical analyses was done using GLMM (according to the actual guidelines) and all p-values were Bonferroni corrected. Clusters were sorted according to the adjusted p values, so that the cluster at the top shows the most significant abundance changes between the two conditions. D. Boxes and whiskers plots representing maximum, minimum, median value, 25 and 75 percentile of different subpopulations of monocytes in healthy donors (CTR, n=8) and COVID-19 patients (COVID, n=7).E. Scatter plots showing the frequency of PD-1+ cells, PD-L1+ cells among total monocytes in healthy controls (CTR, n=8) and COVID-19 patients (COVID, n=7). A scatter plot reporting the median fluorescence intensity (MFI) of PD-L1 in total monocytes is also shown. Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


Figure 3A . Quantification of cytokines and other mediators in plasma obtained from COVID - 19 patients ( COVID , n = 15 ) and healthy controls ( CTR , n = 15 ) . Data represent individual values , mean ± standard error of the mean . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure . B . Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID - 19 patients . Scatter plots showing the quantification of immature and mature monocytes in healthy controls ( CTR , n = 6 ) and COVID - 19 patients ( COVID , n = 7 ) are reported . Mann - Whitney test was used for statistical analysis . Exact p values are reported in the figure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Quantification of cytokines and other mediators in plasma obtained from COVID-19 patients (COVID, n=15) and healthy controls (CTR, n=15). Data represent individual values, mean ± standard error of the mean. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.B. Representative dot plots showing percentages of immature and mature circulating monocytes in CTR and COVID-19 patients. Scatter plots showing the quantification of immature and mature monocytes in healthy controls (CTR, n=6) and COVID-19 patients (COVID, n=7) are reported. Mann-Whitney test was used for statistical analysis. Exact p values are reported in the figure.


Figure 3 ( D ) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein ( Kd = 9 . 5 + / - 2 . 9 µM ) , in strength similar to the binding of the known PTS1 motif from Idp3 ( Kd = 5 . 5 + / - 0 . 5 µM ) . The plots represent the mean of three independent experiments , and the error bars represent the standard deviation . ( F ) Subcellular fractionations followed by enzymatic activity assays show that untagged , native , Glr1 is active in peroxisomes , in addition to its known activity in mitochondria . Fum1 and Pox2 were used as markers for mitochondrial , and peroxisomal fractions , respectively . ( H ) Yeast - 2 - hybrid assay shows Glr1 interacts in vivo with both Pex5 full - length and Pex5 N ' domain .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(D) A fluorescence anisotropy experiment demonstrated direct binding of the Pxp3 motif with purified Pex5 protein (Kd = 9.5 +/-2.9 µM), in strength similar to the binding of the known PTS1 motif from Idp3 (Kd = 5.5 +/- 0.5 µM). The plots represent the mean of three independent experiments, and the error bars represent the standard deviation.(F) Subcellular fractionations followed by enzymatic activity assays show that untagged, native, Glr1 is active in peroxisomes, in addition to its known activity in mitochondria. Fum1 and Pox2 were used as markers for mitochondrial, and peroxisomal fractions, respectively.(H) Yeast-2-hybrid assay shows Glr1 interacts in vivo with both Pex5 full-length and Pex5 N' domain.


Figure 4 ( B ) Metabolomic analysis focused on strains with over - expression or deletion of the AYT1 gene shows a significant reduction or accumulation , respectively , in acetyl - glutamate compared to the matched control strain of each mutant . Only metabolites changing significantly in at least one of the two conditions are presented . D - EIP is D - erythro - 1 - ( Imidazole - 4 - yl ) glycerol 3 - phosphate . ( D ) A growth assay in a condition that does not induce high levels of acetyl - CoA in peroxisomes ( glucose ) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital . In stationary phase , the over - expression ( OE ) of AYT1 grows to a lower density than the control strain , plausibly due to burdening of peroxisomal functions . ( E ) A growth assay in a condition expected to elevate acetyl - glutamate levels in peroxisomes ( oleate as a sole carbon source and glutamate as a nitrogen source ) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control . Data information : The growth assays in D and E were done in three biologically independent replicates and error bars were plotted . Note that some error bars are shorter than the symbol , hence are not visible in the graph . ( G ) A β - oxidation activity assay of Δlpx1 , Δfsh3 , and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon - or 18 carbon - free fatty acids in media supplemented with 0 . 5 % glucose shows a significant reduction in β - oxidation activity compared to the wild type and the two single mutants , suggesting an overlapping role for Fsh3 with Lpx1 . This assay was done in three biologically independent replicates . Bars represent the mean and error bars represent the standard deviation .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Metabolomic analysis focused on strains with over-expression or deletion of the AYT1 gene shows a significant reduction or accumulation, respectively, in acetyl-glutamate compared to the matched control strain of each mutant. Only metabolites changing significantly in at least one of the two conditions are presented. D-EIP is D-erythro-1-(Imidazole-4-yl) glycerol 3-phosphate.(D) A growth assay in a condition that does not induce high levels of acetyl-CoA in peroxisomes (glucose) shows that all strains grow similarly to the control until stationary phase when peroxisomes become vital. In stationary phase, the over-expression (OE) of AYT1 grows to a lower density than the control strain, plausibly due to burdening of peroxisomal functions. (E) A growth assay in a condition expected to elevate acetyl-glutamate levels in peroxisomes (oleate as a sole carbon source and glutamate as a nitrogen source) demonstrates that the AYT1 overexpressing strain grows faster and to a higher density than the control. Data information: The growth assays in D and E were done in three biologically independent replicates and error bars were plotted. Note that some error bars are shorter than the symbol, hence are not visible in the graph.(G) A β-oxidation activity assay of Δlpx1, Δfsh3, and Δlpx1Δfsh3 strains supplemented with labeled 8 carbon- or 18 carbon-free fatty acids in media supplemented with 0.5% glucose shows a significant reduction in β-oxidation activity compared to the wild type and the two single mutants, suggesting an overlapping role for Fsh3 with Lpx1.This assay was done in three biologically independent replicates. Bars represent the mean and error bars represent the standard deviation.


