B . Representative electron of mitochondrial ultrastructural changes observed in COVID - 19 monocytes . Panels " a " " c " are representative electron of monocytes from healthy controls , whereas panels " b " , " d " " e " are representative electron of monocytes from COVID - 19 . In panel " f " of mitochondrial , external perimeter , Feret ' s diameter , aspect circularity 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 " .

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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".


B . Heatmaps representing different monocyte clusters identified by FlowSOM , with percentages in healthy donors ( CTR ) COVID - 19 . colors in heat map represent median of arcsinh , 0 - 1 transformed marker expression calculated over from samples , varying from blue for lower expression to red for higher expression . Each cluster has a unique color assigned ( bar on left which colors correspond to those used in panel A ) .

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B. 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 between healthy donors ( CTR , n = 8 ) COVID - 19 ( COVID , n = 7 ) . represents arcsine - square - root transformed frequencies that were subsequently normalized per cluster ( rows ) to mean of zero standard deviation of one . color of heat varies from blue , indicating relative under - representation , to red indicating relative over - representation . Bar numbers at right indicate significant differentially abundant clusters ( green ) adjusted p values ( Differential performed by diffcyt package present in Catalyst package ) . statistical analyses was done using GLMM ( according to actual guidelines ) p - values were Bonferroni corrected . Clusters were sorted according to adjusted p values , so that cluster at top shows most significant abundance changes between two conditions . D . Boxes whiskers plots representing maximum , minimum , median value , 25 75 percentile of different subpopulations of monocytes in healthy donors ( CTR , n = 8 ) COVID - 19 ( COVID , n = 7 ) .

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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 frequency of PD - 1 + , PD - L1 + among total monocytes in healthy controls ( CTR , n = 8 ) COVID - 19 ( COVID , n = 7 ) . A scatter plot reporting median ( MFI ) of PD - L1 in total monocytes is also shown . Data represent values , mean ± standard error of mean . Mann - Whitney was used for statistical . Exact p values are reported in figure .

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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.


A . of cytokines other mediators in plasma obtained from COVID - 19 ( COVID , n = 15 ) healthy controls ( CTR , n = 15 ) . Data represent values , mean ± standard error of mean . Mann - Whitney was used for statistical . Exact p values are reported in figure .

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A. 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 mature circulating monocytes in CTR COVID - 19 . Scatter plots showing of immature mature monocytes in healthy controls ( CTR , n = 6 ) COVID - 19 ( COVID , n = 7 ) are reported . Mann - Whitney was used for statistical . Exact p values are reported in figure .

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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.


( D ) A anisotropy experiment demonstrated direct binding of Pxp3 motif with purified Pex5 ( Kd = 9 . 5 + / - 2 . 9 µM ) , in strength similar to binding of known PTS1 motif from Idp3 ( Kd = 5 . 5 + / - 0 . 5 µM ) . plots represent mean of three independent experiments , error bars represent standard deviation .

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(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 Pox2 were used as markers for mitochondrial , peroxisomal fractions , respectively .

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(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 shows Glr1 interacts in vivo with both Pex5 full - Pex5 N ' domain .

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(H) Yeast-2-hybrid assay shows Glr1 interacts in vivo with both Pex5 full-length and Pex5 N' domain.


( B ) Metabolomic focused on strains with over - expression or deletion of AYT1 gene shows a significant reduction or accumulation , respectively , in acetyl - glutamate compared to matched control of each mutant . Only metabolites changing significantly in at least one of two conditions are presented . D - EIP is D - erythro - 1 - ( Imidazole - 4 - yl ) glycerol 3 - phosphate .

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(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 in a condition that does not induce high of acetyl - CoA in peroxisomes ( glucose ) shows that strains grow similarly to control until stationary phase when peroxisomes become vital . In stationary phase , over - expression ( OE ) of AYT1 grows to a lower density than control , plausibly due to burdening of peroxisomal functions . ( E ) A growth in a condition expected to elevate acetyl - glutamate in peroxisomes ( oleate as a sole carbon source glutamate as a nitrogen source ) demonstrates that AYT1 overexpressing grows faster to a higher density than control . Data information : growth assays in D E were done in three biologically independent replicates error bars were plotted . Note that some error bars are shorter than symbol , hence are not visible in graph .

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(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 of Δlpx1 , Δfsh3 , Δlpx1Δfsh3 strains supplemented with labeled 8 carbon - or 18 carbon - free fatty in media supplemented with 0 . 5 % glucose shows a significant reduction in β - oxidation activity compared to wild type two single mutants , suggesting an overlapping role for Fsh3 with Lpx1 . This was done in three biologically independent replicates . Bars represent mean error bars represent standard deviation .

