( A - C ) Atg7 + / + and Atg7 - / - MEFs stably expressing GFP ‐ Gal3 ( A , C ) or GFP ‐ Ub ( B ) were treated with 1000 μM LLOMe or 250 μg / ml silica for 3 h . Cells were subjected to immunocytochemistry using the following antibodies : anti ‐ LC3 and anti ‐ Lamp1 ( A ) , anti ‐ p62 and anti ‐ Lamp1 ( B ) , or anti ‐ FK2 and anti ‐ p62 ( C ) . Bars : 10 μm .

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(A-C) Atg7+/+ and Atg7-/- MEFs stably expressing GFP‐Gal3 (A, C) or GFP‐Ub (B) were treated with 1000 μM LLOMe or 250 μg/ml silica for 3 h. Cells were subjected to immunocytochemistry using the following antibodies: anti‐LC3 and anti‐Lamp1 (A), anti‐p62 and anti‐Lamp1 (B), or anti‐FK2 and anti‐p62 (C). Bars: 10 μm.


( A , B ) NIH3T3 cells stably expressing GFP ‐ Gal3 and either empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI ( blue ) ( A ) . The number of GFP ‐ Gal3 or Lamp1 puncta per cell was quantified using G ‐ Count ( see also Supplementary Figure S4A and B ) . Then , the percent of GFP ‐ Gal3 ‐ positive Lamp1 puncta was determined ( B ) . The data represent means ± s . d . At least 70 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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(A, B) NIH3T3 cells stably expressing GFP‐Gal3 and either empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were fixed at the indicated time points and subjected to immunocytochemistry for Lamp1 and DAPI (blue) (A). The number of GFP‐Gal3 or Lamp1 puncta per cell was quantified using G‐Count (see also Supplementary Figure S4A and B). Then, the percent of GFP‐Gal3‐positive Lamp1 puncta was determined (B). The data represent means±s.d. At least 70 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( C - E ) HeLa cells transfected with tfGal3 and either One ‐ STrEP ‐ FLAG ‐ tagged Atg4BC74A ( Atg4B mutant ) or empty vector ( control ) were observed by confocal microscopy after treatment as shown in Figure 2A and B . The number of GFP ± RFP + or GFP + RFP + puncta per cell was quantified using G ‐ Count ( D ) . Then , the percent of GFP + RFP + tfGal3 puncta was calculated ( E ) . The data represent means ± s . d . At least 30 cells were counted ( n = 3 ) . Bar : 10 μm .

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(C-E) HeLa cells transfected with tfGal3 and either One‐STrEP‐FLAG‐tagged Atg4BC74A (Atg4B mutant) or empty vector (control) were observed by confocal microscopy after treatment as shown in Figure 2A and B. The number of GFP±RFP+ or GFP+RFP+ puncta per cell was quantified using G‐Count (D). Then, the percent of GFP+RFP+tfGal3 puncta was calculated (E). The data represent means±s.d. At least 30 cells were counted (n=3). Bar: 10 μm.


( F - H ) NIH3T3 cells stably expressing empty vector ( control ) or mStrawberry ‐ Atg4BC74A ( Atg4B mutant ) were treated with 1000 μM LLOMe for 1 h . After LLOMe washout , cells were cultured in the presence or absence of both 10 μg / ml E64d and Pepstatin A , fixed at the indicated time points , and subjected to immunocytochemistry for Gal3 ( green ) and DAPI ( blue ) ( F ) . The number of endogenous Gal3 puncta per control ( G ) or Atg4B ‐ mutant ( H ) cells was quantified by G ‐ Count . The data represent means ± s . d . At least 50 cells were counted ( n = 3 ) . Bars : 20 μm . Source data for this figure is available on the online supplementary information page .