Figure 5 ( E ) Western blot analysis of Fbp1 - HA levels in mutants Δpex3 ( no peroxisomes ) , Δpex5 ( abolished targeting of GID subunits to peroxisomes ) , and Δpex7 ( no β - oxidation due to loss of Pot1 targeting ) demonstrates that two hours after the transition from glucose to ethanol ( gluconeogenic conditions ) , control and Δpex7 cells upregulated Fbp1 - HA levels , while for Δpex3 and Δpex5 cells Fbp1 - HA levels remain low . Antibodies were used against the HA tag ( for Fbp1 ) and Actin as a loading control .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(E) Western blot analysis of Fbp1-HA levels in mutants Δpex3 (no peroxisomes), Δpex5 (abolished targeting of GID subunits to peroxisomes), and Δpex7 (no β-oxidation due to loss of Pot1 targeting) demonstrates that two hours after the transition from glucose to ethanol (gluconeogenic conditions), control and Δpex7 cells upregulated Fbp1-HA levels, while for Δpex3 and Δpex5 cells Fbp1-HA levels remain low. Antibodies were used against the HA tag (for Fbp1) and Actin as a loading control.


Figure 3 ( G ) Size - exclusion chromatography profiles and corresponding SDS - PAGE of the indicated samples . Two mutant RZZ complexes containing mutations in ROD L110F - L119F - L120K or I191M are indicated respectively as FFK and I / M . Note that in the bottom SDS - PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker . For all other shown SDS - PAGE gels , the marker precedes the first fraction .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(G) Size-exclusion chromatography profiles and corresponding SDS-PAGE of the indicated samples. Two mutant RZZ complexes containing mutations in ROD L110F-L119F-L120K or I191M are indicated respectively as FFK and I/M. Note that in the bottom SDS-PAGE the first and second lane were deliberately inverted and contain the first eluted fraction and the molecular weight marker. For all other shown SDS-PAGE gels, the marker precedes the first fraction.


Figure 4 ( B ) Coomassie and ProQ Diamond - stained gels on mChRZZSF ( 4 µM ) treated with the indicated kinases ( 1 µM ) for 15 hours at 20ºC . Rev = reversine , used at 10 µM . ( D ) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis . Subsequently , cells were immediately treated with 500 nM reversine , 3 . 3 µM nocodazole , and 10 µM MG132 for 1 hour and imaged while in mitosis . CREST serum was used to visualize kinetochores and DAPI to stain DNA . Scale bar : 10 µm . Three biological replicates were performed . ( E ) Cells treated as for panel D were treated for visualization of Spindly . Scale bar : 10 µm . ( F ) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals , normalized to the reversine - untreated control , in the indicated number of cells from the experiment shown in panels C - D . Red lines indicate mean and standard deviation . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 . ( G ) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells . RNAi treatment was performed for 48 h with 50 nM siRNA . Before fixation , cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis . Subsequently , cells were immediately treated with 3 . 3 μM nocodazole for an additional hour . CREST serum was used to visualize kinetochores and DAPI to stain DNA . Scale bar : 10 µm . Three biological replicates were performed . ( H ) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G . Red lines indicate median with interquartile range . ( I ) Levels of CENP - E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly . Three biological replicates were performed . ( J ) Scatter dot plots representing normalized total area of the CENP - E and Zwilch signals , normalized to the RNAi negative control , in the indicated number of cells from the experiment shown in panel I . Red lines indicate mean and standard deviation . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