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(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.


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

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(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.


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

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(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.


( B ) Coomassie ProQ Diamond - stained gels on mChRZZSF ( 4 µM ) treated with indicated kinases ( 1 µM ) for 15 hours at 20ºC . Rev = reversine , used at 10 µM .

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(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 ) of Zwilch at kinetochores of HeLa that had been previously synchronized in G2 phase with 9 μM RO3306 for 16 h then released into mitosis . Subsequently , were immediately treated with 500 nM reversine , 3 . 3 µM nocodazole , 10 µM MG132 for 1 hour imaged while in mitosis . CREST serum was used to visualize kinetochores DAPI to stain . Scale bar : 10 µm . Three biological replicates were performed . ( E ) treated as for panel D were treated for visualization of Spindly . Scale bar : 10 µm .

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(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 of Zwilch Spindly signals , normalized to reversine - untreated control , in indicated number of from experiment shown in panels C - D . Red lines indicate mean standard deviation . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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(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 showing effects of a knockdown of endogenous Spindly in HeLa . RNAi treatment was performed for 48 h with 50 nM siRNA . Before fixation , were synchronized in G2 phase with 9 μM RO3306 for 16 h then released into mitosis . Subsequently , were immediately treated with 3 . 3 μM nocodazole for an additional hour . CREST serum was used to visualize kinetochores DAPI to stain . Scale bar : 10 µm . Three biological replicates were performed .

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(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 ratios of Spindly over CREST for kinetochores of from experiment shown in panel G . Red lines indicate median with interquartile range .

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(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 ) of CENP - E Zwilch were assessed in control in treated as in panel G to knockdown Spindly . Three biological replicates were performed . ( J ) Scatter dot plots representing normalized total of CENP - E Zwilch signals , normalized to RNAi negative control , in indicated number of from experiment shown in panel I . Red lines indicate mean standard deviation . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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(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.


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

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(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


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

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(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.


( A ) - exclusion chromatography SDS - PAGE of elution fractions of indicated samples . Each Spindly construct was incubated with RZZ in absence ( dotted lines ) or in presence ( continuous lines ) of farnesyl transferase farnesyl pyrophosphate . Elution shifts of SpindlyF of resulting RZZSF complexes is indicative of successful interaction . Two technical replicates were performed .

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(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 of fixed HeLa electroporated with full - mChSpindly mChSpindly354 - 605 constructs in depleted of endogenous Spindly by RNAi . Spindly localization was detected with an antibody against 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 . Scale bar : 10 µm . Experiments were replicated three times .

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(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 ratios of indicated Spindly constructs over CREST for kinetochores of from experiment shown in panel D . Red lines indicate median with interquartile range . Statistical was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . 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 was performed with a nonparametric t - comparing two unpaired groups ( Mann - Whitney ) . Symbols indicate : n . s . = p > 0 . 05 , ∗ = p ≤ 0 . 05 , ∗ ∗ = p ≤ 0 . 01 , ∗ ∗ ∗ = p ≤ 0 . 001 , ∗ ∗ ∗ ∗ = p ≤ 0 . 000 .

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(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.


A . Schematic representation of experimental protocol . Abbreviations : LCM , capture microdissection . B , C . RT - PCR performed on extracted from axons collected by LCM ( Ax ) or from eyes . ( B ) β - Actin mRNA is present both in eyes axons , while MAP2 H4 are present only in eye sample , suggesting absence of contamination from bodies or other in LCM axonal samples ( Bellon et al , 2017 ) . Abbreviations : Ax , axonal sample ; Eye , stage 37 / 38 eye ; LCM , capture microdissection ; - , PCR no template control ; NT , RT no template control .

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A. 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 absence ( solid ) presence ( dashed ) of increasing target . Each melting curve represents average of three separate replicates . Yellow boxes indicate range of working temperatures for ex vivo trafficking experiments . Abbreviations : MB , molecular beacon ; Tm , MB melting .

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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 . of expression of pre - mir - 181a - 1 using 2 - ΔCt method U6 as normalizer from small total fraction ( < ~ 150 nt ) . Each data point corresponds to one independent experiment . n = 3 independent experiments . Values are mean ± . Abbreviations : ns , not significant .