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(F-H) NIH3T3 cells stably expressing empty vector (control) or mStrawberry‐Atg4BC74A (Atg4B mutant) were treated with 1000 μM LLOMe for 1 h. After LLOMe washout, cells were cultured in the presence or absence of both 10 μg/ml E64d and Pepstatin A, fixed at the indicated time points, and subjected to immunocytochemistry for Gal3 (green) and DAPI (blue) (F). The number of endogenous Gal3 puncta per control (G) or Atg4B‐mutant (H) cells was quantified by G‐Count. The data represent means±s.d. At least 50 cells were counted (n=3). Bars: 20 μm.Source data for this figure is available on the online supplementary information page.


( A - F ) HeLa cells stably expressing GFP ‐ LC3 were transfected with mStrawberry ‐ Gal3 , and treated with 1000 μM LLOMe for 1 h . Then , cells were fixed and observed by confocal microscopy ( A ) . The same specimens were further examined by transmission electron microscopy ( B - F ) . Green : LC3 ; magenta : Gal3 ; blue : DAPI .

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(A-F) HeLa cells stably expressing GFP‐LC3 were transfected with mStrawberry‐Gal3, and treated with 1000 μM LLOMe for 1 h. Then, cells were fixed and observed by confocal microscopy (A). The same specimens were further examined by transmission electron microscopy (B-F). Green: LC3; magenta: Gal3; blue: DAPI.


( G - I ) NIH3T3 cells stably expressing CFP ‐ Gal3 and YFP ‐ LC3 , and either empty vector ( control ) ( G , H ) or mStrawberry ‐ Atg4BC74A ( I ) were treated with 1000 μM LLOMe for 2 h , fixed , and observed by confocal microscopy . The electron micrographs were taken in the same sample field as the transmission electron microscope . Green : LC3 ; blue : Gal3 and DAPI ; black arrow : single membrane ; white arrow : autophagosome ; white arrowhead : ER membrane .

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(G-I) NIH3T3 cells stably expressing CFP‐Gal3 and YFP‐LC3, and either empty vector (control) (G, H) or mStrawberry‐Atg4BC74A (I) were treated with 1000 μM LLOMe for 2 h, fixed, and observed by confocal microscopy. The electron micrographs were taken in the same sample field as the transmission electron microscope. Green: LC3; blue: Gal3 and DAPI; black arrow: single membrane; white arrow: autophagosome; white arrowhead: ER membrane.


( A ) Plasmacreatinine and ureanitrogen in Atg5F / F and Atg5F / F ; KAPmice treated with vehicle or UA + OA . The values displayed represent means ± s . e . Statistically significant differences ( * P0 . 05 ) are indicated . F / F : Atg5F / Fmice ; F / F ; KAP : Atg5F / F ; KAPmice .

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(A) Plasmacreatinine and ureanitrogen in Atg5F/F and Atg5F/F;KAPmice treated with vehicle or UA+OA. The values displayed represent means±s.e. Statistically significant differences (*P0.05) are indicated. F/F: Atg5F/Fmice; F/F;KAP: Atg5F/F;KAPmice.


( B , C ) PAS ‐ stained renal cortical region of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( n = 4 - 7 ) . Bars : 40 μm ( B ) . Kidney injury score of Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA ( C ) .

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(B, C) PAS‐stained renal cortical region of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (n=4-7). Bars: 40 μm (B). Kidney injury score of Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA (C).


( D ) Kidney cortexes from Atg5F / F and Atg5F / F ; KAP mice treated with vehicle or UA + OA were subjected to immunohistocheminal analysis of Lamp1 and LC3 . Green : LC3 ; magenta : Lamp1 ; blue : DAPI . F / F : Atg5F / F mice ; F / F ; KAP : Atg5F / F ; KAP mice . Bar : 5 μm .

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(D) Kidney cortexes from Atg5F/F and Atg5F/F;KAP mice treated with vehicle or UA+OA were subjected to immunohistocheminal analysis of Lamp1 and LC3. Green: LC3; magenta: Lamp1; blue: DAPI. F/F: Atg5F/F mice; F/F;KAP: Atg5F/F;KAP mice. Bar: 5 μm.