O O B-PANEL_START O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Coomassie and ProQ Diamond-stained gels on mChRZZSF (4 µM) treated with the indicated kinases (1 µM) for 15 hours at 20ºC. Rev = reversine, used at 10 µM.(D) Levels of Zwilch at kinetochores of HeLa cells that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 500 nM reversine, 3.3 µM nocodazole, and 10 µM MG132 for 1 hour and imaged while in mitosis. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed. (E) Cells treated as for panel D were treated for visualization of Spindly. Scale bar: 10 µm.(F) Scatter dot plots representing normalized total area of the Zwilch and Spindly signals, normalized to the reversine-untreated control, in the indicated number of cells from the experiment shown in panels C-D. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.(G) Representative images showing the effects of a knockdown of the endogenous Spindly in HeLa cells. RNAi treatment was performed for 48 h with 50 nM siRNA. Before fixation, cells were synchronized in G2 phase with 9 μM RO3306 for 16 h and then released into mitosis. Subsequently, cells were immediately treated with 3.3 μM nocodazole for an additional hour. CREST serum was used to visualize kinetochores and DAPI to stain DNA. Scale bar: 10 µm. Three biological replicates were performed.(H) Scatter dot plots representing normalized intensity ratios of Spindly over CREST for individual kinetochores of cells from the experiment shown in panel G. Red lines indicate median with interquartile range.(I) Levels of CENP-E and Zwilch were assessed in control cells and in cells treated as in panel G to knockdown Spindly. Three biological replicates were performed. (J) Scatter dot plots representing normalized total area of the CENP-E and Zwilch signals, normalized to the RNAi negative control, in the indicated number of cells from the experiment shown in panel I. Red lines indicate mean and standard deviation. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


Figure 5 ( E ) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS - PAGE separation of reactions . Data information : Two technical replicates for panels ,

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(E) The phosphorylation state of RZZ and SpindlyF was monitored by ProQ Diamond after SDS-PAGE separation of reactions. Data information: Two technical replicates for panels , E


Figure 6 ( D ) Heat - induced mChRZZGFPSF and MPS1 - induced rings of mChRZZmChSF have similar diameters . As shown in panel B , heat - and MPS1 - induced rings of mChRZZmChSF also have very similar dimensions . Data information : Experiments in D were carried out once .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(D) Heat-induced mChRZZGFPSF and MPS1-induced rings of mChRZZmChSF have similar diameters. As shown in panel B, heat- and MPS1-induced rings of mChRZZmChSF also have very similar dimensions. Data information: Experiments in D were carried out once.


Figure 7 ( A ) Size - exclusion chromatography and SDS - PAGE of elution fractions of the indicated samples . Each Spindly construct was incubated with RZZ in absence ( dotted lines ) or in presence ( continuous lines ) of farnesyl transferase and farnesyl pyrophosphate . Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction . Two technical replicates were performed . ( D ) Representative images of fixed HeLa cells electroporated with full - length mChSpindly and mChSpindly354 - 605 constructs in cells depleted of endogenous Spindly by RNAi . Spindly localization was detected with an antibody against the C - terminal region of Spindly . Corona expansion or lack thereof were monitored through CENP - E . CREST serum was used to visualize kinetochores , DAPI to stain for DNA . Scale bar : 10 µm . Experiments were replicated three times . ( E ) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D . Red lines indicate median with interquartile range . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 . ( G ) Kinetochore intensities were quantified like in panel E . Red lines indicate median with interquartile range . Statistical analysis was performed with a nonparametric t - test comparing two unpaired groups ( Mann - Whitney test ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Size-exclusion chromatography and SDS-PAGE of elution fractions of the indicated samples. Each Spindly construct was incubated with RZZ in absence (dotted lines) or in presence (continuous lines) of farnesyl transferase and farnesyl pyrophosphate. Elution shifts of SpindlyF and of the resulting RZZSF complexes is indicative of successful interaction. Two technical replicates were performed.(D) Representative images of fixed HeLa cells electroporated with full-length mChSpindly and mChSpindly354-605 constructs in cells depleted of endogenous Spindly by RNAi. Spindly localization was detected with an antibody against the C-terminal region of Spindly. Corona expansion or lack thereof were monitored through CENP-E. CREST serum was used to visualize kinetochores, DAPI to stain for DNA. Scale bar: 10 µm. Experiments were replicated three times.(E) Scatter dot plots representing normalized intensity ratios of the indicated Spindly constructs over CREST for individual kinetochores of cells from the experiment shown in panel D. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000. (G) Kinetochore intensities were quantified like in panel E. Red lines indicate median with interquartile range. Statistical analysis was performed with a nonparametric t-test comparing two unpaired groups (Mann-Whitney test). Symbols indicate: n.s. = p > 0.05, ∗ = p ≤ 0.05, ∗∗ = p ≤ 0.01, ∗∗∗ = p ≤ 0.001, ∗∗∗∗ = p ≤ 0.000.