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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 normalized to axon ( µm ) . Each data point corresponds to one axon . Total number of axons analyzed : 928 , 61 axons ( WT ) , 226 , 15 axons ( co - MO ) , 208 , 35 axons ( MO ) . n = 4 independent experiments . Values are mean ± . Abbreviations : ns , not significant ; co - MO , control morpholino ; pri - miR - MO , morpholino blocking pre - miR - 181a - 1 processing by targeting Drosha cleavage site .

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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 are indicated ( arrows ) . Dashed lines delineate axons . Abbreviations : MB , molecular beacon . Scale bars : 5µm .

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J. Representative axons. MB puncta are indicated (white arrows). Dashed white lines delineate axons. Abbreviations: MB, molecular beacon. Scale bars: 5µm.


M . Frequency ( in ) of colocalization between MB cy5 - pre - miR - 181a - 1 ( # MB + / pre - miR + ) . Each data point corresponds to one axon . Total number of axons analyzed : 354 , 32 axons ( MB ) , 337 , 32 axons ( cy5 - pre - miR - 181a - 1 ) . n = 5 independent experiments . Values are mean ± . Abbreviations : MB , molecular beacon .

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

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 . , red blue arrows indicate , respectively , co - localized , single MB , single pre - miR - 181a - 1 . Scale bars : 5µm .

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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.


B . Representative image of a single distal RGC axon from MB - electroporated retina . Growth cone wrist central domain are indicated with arrow star , respectively . Dashed delineates axon . Abbreviations : MB , molecular beacon . Scale bars : 5µm . C . Illustrative kymograph . Scale bars : 5µm .

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B. 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 ) of MB ( endo ) cy3 - pre - miR - 181a - 1 ( exo ) along RGC axon shaft . Each data point corresponds to one independent experiment . Total number of axons analyzed : 353 , 20 axons ( endo ) , 484 , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are mean ± . Abbreviations : MB , molecular beacon ; ns , not significant .

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

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 ) cy3 - pre - miR - 181a - 1 ( exo ) . Each data point corresponds to one punctum . Total number of axons analyzed : 353 , 20 axons ( endo ) , 484 , 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 .

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

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 ) cy3 - pre - miR - 181a - 1 ( exo ) tracked particles were fitted with an anomalous diffusion model α thus calculated ( red ) . Total number of particles axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiment . Values are mean ± . Abbreviations : MB , molecular beacon .

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

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 ) cy3 - pre - miR - 181a - 1 ( exo ) tracked particle . Each data point corresponds to one particle . Total number of particles 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 .

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

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 ( ) of MB ( endo ) cy3 - pre - miR - 181a - 1 ( exo ) tracked particles . Each data point corresponds to one independent experiment . Total number of particles axons analyzed : 67 particles , 20 axons ( endo ) , 82 particles , 29 axons ( exo ) . n = 3 ( endo ) , n = 4 ( exo ) independent experiments . Values are mean ± . Abbreviations : MB , molecular beacon ; ns , not significant .

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

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 ) 30 min after 2 . 4μM Noco bath - application ( bottom panel ; + Noco ) . Stationary in both panels are indicated with black arrows . Scale bars : 5µm .

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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 ) of MB punctum speed . Each data point corresponds to one independent experiment . Total number of axons analyzed : 358 , 25 axons ( - Noco ) ; 503 , 34 axons ( + Noco ) . n = 3 independent experiments . Values are mean ± . Abbreviations : MB , molecular beacon ; ns , not significant ; Noco , nocodazole .

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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 axons analyzed : 358 , 25 axons ( - Noco ) , 503 , 34 axons ( + Noco ) . n = 3 independent experiments . Values are median with interquartile range . Abbreviations : MB , molecular beacon ; ns , not significant ; Noco , nocodazole .

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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.


B . Representative axon where MB - labeled pre - miR - 181a - 1 ( red ) CD63 - eGFP - labeled vesicles ( green ) are co - trafficked ( arrows ) . Abbreviations : MB , molecular beacon . Scale bars : 5µm .

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B. 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 .

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C. Representative kymographs. Abbreviations: CD63, CD63-eGFP; MB, molecular beacon. Scale bars: 5µm.


D . Composite kymograph shown in ( C ) where traces where drawn color coded . Yellow trajectories represent co - trafficking MB - labeled pre - miRNA ( red ) CD63 - eGFP - labeled vesicle ( green ) . Abbreviations : MB , molecular beacon . Scale bars : 5µm .