( E ) Electron micrographs of kidney proximal tubules in Atg5 F / F and Atg5F / F ; KAP mice treated with UA + OA . Yellow arrowhead : autophagosome ; asterisk : lysosome ; Mt : mitochondrion . Bar : 1 μm .

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(E) Electron micrographs of kidney proximal tubules in Atg5 F/F and Atg5F/F;KAP mice treated with UA+OA. Yellow arrowhead: autophagosome; asterisk: lysosome; Mt: mitochondrion. Bar: 1 μm.


Wild - type , apg12 - 1 mutant and Δapg12 cells were cultured in nitrogen - starvation medium containing 1 mM PMSF . After incubation for 6 h , cells were observed under a phase - contrast microscope . Arrows indicate autophagic bodies .

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Wild-type, apg12-1 mutant and Δapg12 cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.


c , Wild - type ( squares ) and Δapg12 ( circles ) were cultured in nitrogen - starvation medium and their viability was determined by phloxine B staining .

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c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.


d , Quantification of autophagic activity of wild - type and Δapg12 cells by alkaline phosphatase ( ALP ) assay before ( black bars ) and after ( white bars ) nitrogen starvation for 4 h . Error bars indicate s . d . of three independent experiments .

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d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.


a , b , Lysates from Δapg12 cells carrying only vector or 3 × HA - APG12 ( a ) , and Δapg5 , apg7 - 1 , apg10 - 1 and Δapg1 cells carrying 3 × HA - Apg12 plasmid ( b ) were immunoblotted using anti - HA antibody . The positions of 3 × HA - Apg12 and the larger product ( asterisks ) are indicated .

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a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.


c , Immunoblot analysis of wild - type and Δapg12 cells harbouring HA - APG5 plasmid .

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c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.


d , Δapg5 Δapg12 cells were co - transformed with Myc - APG12 and HA - APG5 . Their lysates were immunoprecipitated with anti - Myc or anti - HA antibodies and detected by immunoblotting using anti - Myc antibody . The position of the crossreacting IgG heavy chain is

d, Δapg5 Δapg12 cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HA antibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicated


b , Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti - HA antibody .

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b, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.


c , Autophagic activity was measured as described for Fig . 1d .

c, Autophagic activity was measured as described for Fig. 1d.


d , Transport of pro - API to the vacuole was examined by immunoblotting with anti - API antiserum . The positions of pro - API and mature API are indicated .

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d, Transport of pro-API to the vacuole was examined by immunoblotting with anti-API antiserum. The positions of pro-API and mature API are indicated.


Δapg5 cells were transformed with vector alone , wild - type APG5 or APG5K149R , and then immunoblotted with anti - HA ( b )

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)


Autophagic activity was determined by alkaline phosphatase assay ( c ) .

Autophagic activity was determined by alkaline phosphatase assay (c).


Δapg5 cells were transformed with vector alone , wild - type APG5 or APG5K149R , and then immunoblotted anti - API ( d ) .

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Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).


c , In vitro conjugation of Apg5 and Apg12 . A cell lysate of Δapg12 carrying HA - APG5 ( lane 1 ) was incubated with an equal amount of lysate from Δapg5 carrying HA - APG12 ( 2 µ plasmid ) ( lane 2 ) at 30 ° C with ( lane 4 - 6 ) or without ( lane 7 ) 5 mM ATP . Samples were mixed with SDS - sample buffer at the times indicated .

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c, In vitro conjugation of Apg5 and Apg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12 (2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.


Spheroplasts were generated from cells expressing either HA - Apg12 or HA - Apg5 . Their lysates were mixed and layered on top of a 10 - step ( 18 - 54 % w / w ) sucrose gradient , and centrifuged at 174 , 000g for 2 . 5 h ( ref . 29 ) . Fifteen fractions were collected and the positions of free Apg5 , free Apg12 and Apg5 / Apg12 conjugate were examined by western blotting . The peak fractions of alcohol dehydrogenase ( ADH ) ( cytosol ) , ALP ( vacuole ) , Kex2 ( Golgi ) and Sec12 ( endoplasmic reticulum ) are indicated by arrows .