Figure 1A . Schematic representation of the experimental protocol . Abbreviations : LCM , laser capture microdissection . B , C . RT - PCR performed on RNA extracted from axons collected by LCM ( Ax ) or from eyes . ( B ) β - Actin mRNA is present both in eyes and axons , while MAP2 and H4 are present only in the eye sample , suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples ( Bellon et al , 2017 ) . Abbreviations : Ax , axonal sample ; Eye , stage 37 / 38 eye ; LCM , laser capture microdissection ; - , PCR no template control ; NT , RT no template control . F . Thermal denaturation profiles of MB , in the absence ( solid line ) and presence ( dashed line ) of increasing target concentration . Each melting curve represents the average of three separate replicates . Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments . Abbreviations : MB , molecular beacon ; Tm , MB melting temperature . H . Quantification of the expression levels of pre - mir - 181a - 1 using the 2 - ΔCt method and U6 as normalizer from small total RNA fraction ( < ~ 150 nt ) . Each data point corresponds to one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant . I . Total number of MB puncta normalized to axon length ( µm ) . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 928 puncta , 61 axons ( WT ) , 226 puncta , 15 axons ( co - MO ) , 208 puncta , 35 axons ( MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino ; pri - miR - MO , morpholino blocking pre - miR - 181a - 1 processing by targeting the Drosha cleavage site . J . Representative axons . MB puncta are indicated ( white arrows ) . Dashed white lines delineate axons . Abbreviations : MB , molecular beacon . Scale bars : 5µm . M . Frequency ( in percentage ) of puncta colocalization between MB and cy5 - pre - miR - 181a - 1 ( # MB + / pre - miR + ) . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 354 puncta , 32 axons ( MB ) , 337 puncta , 32 axons ( cy5 - pre - miR - 181a - 1 ) . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon . N . Representative image of RGC axons . White , red and blue arrows indicate , respectively , co - localized , single MB , and single pre - miR - 181a - 1 puncta . Scale bars : 5µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Schematic representation of the experimental protocol. Abbreviations: LCM, laser capture microdissection. B, C. RT-PCR performed on RNA extracted from axons collected by LCM (Ax) or from eyes. (B) β-Actin mRNA is present both in eyes and axons, while MAP2 and H4 are present only in the eye sample, suggesting the absence of contamination from cell bodies or other cells in LCM axonal samples (Bellon et al, 2017). Abbreviations: Ax, axonal sample; Eye, stage 37/38 eye; LCM, laser capture microdissection; -, PCR no template control; NT, RT no template control. F. Thermal denaturation profiles of MB, in the absence (solid line) and presence (dashed line) of increasing target concentration. Each melting curve represents the average of three separate replicates. Yellow boxes indicate the range of working temperatures for ex vivo trafficking experiments. Abbreviations: MB, molecular beacon; Tm, MB melting temperature.H. Quantification of the expression levels of pre-mir-181a-1 using the 2-ΔCt method and U6 as normalizer from small total RNA fraction (<~150 nt). Each data point corresponds to one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.I. Total number of MB puncta normalized to axon length (µm). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 928 puncta, 61 axons (WT), 226 puncta, 15 axons (co-MO), 208 puncta, 35 axons (MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; pri-miR-MO, morpholino blocking pre-miR-181a-1 processing by targeting the Drosha cleavage site.J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm.M. Frequency (in percentage) of puncta colocalization between MB and cy5-pre-miR-181a-1 (#MB+/pre-miR+). Each data point corresponds to one axon. Total number of puncta and axons analyzed: 354 puncta, 32 axons (MB), 337 puncta, 32 axons (cy5-pre-miR-181a-1). n=5 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon.N. Representative image of RGC axons. White, red and blue arrows indicate, respectively, co-localized, single MB, and single pre-miR-181a-1 puncta. Scale bars: 5µm.