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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 ) CD63 - eGFP - positive vesicle ( green arrow ) co - trafficked along axon shaft to growth cone ( delineated with dashed lines ) wrist ( arrowhead ) central domain ( star ) . Scale bars : 5µm .

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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 ) of MB - labeled pre - miR - 181a - 1 co - trafficked with CD63 - eGFP - positive vesicles . Each data point corresponds to one axon . Total number of axons analyzed : 253 , 22 axons ( MB + ) , 306 , 22 axons ( CD63 + ) . n = 5 independent experiments . Values are mean ± . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon .

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

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 ) of MB - labeled pre - miR - 181a - 1 co - trafficked with CD63 - eGFP - positive vesicles . Each data point corresponds to one axon . Total number of axons analyzed : 92 , 22 axons ( anterograde ) , 78 , 22 axons ( retrograde ) . n = 5 independent experiments . Values are mean ± . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon .

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

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 dissociated RGC . Abbreviations : CD63 , CD63 - eGFP . Scale bars : 5µm .

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I. Representative image of an electroporated and dissociated RGC. Abbreviations: CD63, CD63-eGFP. Scale bars: 5µm.


K , L . of MB ( K ) or CD63 - eGFP ( L ) measured in axons 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 ± . Abbreviations : CD63 , CD63 - eGFP ; MB , molecular beacon ; GC , growth cone .

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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.


C . Representative of co - electroporated axons . Dashed lines delineate axons . arrows indicate colocalized . Colored arrows represent non - colocalized signal as follows : magenta for CD63 yellow for Lamp1 or Rab5a . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP . Scale bars : 5μm .

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C. 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 ) of CD63 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of , axons , independent experiments analyzed : 540 , 23 axons , n = 4 ( CD63 / Rab7a ) , 416 , 18 axons , n = 3 ( CD63 / LysoTracker ) , 584 , 17 axons , n = 2 ( CD63 / Lamp1 ) , 649 , 19 axons , n = 3 ( CD63 / Rab5a ) . Values are mean ± . Abbreviations : CD63 , CD63 - eGFP or CD63 - mRFP ; Lamp1 , Lamp1 - eGFP ; Rab5a , Rab5a - eGFP or Rab5a - mRFP ; Rab7a , Rab7a - eGFP or Rab7a - mRFP .

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

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 of co - electroporated axons . Dashed lines delineate axons . arrows indicate colocalized . Colored arrows represent non - colocalized signal as follows : cyan for pre - miR - 181a - 1 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 .

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 ) of pre - miR - 181a - 1 colocalization with specific organelle markers . Each data point corresponds to one axon . Total number of , axons independent experiments analyzed : 825 , 41 axons , n = 8 ( pre - miR - 181a - 1 / CD63 ) , 732 , 33 axons , n = 7 ( pre - miR - 181a - 1 / Rab7a ) , 681 , 29 axons , n = 5 ( pre - miR - 181a - 1 / LysoTracker ) , 501 , 17 axons , n = 2 ( pre - miR - 181a - 1 / Lamp1 ) , 791 , 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 .

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 of co - electroporated axons . Dashed lines delineate axons . arrows indicate colocalized . Colored arrows represent non - colocalized signal as follows : magenta for CD63 , cyan for pre - miR - 181a - 1 , yellow for Rab7a 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 .

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 ) of pre - miR - 181a - 1 colocalization with vesicles double positive for organelle markers . Each data point corresponds to one axon . Total number of , axons independent experiments analyzed : 745 , 23 axons , n = 5 ( CD63 / pre - miR - 181a - 1 / Rab7a ) , 577 , 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 .

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 , axons growth cones analyzed : 99 , 16 axons 10 growth cones ( MB ) , 291 , 36 axons 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O

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.


A . Schematic representation of experimental protocol . B . Representative Xenopus growth cones stained with anti - Dicer anti - Ago2 . dashed lines delineate growth cones . Scale bars : 5µm .

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

A. 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 in schematic ) comprising optic nerve ( ON ) stained with anti - neurofilament anti - HA antibodies to detect RGC axons ( FLAG - HA2 ) - Dicer , respectively . Note absence of HA signal in wild type ( WT ) mice . dashed delineates ON . Zoom of triple stained ON ( right panel ) : Dicer signal is detected inside ON 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 .

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

C. 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 dashed red in schematic , maximal projection of 5 µm depth ) . Note presence of Dicer within SGS marked with neurofilament . n = 3 independent experiments . Abbreviations : WT , wild type ; P0 , postnatal day 0 . Scale bars : 30µm .