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Spheroplasts were generated from cells expressing either HA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12 conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.


b ) Caco - 2 cells were infected with SARS - CoV - 2 as per Figure 1A . Cells were washed , fixed and stained with DAPI ( blue ) to visualise cell nuclei and incubated with anti - N protein ( red ) to visualise SARS - CoV - 2 infected cells . Scale bar denotes 10 µM .

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b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.


c ) Infection rates were calculated using automated imaging software ( see materials and methods ) calculated from six fields of view per time point per biological replicate . Data point indicates the sample means and the error bars the standard error of the mean ( SEM ) ( n = 3 ) . d ) SARS - CoV - 2 detected proteins plotted as a function of time where each data point denotes a different replicate ( n = 3 ) .

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c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).


a ) Total number of significantly up - or down - regulated proteins ( | z - score | > 1 . 96 , q - value < 0 . 05 ) per time point of SARS - CoV - 2 infected Caco - 2 cells as described in 1A . Proteins that were significantly changed in abundance ( left ) and stability ( right ) are sub - grouped according to either down - regulation / destabilization ( red ) or up - regulation / stabilisation ( turquoise ) .

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a) Total number of significantly up- or down-regulated proteins (|z-score| >1.96, q-value <0.05) per time point of SARS-CoV-2 infected Caco-2 cells as described in 1A. Proteins that were significantly changed in abundance (left) and stability (right) are sub-grouped according to either down-regulation/destabilization (red) or up-regulation/stabilisation (turquoise).


c ) SAFB phosphopeptide abundance changes during SARS - CoV - 2 infection . Vero cell phosphopeptide data from ( Bouhaddou et al , 2020 ) . Data previously acquired from Vero cells infected with SARS - CoV - 2 ( Bouhaddou et al , 2020 ) . S604 phosphosite that temporally coincides with SAFB thermal destabilisation ( Potel et al , 2020 ) . Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS - CoV - 2 infected Caco - 2 cells as described in 1A ( n = 3 ) . Asterisk denotes significant regulation ( see methods ) .

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c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods).


D ) Thermal stability profile for all unmodified SAFB peptides ( green ) and the phosphorylated S604 ( pS604 ) ( purple ) in HeLa cells . Data from ( Potel et al , 2020 ) .

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D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).


b ) The corresponding HSP90 inhibitor , Tanespimycin , effectively suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Calu - 3 cells were pre - incubated with the compound , followed by the addition of SARS - CoV - 2 and subsequent co - incubation in the presence of the compound for 20 - 24 hours . Viral load was quantified in parallel using antibody - based detection of double stranded RNA ( orange ) . Compound toxicity in Calu - 3 cells was assessed ( purple ) treated with the compound alone for 20 - 24 hours .

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b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.


d ) The Cyp1a1 inhibitor suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Dose response curves were performed as described in 4A . See Appendix Figure S7 for additional tested drugs . Data displayed as described in 5B .

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d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.


B . Coomassie - stained SDS - PAGE ( from left to right ) of Hchim , V1ΔH , VoND , and C .

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B. Coomassie-stained SDS-PAGE (from left to right) of Hchim, V1ΔH, VoND, and C.


C . Representative ATPase activity assay ( ATP regenerating system ) . At the indicated time , 200 nM ConA is added to the assay .

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C. Representative ATPase activity assay (ATP regenerating system). At the indicated time, 200 nM ConA is added to the assay.


C . Growth phenotypes of wild type and c ″ - mutant yeast strains probed on ( 1 ) rich media buffered to pH 5 ( YPD pH 5 ) ; ( 2 ) synthetic dropout ( SD ) plus all amino acids ( SD + all a . a . ) ; ( 3 ) SD minus uracil ( SD - ura ) ; ( 4 , 5 ) YPD buffered to pH 7 plus 4 mM Zn2 + or 60 mM Ca2 + , respectively ; ( 6 ) YPD buffered to pH 7 ; ( 7 ) YPD plus 60 mM Ca2 + .