Figure 2B . Representative image of a single distal RGC axon from MB - electroporated retina . Growth cone wrist and central domain are indicated with white arrow and star , respectively . Dashed white line delineates the axon . Abbreviations : MB , molecular beacon . Scale bars : 5µm . C . Illustrative kymograph . Scale bars : 5µm . D . Frequency distribution ( in percentage ) of MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) puncta along the RGC axon shaft . Each data point corresponds to one independent experiment . Total number of puncta and axons analyzed : 353 puncta , 20 axons ( endo ) , 484 puncta , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon ; ns , not significant . E . Average velocity of MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) puncta . Each data point corresponds to one punctum . Total number of puncta and axons analyzed : 353 puncta , 20 axons ( endo ) , 484 puncta , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are median with interquartile range . Abbreviations : MB , molecular beacon ; ns , not significant ; antero , anterograde movement ; retro , retrograde movement . F . MSD data for MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) tracked particles were fitted with an anomalous diffusion model and α thus calculated ( red ) . Total number of particles and axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiment . Values are mean ± SEM . Abbreviations : MB , molecular beacon . G . MSD alpha coefficient distribution for each single MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) tracked particle . Each data point corresponds to one particle . Total number of particles and axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are median with interquartile range . H . Relative frequency distribution ( percentage ) of MB ( endo ) and cy3 - pre - miR - 181a - 1 ( exo ) tracked particles . Each data point corresponds to one independent experiment . Total number of particles and axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon ; ns , not significant . J . Representative kymographs before Nocodazole ( top panel ; - Noco ) and 30 min after 2 . 4μM Noco bath - application ( bottom panel ; + Noco ) . Stationary puncta in both panels are indicated with black arrows . Scale bars : 5µm . K . Frequency distribution ( in percentage ) of MB punctum speed . Each data point corresponds to one independent experiment . Total number of puncta and axons analyzed : 358 puncta , 25 axons ( - Noco ) ; 503 puncta , 34 axons ( + Noco ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : MB , molecular beacon ; ns , not significant ; Noco , nocodazole . L . Frequency distribution of MB punctum speed . Each data point corresponds to one punctum . Total number of puncta and axons analyzed : 358 puncta , 25 axons ( - Noco ) , 503 puncta , 34 axons ( + Noco ) . n = 3 independent experiments . Values are median with interquartile range . Abbreviations : MB , molecular beacon ; ns , not significant ; Noco , nocodazole .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. Representative image of a single distal RGC axon from MB-electroporated retina. Growth cone wrist and central domain are indicated with white arrow and star, respectively. Dashed white line delineates the axon. Abbreviations: MB, molecular beacon. Scale bars: 5µm. C. Illustrative kymograph. Scale bars: 5µm. D. Frequency distribution (in percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta along the RGC axon shaft. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.E. Average velocity of MB (endo) and cy3-pre-miR-181a-1 (exo) puncta. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 353 puncta, 20 axons (endo), 484 puncta, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; antero, anterograde movement; retro, retrograde movement.F. MSD data for MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles were fitted with an anomalous diffusion model and α thus calculated (red). Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiment. Values are mean ± SEM. Abbreviations: MB, molecular beacon.G. MSD alpha coefficient distribution for each single MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particle. Each data point corresponds to one particle. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are median with interquartile range.H. Relative frequency distribution (percentage) of MB (endo) and cy3-pre-miR-181a-1 (exo) tracked particles. Each data point corresponds to one independent experiment. Total number of particles and axons analyzed: 67 particles, 20 axons (endo), 82 particles, 29 axons (exo). n=3 (endo), n=4 (exo) independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant.J. Representative kymographs before Nocodazole (top panel; - Noco) and 30 min after 2.4μM Noco bath-application (bottom panel; +Noco). Stationary puncta in both panels are indicated with black arrows. Scale bars: 5µm.K. Frequency distribution (in percentage) of MB punctum speed. Each data point corresponds to one independent experiment. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco); 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are mean ± SEM. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.L. Frequency distribution of MB punctum speed. Each data point corresponds to one punctum. Total number of puncta and axons analyzed: 358 puncta, 25 axons (-Noco), 503 puncta, 34 axons (+Noco). n=3 independent experiments. Values are median with interquartile range. Abbreviations: MB, molecular beacon; ns, not significant; Noco, nocodazole.


Figure 3B . Representative axon where MB - labeled pre - miR - 181a - 1 ( red ) and CD63 - eGFP - labeled vesicles ( green ) are co - trafficked ( white arrows ) . Abbreviations : MB , molecular beacon . Scale bars : 5µm . C . Representative kymographs . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon . Scale bars : 5µm . D . Composite kymograph shown in ( C ) where the individual traces where drawn and color coded . Yellow trajectories represent co - trafficking MB - labeled pre - miRNA ( red ) and CD63 - eGFP - labeled vesicle ( green ) . Abbreviations : MB , molecular beacon . Scale bars : 5µm . E . Representative time - lapse depicting MB - labeled pre - miR - 181a - 1 ( red arrow ) and CD63 - eGFP - positive vesicle ( green arrow ) co - trafficked along the axon shaft to the growth cone ( delineated with dashed white lines ) wrist ( white arrowhead ) and central domain ( white star ) . Scale bars : 5µm . F . Frequency ( in percentage ) of MB - labeled pre - miR - 181a - 1 co - trafficked with CD63 - eGFP - positive vesicles . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 253 puncta , 22 axons ( MB + ) , 306 puncta , 22 axons ( CD63 + ) . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon . G . Frequency distribution ( in percentage ) of MB - labeled pre - miR - 181a - 1 co - trafficked with CD63 - eGFP - positive vesicles . Each data point corresponds to one axon . Total number of puncta and axons analyzed : 92 puncta , 22 axons ( anterograde ) , 78 puncta , 22 axons ( retrograde ) . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon . I . Representative image of an electroporated and dissociated RGC . Abbreviations : CD63 , CD63 - eGFP . Scale bars : 5µm . K , L . Fluorescence intensity of MB ( K ) or CD63 - eGFP ( L ) measured in axons and growth cone central domains . Each data point corresponds to one axon or a growth cone central domain . Total number of axons analyzed : 16 . n = 5 independent experiments . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon ; GC , growth cone .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Representative axon where MB-labeled pre-miR-181a-1 (red) and CD63-eGFP-labeled vesicles (green) are co-trafficked (white arrows). Abbreviations: MB, molecular beacon. Scale bars: 5µm.C. Representative kymographs. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon. Scale bars: 5µm.D. Composite kymograph shown in (C) where the individual traces where drawn and color coded. Yellow trajectories represent co-trafficking MB-labeled pre-miRNA (red) and CD63-eGFP-labeled vesicle (green). Abbreviations: MB, molecular beacon. Scale bars: 5µm.E. Representative time-lapse depicting MB-labeled pre-miR-181a-1 (red arrow) and CD63-eGFP-positive vesicle (green arrow) co-trafficked along the axon shaft to the growth cone (delineated with dashed white lines) wrist (white arrowhead) and central domain (white star). Scale bars: 5µm.F. Frequency (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 253 puncta, 22 axons (MB+), 306 puncta, 22 axons (CD63+). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.G. Frequency distribution (in percentage) of MB-labeled pre-miR-181a-1 co-trafficked with CD63-eGFP-positive vesicles. Each data point corresponds to one axon. Total number of puncta and axons analyzed: 92 puncta, 22 axons (anterograde), 78 puncta, 22 axons (retrograde). n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon.I. Representative image of an electroporated and dissociated RGC. Abbreviations: CD63, CD63-eGFP. Scale bars: 5µm.K,L. Fluorescence intensity of MB (K) or CD63-eGFP (L) measured in axons and growth cone central domains. Each data point corresponds to one axon or a growth cone central domain. Total number of axons analyzed: 16. n=5 independent experiments. Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon; GC, growth cone.