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

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.


of miRNA pre - miRNA expression using 2 - ΔCt method 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 ± . Abbreviations : ns , not significant .

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

Quantification 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.


of miRNA pre - miRNA expression using 2 - ΔCt method 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 ± . 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 ) of collapsed growth cones from stage 37 / 38 , following a 10 min Sema3A bath application . used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 5p ) ( E ) Total number of counted growth cones is reported in column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± . Abbreviations : ns , not significant ; co - MO , control morpholino .

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

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 ) of collapsed growth cones from stage 37 / 38 , following a 10 min Sema3A bath application . used : 200ng / mL ( Sema3A ) , 2µM ( MOs - 3p ) ( F ) . Total number of counted growth cones is reported in column . Each data point represents one independent experiment . n = 3 independent experiments . Values are mean ± . Abbreviations : ns , not significant ; co - MO , control morpholino .

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

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.


B . Representative of RGC axons within optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . C . 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 ± . Abbreviations : co - MO , control morpholino .

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

B. 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 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 of RGC axons within optic tectum . arrows indicate axons targeted both by MO ( green ) miRNA mimics ( red ) ( bottom ) . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . E . of misprojecting axons . number reported on bars are 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 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 ± . Abbreviations : co - MO , control morpholino ; ns , not significant .

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

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 of RGC axons within optic tectum . A subset of aberrantly projecting axons are indicated ( red arrows ) . Note that a few straying axons are always observed within wild type tectum . Abbreviations : co - MO , control morpholino . Scale bars : 20µm . G . 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 ± . Abbreviations : co - MO , control morpholino .

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 ) of amount of time spent on black background . Each data point corresponds to one . Total number of analyzed : 43 ( co - MO ) , 45 ( miR - 181 - MO ) . n = 4 independent experiments . Values are mean ± . Abbreviations : co - MO , control morpholino .

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

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.


( in ) of axonal recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo in vivo . Concentrations used : 200ng / mL Sema3A ; 100µM cycloheximide Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to bleached growth cone ( Wong et al , 2017 ) . ( B ) 200ng / mL Sema3A was bath applied with or without 100µM cycloheximide ( CHX , a translational blocker ) . Mutating TUBB3 3 ' UTR did not affect recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate constitutive TUBB3 expression in distal axons .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons.


( in ) of axonal recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo in vivo . Concentrations used : 200ng / mL Sema3A ; Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to bleached growth cone ( Wong et al , 2017 ) . , isolated axons ( C ) were used for FRAP .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). , isolated axons (C) were used for FRAP analysis.


( in ) of axonal recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo in vivo . Concentrations used : 200ng / mL Sema3A ; 2µM co - MO or MOs - 3p . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to bleached growth cone ( Wong et al , 2017 ) . isolated transfected axons ( D ) were used for FRAP .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). isolated transfected axons (D) were used for FRAP analysis.


( in ) of axonal recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo in vivo . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to bleached growth cone ( Wong et al , 2017 ) . In vivo ( E ) , whole with exposed brain were used . electroporated eye was removed prior to mounting to eliminate somatic contribution . ( E ) red on brain schematic indicate Sema3A expressing territories . Numbers of axons analyzed are reported between brackets .

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). In vivo (E), whole embryos with exposed brain were used. The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution. (E) The red area on the brain schematic indicate Sema3A expressing territories. Numbers of axons analyzed are reported between brackets.


( in ) of axonal recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP . APP , Amyloid beta precursor

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. APP, Amyloid beta precursor protein


( in ) of axonal recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP . THBS1 , Thrombospondin

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

Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. THBS1, Thrombospondin 1


H , I . Representative growth cones depicting Venus as a . Dashed lines delineate growth cones . Abbreviations : wt , wild type ; mut , miR - 181 - 5p responsive elements mutated ; CHX , cycloheximide ; TUBB3 , tubulin beta 3 class III . Scale bars : 10µm .

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

H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.


B . Schematic representation of experimental paradigm . Representative of local translation events of TUBB3 in growth cone . Dashed delineates growth cone . Each arrow points to a puro - PLA . Abbreviations : TUBB3 , tubulin beta 3 class III ; Puro , puromycin . Scale bars : 5 μm . of number of per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 30 ( PBS ) , 36 ( Sema3A ) . n = 2 independent experiments . Values are median with interquartile range .

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

B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range.