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C. Growth phenotypes of wild type and c″-mutant yeast strains probed on (1) rich media buffered to pH 5 (YPD pH 5); (2) synthetic dropout (SD) plus all amino acids (SD + all a.a.); (3) SD minus uracil (SD - ura); (4,5) YPD buffered to pH 7 plus 4 mM Zn2+ or 60 mM Ca2+, respectively; (6) YPD buffered to pH 7; (7) YPD plus 60 mM Ca2+.


B . Mass spectrometry analysis of V1Hchim . Protein IDs are plotted as a function of overall peptide spectrum matches ( PSM ) and the posterior error probability ( PEP ) scores .

O O O O O O O O O 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. Mass spectrometry analysis of V1Hchim. Protein IDs are plotted as a function of overall peptide spectrum matches (PSM) and the posterior error probability (PEP) scores.


A . Size exclusion chromatography elution profile ( 1 . 6 cm × 50 cm Superdex S75 ) and SDS - PAGE of full - length N - terminally 7 × His tagged yeast Oxr1 . B . Inhibition of V1ΔH and V1Hchim ' s MgATPase activity by Oxr1 in absence and presence of subunit C . The data from one representative of two experiments using two different preparations are shown . Individual data points ( n = 2 ) and mean from one representative of two biological replicates are shown with error bars representing S . E . M . . C . Oxr1 binds wild type V1 . Samples of V1 , Oxr1 , and mixtures of V1 with a 2 - fold molar ratio of Oxr1 in absence or presence of a 3 - fold molar ratio of subunit C were subjected to size exclusion chromatography on a 1 cm × 30 cm Superose 6 Increase column . SDS - PAGE is shown for all four samples and immunoblot analysis of column fractions using a penta - His antibody is shown for samples containing Oxr1 and Oxr1 plus C . Note that incubation of V1 with Oxr1 in presence of excess subunit C leads to partial release of subunit H ( highlighted by the rectangular box in the V1 + Oxr1 + C gel ) . Also , some degradation of Oxr1 ' s unstructured N - terminus is observed as indicated by the asterisk ( only the full - length protein is detected by the αHis - tag antibody ) . The data from one representative of two experiments using two different preparations are shown .

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A. Size exclusion chromatography elution profile (1.6 cm × 50 cm Superdex S75) and SDS-PAGE of full-length N-terminally 7×His tagged yeast Oxr1. B. Inhibition of V1ΔH and V1Hchim's MgATPase activity by Oxr1 in absence and presence of subunit C. The data from one representative of two experiments using two different preparations are shown. Individual data points (n=2) and mean from one representative of two biological replicates are shown with error bars representing S.E.M. . C. Oxr1 binds wild type V1. Samples of V1, Oxr1, and mixtures of V1 with a 2-fold molar ratio of Oxr1 in absence or presence of a 3-fold molar ratio of subunit C were subjected to size exclusion chromatography on a 1 cm × 30 cm Superose 6 Increase column. SDS-PAGE is shown for all four samples and immunoblot analysis of column fractions using a penta-His antibody is shown for samples containing Oxr1 and Oxr1 plus C. Note that incubation of V1 with Oxr1 in presence of excess subunit C leads to partial release of subunit H (highlighted by the rectangular box in the V1 + Oxr1 + C gel). Also, some degradation of Oxr1's unstructured N-terminus is observed as indicated by the asterisk (only the full-length protein is detected by the αHis-tag antibody). The data from one representative of two experiments using two different preparations are shown.


D . Dissociation kinetics of Hwt and Hchim from V1ΔH in presence of Oxr1 and subunit C as probed by BLI . Recombinant wild type ( left ) and chimeric subunit H ( right ) with an N - terminal fusion to maltose binding protein ( MBP ) was immobilized on anti - mouse Fc ( AMC ) BLI sensors via an αMBP monoclonal antibody . The sensors were then dipped into wells with V1ΔH followed by wells with buffer in absence or presence of Oxr1 and C to monitor the kinetics of V1ΔH dissociation . Only the dissociation phase is shown . The data from one representative of three experiments using two different preparations are shown .