Figure 4C . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : magenta for CD63 and yellow for Lamp1 or Rab5a . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP . Scale bars : 5μm . D . Frequency ( in percentage ) of CD63 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of puncta , axons , and independent experiments analyzed : 540 puncta , 23 axons , n = 4 ( CD63 / Rab7a ) , 416 puncta , 18 axons , n = 3 ( CD63 / LysoTracker ) , 584 puncta , 17 axons , n = 2 ( CD63 / Lamp1 ) , 649 puncta , 19 axons , n = 3 ( CD63 / Rab5a ) . Values are mean ± SEM . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP . E . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : cyan for pre - miR - 181a - 1 and yellow for Lamp1 or Rab5a . Abbreviations : Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . Scale bars : 5μm . F . Frequency ( in percentage ) of pre - miR - 181a - 1 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of puncta , axons and independent experiments analyzed : 825 puncta , 41 axons , n = 8 ( pre - miR - 181a - 1 / CD63 ) , 732 puncta , 33 axons , n = 7 ( pre - miR - 181a - 1 / Rab7a ) , 681 puncta , 29 axons , n = 5 ( pre - miR - 181a - 1 / LysoTracker ) , 501 puncta , 17 axons , n = 2 ( pre - miR - 181a - 1 / Lamp1 ) , 791 puncta , 25 axons , n = 3 ( pre - miR - 181a - 1 / Rab5a ) . Values are median with interquartile range . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP , Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . G . Representative images of co - electroporated axons . Dashed white lines delineate axons . White arrows indicate colocalized puncta . Colored arrows represent non - colocalized signal as follows : magenta for CD63 , cyan for pre - miR - 181a - 1 , yellow for Rab7a and blue for LysoTracker . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . Scale bar : 5μm . H . Frequency ( in percentage ) of pre - miR - 181a - 1 colocalization with vesicles double positive for organelle markers . Each data point corresponds to one axon . Total number of puncta , axons and independent experiments analyzed : 745 puncta , 23 axons , n = 5 ( CD63 / pre - miR - 181a - 1 / Rab7a ) , 577 puncta , 18 axons , n = 3 ( CD63 / pre - miR - 181a - 1 / LysoTracker ) . Values are median with interquartile range . Abbreviations : ns , not significant ; CD63 , CD63 - eGFP or CD63 - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 or cy5 - pre - miR - 181a - 1 . I . Representative 3D - STED super - resolution image . Total number of puncta , axons and growth cones analyzed : 99 puncta , 16 axons and 10 growth cones ( MB ) , 291 puncta , 36 axons and 15 growth cones ( cy3 - pre - miR - 181a - 1 ) . n = 2 ( MB ) , n = 3 ( cy3 - pre - miR - 181a - 1 ) independent experiments . Abbreviations : CD63 , CD63 - eGFP ; pre - miR - 181a - 1 , cy3 - pre - miR - 181a - 1 ; MB ; molecular beacon .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63 and yellow for Lamp1 or Rab5a. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP. Scale bars: 5μm.D. Frequency (in percentage) of CD63 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons, and independent experiments analyzed: 540 puncta, 23 axons, n=4 (CD63/Rab7a), 416 puncta, 18 axons, n=3 (CD63/LysoTracker), 584 puncta, 17 axons, n=2 (CD63/Lamp1), 649 puncta, 19 axons, n=3 (CD63/Rab5a). Values are mean ± SEM. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP.E. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: cyan for pre-miR-181a-1 and yellow for Lamp1 or Rab5a. Abbreviations: Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bars: 5μm.F. Frequency (in percentage) of pre-miR-181a-1 colocalization with specific organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 825 puncta, 41 axons, n=8 (pre-miR-181a-1/CD63), 732 puncta, 33 axons, n=7 (pre-miR-181a-1/Rab7a), 681 puncta, 29 axons, n=5 (pre-miR-181a-1/LysoTracker), 501 puncta, 17 axons, n=2 (pre-miR-181a-1/Lamp1), 791 puncta, 25 axons, n=3 (pre-miR-181a-1/Rab5a). Values are median with interquartile range. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP, Lamp1, Lamp1-eGFP; Rab5a, Rab5a-eGFP or Rab5a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.G. Representative images of co-electroporated axons. Dashed white lines delineate axons. White arrows indicate colocalized puncta. Colored arrows represent non-colocalized signal as follows: magenta for CD63, cyan for pre-miR-181a-1, yellow for Rab7a and blue for LysoTracker. Abbreviations: CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1. Scale bar: 5μm.H. Frequency (in percentage) of pre-miR-181a-1 colocalization with vesicles double positive for organelle markers. Each data point corresponds to one axon. Total number of puncta, axons and independent experiments analyzed: 745 puncta, 23 axons, n=5 (CD63/pre-miR-181a-1/Rab7a), 577 puncta, 18 axons, n=3 (CD63/pre-miR-181a-1/LysoTracker). Values are median with interquartile range. Abbreviations: ns, not significant; CD63, CD63-eGFP or CD63-mRFP; Rab7a, Rab7a-eGFP or Rab7a-mRFP; pre-miR-181a-1, cy3-pre-miR-181a-1 or cy5-pre-miR-181a-1.I. Representative 3D-STED super-resolution image. Total number of puncta, axons and growth cones analyzed: 99 puncta, 16 axons and 10 growth cones (MB), 291 puncta, 36 axons and 15 growth cones (cy3-pre-miR-181a-1). n=2 (MB), n=3 (cy3-pre-miR-181a-1) independent experiments. Abbreviations: CD63, CD63-eGFP; pre-miR-181a-1, cy3-pre-miR-181a-1; MB; molecular beacon.