D . Schematic representation of experimental paradigm . Concentrations used : 2 μM co - MO ; 2 μM MOs - 3p ; 200ng / mL Sema3A . Representative of local translation events of TUBB3 in growth cone of transfected axons . Dashed delineates growth cone . arrows represent number of puro - PLA per growth cone . Abbreviations : co - MO , control morpholino . Scale bars : 5 μm . E . of number of per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 32 ( co - MO + PBS ) , 45 ( co - MO + Sema3A ) , 27 ( MOs - 3p + Sema3A ) . n = 2 independent experiments . Values are median with interquartile range . Abbreviations : co - MO , control morpholino ; ns , not significant .

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D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant.


G . of collapsed growth cones in axons transfected with co - MO , MOs - 3p + co - MO MOs - 3p + MOs - TUBB3 . Each data point corresponds to one independent experiment . Total number of growth cones counted : 152 ( co - MO ) , 155 ( MOs - 3p + coMO ) , 179 ( MOs - 3p + MOs - TUBB3 ) . n = 3 independent experiments . Values are mean ± . Abbreviations : ns , not significant ; co - MO , control morpholino ; TUBB3 , tubulin beta 3 class III .

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G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III.


Left graph reports volume in NSG mice injected with control ( shCTR ) antimiR - 9 FaDu followed for up to 35 days ( n = 5 mice / group ) . Data represent mean ( ± SD ) unpaired t - was used to verify statistical significance at each time point . On right , typical of explanted tumors formed by control ( shCTR ) antimiR - 9 FaDu at necropsy .

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Left graph reports the tumor volume in NSG mice injected with control (shCTR) and antimiR-9 FaDu cells followed for up to 35 days (n=5 mice/group). Data represent the mean (±SD) and unpaired t-test was used to verify the statistical significance at each time point. On the right, typical images of explanted tumors formed by control (shCTR) and antimiR-9 FaDu cells at necropsy.


Typical of Hematoxylin & Eosin ( H & E - upper ) Ki67 expression ( Ki67 - bottom ) evaluated by immunohistochemistry ( ) in tumors explanted from mice treated as in H . 4X 20X magnification are shown . Right graph reports of Ki67 positive . Each dot represents one . Data are expressed as mean ( ± SD ) unpaired t - was used to verify statistical significance .

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Typical images of Hematoxylin&Eosin (H&E - upper images) and Ki67 expression (Ki67 - bottom images) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in H. 4X and 20X magnification are shown. Right graph reports the percentage of Ki67 positive cells. Each dot represents one tumor. Data are expressed as mean (±SD) and unpaired t-test was used to verify the statistical significance.


qRT - PCR analyses of normalized miR - 9 expression , expressed as fold over untreated condition , in control ( shCTR ) antimiR - 9 FaDu treated with Cetuximab ( CTX ) for up to four hours . Data represent mean ( ± SD ) of three independent experiments performed in duplicate two - way ANOVA was used to verify statistical significance .

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qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.


Graph reporting viability of control ( shCTR ) antimiR - 9 FaDu treated with increasing of Gefitinib as indicated evaluated using MTS . Data represent mean ( ± SD ) of two independent experiments performed in sextuplicate , unpaired t - was used to verify statistical significance per each dose .

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Graph reporting the cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and unpaired t-test was used to verify the statistical significance per each dose.


qRT - PCR analyses of Sp1 expression in control ( shCTR ) antimiR - 9 FaDu transiently transduced with PGK - miR - 9 or control vector as indicated . Data represent mean ( ± SD ) of three independent experiments performed in duplicate two - way ANOVA with Sidak ' s multiple comparison was used to verify statistical significance . qRT - PCR analyses of Sp1 expression in CAL27 transfected with pcDNA miR - 9 or control vector . Data represent mean ( ± SD ) of three independent experiments performed in duplicate unpaired t - was used to verify statistical significance .

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qRT-PCR analyses of Sp1 expression in control (shCTR) and antimiR-9 FaDu cells transiently transduced with PGK-miR-9 or control vector as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. qRT-PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR-9 or control vector. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.


Graphs reporting viability of FaDu described in G treated with increasing of Gefitinib ( GEFI - left panel ) or Cetuximab ( CTX - right panel ) as indicated evaluated using MTS . Data represent mean ( ± SD ) of two independent experiments performed in sextuplicate , two - way ANOVA with Sidak ' s multiple comparison was used to verify statistical significance .

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Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.


Graph reporting normalized Luciferase activity of Sp1 promoter fragments in control ( shCTR ) or antimiR - 9 FaDu . Data represent mean ( ± SD ) of three independent experiments performed in duplicate unpaired t was used to verify statistical significance .