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D. Dissociation kinetics of Hwt and Hchim from V1ΔH in presence of Oxr1 and subunit C as probed by BLI. Recombinant wild type (left) and chimeric subunit H (right) with an N-terminal fusion to maltose binding protein (MBP) was immobilized on anti-mouse Fc (AMC) BLI sensors via an αMBP monoclonal antibody. The sensors were then dipped into wells with V1ΔH followed by wells with buffer in absence or presence of Oxr1 and C to monitor the kinetics of V1ΔH dissociation. Only the dissociation phase is shown. The data from one representative of three experiments using two different preparations are shown.


E . Oxr1 mediated inhibition of assembly of holo V - ATPase from V1Hchim , subunit C and VoND . The data from one representative of two experiments using two different preparations are shown . Individual data points ( n = 2 ) and mean from one representative of two biological replicates are shown with error bars representing S . E . M . .

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E. Oxr1 mediated inhibition of assembly of holo V-ATPase from V1Hchim, subunit C and VoND. The data from one representative of two experiments using two different preparations are shown. Individual data points (n=2) and mean from one representative of two biological replicates are shown with error bars representing S.E.M. .


F . Oxr1 prevents binding of V1Hchim to VoND as probed by BLI . Streptavidin coated BLI sensors were loaded with Vo in lipid nanodiscs containing biotinylated MSP , and sensors were then dipped into wells containing recombinant C and V1Hchim in absence and presence of Oxr1 . Only the V1Hchim association and dissociation phase from one representative of two experiments using two different preparations is shown .

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F. Oxr1 prevents binding of V1Hchim to VoND as probed by BLI. Streptavidin coated BLI sensors were loaded with Vo in lipid nanodiscs containing biotinylated MSP, and sensors were then dipped into wells containing recombinant C and V1Hchim in absence and presence of Oxr1. Only the V1Hchim association and dissociation phase from one representative of two experiments using two different preparations is shown.


G . Oxr1 mediated dissociation of holo V - ATPase as analyzed by negative stain electron microscopy . V1HchimVoND was incubated without and with a 3 - fold molar ratio of Oxr1 for 16 h . Samples were then spotted on carbon coated copper grids , stained with 1 % uranyl acetate and observed by transmission electron microscopy . Number of assembled V1Vo and disassembled V1 and Vo particles were counted on a total of 20 micrographs each . The horizontal dotted lines indicate the mean . A few holoenzyme and free V1 and VoND particles are highlighted by white circles and boxes , respectively . Bar = 50 nm . The data from one representative of two experiments using two different preparations are shown .

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G. Oxr1 mediated dissociation of holo V-ATPase as analyzed by negative stain electron microscopy. V1HchimVoND was incubated without and with a 3-fold molar ratio of Oxr1 for 16 h. Samples were then spotted on carbon coated copper grids, stained with 1% uranyl acetate and observed by transmission electron microscopy. Number of assembled V1Vo and disassembled V1 and Vo particles were counted on a total of 20 micrographs each. The horizontal dotted lines indicate the mean. A few holoenzyme and free V1 and VoND particles are highlighted by white circles and boxes, respectively. Bar = 50 nm. The data from one representative of two experiments using two different preparations are shown.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data.


SIRT1 activity is required for resveratrol - induced autophagy but not for spermidine - mediated autophagy induction in mammalian cultured cells . ( A - C ) Human colon carcinoma HCT 116 cells were left untransfected ( Co , control ) or transfected with an irrelevant siRNA ( UNR , unrelated ) or siRNAs specific for ATG5 , ATG7 , or SIRT1 and then retransfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and left untreated or treated for 4 h with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) . The same experiment was performed in the presence of bafilomycin A1 ( BafA1 ) , which inhibits the fusion between lysosomes and autophagosomes , to evaluate the autophagic flux . ( A ) Representative images . ( B ) Quantitative data . ( C ) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1 - specific siRNA showing LC3 lipidation after treatment with 100 - µM spermidine in the presence or absence of bafilomycin A1 .