Figure 5A . Schematic representation of the experimental protocol . B . Representative Xenopus growth cones stained with anti - Dicer and anti - Ago2 . White dashed lines delineate growth cones . Scale bars : 5µm . C . Representative E13 . 5 mice brain cross - section ( dashed red line in the schematic ) comprising the optic nerve ( ON ) stained with anti - neurofilament and anti - HA antibodies to detect RGC axons and ( FLAG - HA2 ) - Dicer , respectively . Note the absence of HA signal in wild type ( WT ) mice . The white dashed line delineates the ON . Zoom of the triple stained ON ( right panel ) : Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se ( arrowheads ) . n = 3 independent experiments . Abbreviations : WT , wild type ; HA , ( FLAG - HA2 ) - Dicer ; E13 . 5 , embryonic day 13 . 5 ; ONH , optic nerve head ; ON , optic nerve . Scale bars : 30µm . D . Representative P0 superior colliculus stratum griseum superficiale ( SGS ) ( sagittal sectioning of P0 brains along the dashed red line in the schematic , maximal projection of 5 µm depth ) . Note the presence of Dicer within the SGS marked with neurofilament . n = 3 independent experiments . Abbreviations : WT , wild type ; P0 , postnatal day 0 . Scale bars : 30µm .

O B-PANEL_START O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Schematic representation of the experimental protocol. B. Representative Xenopus growth cones stained with anti-Dicer and anti-Ago2. White dashed lines delineate growth cones. Scale bars: 5µm. C. Representative E13.5 mice brain cross-section (dashed red line in the schematic) comprising the optic nerve (ON) stained with anti-neurofilament and anti-HA antibodies to detect RGC axons and (FLAG-HA2)-Dicer, respectively. Note the absence of HA signal in wild type (WT) mice. The white dashed line delineates the ON. Zoom of the triple stained ON (right panel): Dicer signal is detected inside ON cells surrounding axon bundles but not in axons per se (arrowheads). n=3 independent experiments. Abbreviations: WT, wild type; HA, (FLAG-HA2)-Dicer; E13.5, embryonic day 13.5; ONH, optic nerve head; ON, optic nerve. Scale bars: 30µm.D. Representative P0 superior colliculus stratum griseum superficiale (SGS) (sagittal sectioning of P0 brains along the dashed red line in the schematic, maximal projection of 5 µm depth). Note the presence of Dicer within the SGS marked with neurofilament. n=3 independent experiments. Abbreviations: WT, wild type; P0, postnatal day 0. Scale bars: 30µm.