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Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control (shCTR) or antimiR-9 FaDu cells. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.


On left , WB analyses of KLF5 Sp1 expression in control ( shCTR ) antimiR - 9 FaDu . Histone H3 expression was used as loading control . On right , analyses of KLF5 SP1 expression normalized on H3 loading control . Data represent mean ( ± SD ) of three biological replicates unpaired t was used to verify statistical significance .

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On the left, WB analyses of KLF5 and Sp1 expression in control (shCTR) and antimiR-9 FaDu. Histone H3 expression was used as loading control. On the right, protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control. Data represent the mean (±SD) of three biological replicates and unpaired t test was used to verify the statistical significance.


Upper panel shows WB analyses of KLF5 Sp1 expression in FaDu . Actin expression was used as loading control . Lower graph shows SP1 normalized over actin expression . Data represents mean ( ± SD ) of three independent experiments unpaired t was used to verify statistical significance .

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Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells. Actin expression was used as loading control. Lower graph shows the SP1 protein quantification normalized over actin expression. Data represents the mean (±SD) of three independent experiments and unpaired t test was used to verify the statistical significance.


Left , WB analyses of indicated expression in control ( shCTR ) antimiR - 9 FaDu not irradiated ( NIR ) or treated with 5Gy IR allowed to repair for indicated hours ( h ) . Tubulin was used as loading control . On right , analyses of KLF5 Sp1 expression normalized on tubulin loading control . On right , graphs report of indicated normalized on tubulin expression . Data are expressed as mean ( ± SD ) of three independent experiments unpaired t was used to calculate statistical significance at time point .

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Left, WB analyses of the indicated protein expression in control (shCTR) and antimiR-9 FaDu cells not irradiated (NIR) or treated with 5Gy IR and allowed to repair for the indicated hours (h). Tubulin was used as loading control. On the right, protein quantification analyses of KLF5 and Sp1 expression normalized on tubulin loading control. On the right, graphs report the quantification of the indicated proteins normalized on tubulin expression. Data are expressed as mean (±SD) of three independent experiments and unpaired t test was used to calculate the statistical significance at time point.


Left panel reports typical immunofluorescence of control ( shCTR ) or antimiR - 9 FaDu , not irradiated ( NIR ) analyzed 1 , 4 or 8 hours after 2Gy IR ( γH2AX green , pS10 - H3 red , nuclei in blue ) . Graphs report of γH2AX ( middle ) pS10 - H3 ( right ) positive . Data represent mean ( ± SD ) of three independent experiments in which at least 10 randomly selected fields were evaluated . Unpaired t was used to calculate statistical significance at each time point .

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Left panel reports typical immunofluorescence images of control (shCTR) or antimiR-9 FaDu cells, not irradiated (NIR) and analyzed 1, 4 or 8 hours after 2Gy IR (γH2AX green, pS10-H3 red, nuclei in blue). Graphs report the percentage of γH2AX (middle) and pS10-H3 (right) positive cells. Data represent the mean (±SD) of three independent experiments in which at least 10 randomly selected fields were evaluated. Unpaired t test was used to calculate the statistical significance at each time point.


Graph reports volume of tumors described in A ( n = 10 mice / group ) . Data represent mean ( ± SD ) two - way ANOVA was used to verify statistical significance .

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Graph reports the tumor volume of tumors described in A (n=10 mice/group). Data represent the mean (±SD) and two-way ANOVA was used to verify the statistical significance.


Typical of Hematoxylin & Eosin ( H & E ) ( C ) Ki67 expression ( Ki67 ) ( D ) evaluated by immunohistochemistry ( ) in tumors explanted from mice treated as in A / B .

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Typical images of Hematoxylin&Eosin (H&E) (C) and Ki67 expression (Ki67) (D) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in A/B.


Kaplan - Mayer curve evaluating overall survival of HNSCC treated with RT + Cetuximab ( CTX ) combination included in TCGA dataset , segregated on expression of miR - 9 in ( Low expression < 75819 reads n = 8 ; High expression ≥ 75819 reads n = 23 ) . Number of evaluated samples ( n ) p value are reported in graph . Statistical significance was calculated with long - rank .

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Kaplan-Mayer curve evaluating the overall survival of HNSCC patients treated with RT+Cetuximab (CTX) combination included in the TCGA dataset, segregated on the expression of miR-9 in the primary tumor (Low expression <75819 reads n=8; High expression ≥75819 reads n=23). Number of evaluated samples (n) and p value are reported in the graph. Statistical significance was calculated with long-rank test.