Figure 6Quantification of miRNA and pre - miRNA expression levels using the 2 - ΔCt method and U6 as normalizer , upon Sema3A ( B ) stimulation . Data are normalized to PBS control . Each data point represents a single RT - qPCR reaction . n = 3 ( B ) independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant . Quantification of miRNA and pre - miRNA expression levels using the 2 - ΔCt method and U6 as normalizer , upon Slit2 ( C ) stimulation . Data are normalized to PBS control . Each data point represents a single RT - qPCR reaction . n = 4 ( C ) independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant . Frequency ( in percentage ) of collapsed growth cones from stage 37 / 38 embryos , following a 10 min Sema3A bath application . Concentration used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 5p ) ( E ) Total number of counted growth cones is reported in the column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino . Frequency ( in percentage ) of collapsed growth cones from stage 37 / 38 embryos , following a 10 min Sema3A bath application . Concentration used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 3p ) ( F ) . Total number of counted growth cones is reported in the column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Sema3A (B) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=3 (B) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.Quantification of miRNA and pre-miRNA expression levels using the 2-ΔCt method and U6 as normalizer, upon Slit2 (C) stimulation. Data are normalized to PBS control. Each data point represents a single RT-qPCR reaction. n=4 (C) independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant.Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-5p) (E) Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.Frequency (in percentage) of collapsed growth cones from stage 37/38 embryos, following a 10 min Sema3A bath application. Concentration used: 200ng/mL (Sema3A), 2µM (MOs-3p) (F). Total number of counted growth cones is reported in the column. Each data point represents one independent experiment. n=3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino.


Figure 7B . Representative images of RGC axons within the optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within the wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . C . Quantification of misprojecting axons . Each data point corresponds to one brain . Total number of brains analyzed : 45 brains ( co - MO ) , 52 brains ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino . D . Schematic representation of the experimental paradigm . Concentrations used : 0 . 5μg / μL pCS2 - mCherry or pCS2 - eGFP plasmids ; 250μM miR - 181 - MO or control MO cocktail ; 50μM miR - 181 or control mimics ( top ) . Representative images of RGC axons within the optic tectum . White arrows indicate axons targeted both by MO ( green ) and miRNA mimics ( red ) ( bottom ) . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . E . Quantification of misprojecting axons . The number reported on the bars are the total number of co - electroporated axons . Note how miR - 181 mimics rescued aberrant misprojection of morphant axons in vivo . Each data point corresponds to one independent experiment . Total number of axons and brains analyzed : 96 axons , 4 brains ( co - MO + ctrl mimics ) , 101 axons , 4 brains ( miR - 181 - MO + ctrl mimics ) , 134 axons , 4 brains ( miR - 181 - MO + miR - 181 mimics ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino ; ns , not significant . F . Representative images of RGC axons within the optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within the wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . G . Quantification of misprojecting axons . Each data point corresponds to one brain . Total number of brains analyzed : 21 brains ( co - MO ) , 31 brains ( miR - 181 - MO ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino . I . Frequency ( in percentage ) of the amount of time embryos spent on black background . Each data point corresponds to one embryo . Total number of embryos analyzed : 43 embryos ( co - MO ) , 45 embryos ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± SEM . Abbreviations : co - MO , control morpholino .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7B. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. C. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 45 brains (co-MO), 52 brains (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino. D. Schematic representation of the experimental paradigm. Concentrations used: 0.5μg/μL pCS2-mCherry or pCS2-eGFP plasmids; 250μM miR-181-MO or control MO cocktail; 50μM miR-181 or control mimics (top). Representative images of RGC axons within the optic tectum. White arrows indicate axons targeted both by MO (green) and miRNA mimics (red) (bottom). Abbreviations: co-MO, control morpholino. Scale bars: 20µm. E. Quantification of misprojecting axons. The number reported on the bars are the total number of co-electroporated axons. Note how miR-181 mimics rescued aberrant misprojection of morphant axons in vivo. Each data point corresponds to one independent experiment. Total number of axons and brains analyzed: 96 axons, 4 brains (co-MO+ctrl mimics), 101 axons, 4 brains (miR-181-MO + ctrl mimics), 134 axons, 4 brains (miR-181-MO+miR-181 mimics). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino; ns, not significant. F. Representative images of RGC axons within the optic tectum. A subset of aberrantly projecting axons are indicated (red arrows). Note that a few straying axons are always observed within the wild type tectum. Abbreviations: co-MO, control morpholino. Scale bars: 20µm. G. Quantification of misprojecting axons. Each data point corresponds to one brain. Total number of brains analyzed: 21 brains (co-MO), 31 brains (miR-181-MO). n=3 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino. I. Frequency (in percentage) of the amount of time embryos spent on black background. Each data point corresponds to one embryo. Total number of embryos analyzed: 43 embryos (co-MO), 45 embryos (miR-181-MO). n=4 independent experiments. Values are mean ± SEM. Abbreviations: co-MO, control morpholino.