Kaplan - Mayer curve evaluating progression free survival of HNSCC treated with RT + Cetuximab ( CTX ) combination at CRO - Aviano National Cancer Institute at University Cattolica segregated on miR - 9 expression in tumors , defined as expression in above ( high expression n = 18 ) or below ( low expression n = 17 ) median expression , as defined by ddPCR . Hazard ( HR ) statistical significance were calculated with long - rank ( Mentel - Cox ) are reported in graph .

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Kaplan-Mayer curve evaluating the progression free survival of HNSCC patients treated with RT+Cetuximab (CTX) combination at the CRO-Aviano National Cancer Institute and at the University Cattolica segregated based on miR-9 expression in primary tumors, defined as the expression in above (high expression n=18) or below (low expression n=17) the median expression, as defined by ddPCR. Hazard Ratio (HR) and statistical significance were calculated with long-rank (Mentel-Cox) test and are reported in the graph.


( A ) A titration of anti - MHV polyclonal serum was added either directly to media ( extracellular ) or electroporated into L929 ( intracellular ) , then were infected with MHV - A59 . kinetics of growth was monitored by measuring total . Data information : Data was analysed using a Students t - . Error bars depict mean + / - . data represents at least two independent replicates .

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(A) A titration of anti-MHV polyclonal serum was added either directly to media (extracellular) or electroporated into L929 cells (intracellular), then cells were infected with MHV-A59. The kinetics of cell growth was monitored by measuring total cell area. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( D ) Kinetics of growth following MHV - A59 infection in presence of a titration of electroporated anti - N antibody . Data information : Data was analysed using a Students t - . Error bars depict mean + / - . data represents at least two independent replicates .

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(D) Kinetics of cell growth following MHV-A59 infection in the presence of a titration of electroporated anti-N antibody. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( G & H ) WT or TRIM21 KO L929 were infected with MHV - A59 in presence of electroporated polyclonal antiserum ( G ) or anti - N antibody ( H ) quantified by 48h later . Co - electroporation of A / G with anti - N antibody into WT mimics TRIM21 KO phenotype . Data information : Data was analysed using a Students t - . Error bars depict mean + / - . data represents at least two independent replicates .

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(G&H) WT or TRIM21 KO L929 cells were infected with MHV-A59 in the presence of electroporated polyclonal antiserum (G) or anti-N antibody (H) and quantified by cell area 48h later. Co-electroporation of Protein A/G with anti-N antibody into WT cells mimics the TRIM21 KO phenotype. Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( I ) L929 were infected with MHV - A59 for 4h , then cyclohexamide ( CHX ) was added to block further viral synthesis . After 5h , were electroporated with anti - N or anti - GFP antibodies left for a further 3h before being western blotted for N ( * denotes a non - specific band ) . Data information : Data was analysed using a Students t - . Error bars depict mean + / - . data represents at least two independent replicates .

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(I) L929 Cells were infected with MHV-A59 for 4h, then cyclohexamide (CHX) was added to block further viral protein synthesis. After 5h, cells were electroporated with anti-N or anti-GFP antibodies and left for a further 3h before being western blotted for cellular N protein levels (*denotes a non-specific band). Data information: Data was analysed using a Students t-test. Error bars depict the mean +/- SEM. All data represents at least two independent replicates.


( A & B ) Vero OE ACE2 TMPRSS2 were infected with SARS - CoV - 2 in presence of IgG or anti - N antibodies added directly into media or electroporated into . Viral replication was then determined by RT - qPCR ( A ) or plaque ( B ) . Electroporation of anti - N antibodies significantly inhibits SARS - CoV - 2 replication ( * * * = p < 0 . 0002 ) . Data information : data represents at least three independent replicates . Error bars depict mean + / - . Statistical comparisons were performed using a one - way ANOVA .

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(A&B) Vero cells OE ACE2 and TMPRSS2 were infected with SARS-CoV-2 in the presence of IgG or anti-N antibodies added directly into media or electroporated into cells. Viral replication was then determined by RT-qPCR (A) or plaque assay (B). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication (*** = p < 0.0002). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way ANOVA.


( C & D ) As with A & B , except with a titration of electroporated anti - N antibodies . Data information : data represents at least three independent replicates . Error bars depict mean + / - . Statistical comparisons were performed using a one - way ANOVA .

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(C&D) As with A&B, except with a titration of electroporated anti-N antibodies. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way ANOVA.