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SIRT1 activity is required for resveratrol-induced autophagy but not for spermidine-mediated autophagy induction in mammalian cultured cells. (A-C) Human colon carcinoma HCT 116 cells were left untransfected (Co, control) or transfected with an irrelevant siRNA (UNR, unrelated) or siRNAs specific for ATG5, ATG7, or SIRT1 and then retransfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and left untreated or treated for 4 h with 100-µM resveratrol (Resv) or spermidine (Spd). The same experiment was performed in the presence of bafilomycin A1 (BafA1), which inhibits the fusion between lysosomes and autophagosomes, to evaluate the autophagic flux. (A) Representative images. (B) Quantitative data. (C) Representative immunoblots of HCT 116 cells transfected either with an unrelated siRNA or with a SIRT1-specific siRNA showing LC3 lipidation after treatment with 100-µM spermidine in the presence or absence of bafilomycin A1.


( D - F ) HCT 116 cells were left transfected with a GFP - LC3 plasmid , cultured in complete medium for 24 h , and treated with either vehicle ( Co ) , 100 - µM resveratrol , or 100 - µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h . ( D ) Representative images . ( E ) Quantitative data . ( B and E ) Bars depict the percentages of cells showing accumulation of GFP - LC3 in puncta ( GFP - LC3vac ; means ± SEM ; n = 3 ; * , P < 0 . 05 ) . ( F ) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100 - µM spermidine in the presence or absence of EX527 . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D-F) HCT 116 cells were left transfected with a GFP-LC3 plasmid, cultured in complete medium for 24 h, and treated with either vehicle (Co), 100-µM resveratrol, or 100-µM spermidine in the presence or absence of the SIRT1 inhibitor EX527 for 4 h. (D) Representative images. (E) Quantitative data. (B and E) Bars depict the percentages of cells showing accumulation of GFP-LC3 in puncta (GFP-LC3vac; means ± SEM; n = 3; *, P < 0.05). (F) Representative immunoblots showing LC3 lipidation in HCT 116 cells treated with 100-µM spermidine in the presence or absence of EX527. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


The lifespan - extending and autophagy - inducing effects of spermidine in yeast are not mediated by Sir2 . ( A - E ) EGFP - Atg8p was ectopically expressed in wild - type ( WT ) or Δsir2 S . cerevisiae undergoing chronological aging on small synthetic 2 % glucose media with or without ( Co , control ) supplementation of 4 - mM spermidine ( Spd ) . ( A ) Representative images . EGFP - Atg8p localization ( bottom ) was visualized by fluorescence microscopy . Yeast cells undergoing autophagy ( in which EGFP - Atg8p exhibits a prominent vacuolar localization ) are indicated by arrows . Yeast morphology was monitored by differential interference contrast ( DIC ; top ) .

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The lifespan-extending and autophagy-inducing effects of spermidine in yeast are not mediated by Sir2. (A-E) EGFP-Atg8p was ectopically expressed in wild-type (WT) or Δsir2 S. cerevisiae undergoing chronological aging on small synthetic 2% glucose media with or without (Co, control) supplementation of 4-mM spermidine (Spd). (A) Representative images. EGFP-Atg8p localization (bottom) was visualized by fluorescence microscopy. Yeast cells undergoing autophagy (in which EGFP-Atg8p exhibits a prominent vacuolar localization) are indicated by arrows. Yeast morphology was monitored by differential interference contrast (DIC; top).


( B ) Representative immunoblots against EGFP . Free EGFP indicates the vacuolar degradation of EGFP - Atg8p fusion , thereby representing the autophagic flux . Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine - mediated autophagy induction .

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(B) Representative immunoblots against EGFP. Free EGFP indicates the vacuolar degradation of EGFP-Atg8p fusion, thereby representing the autophagic flux. Notice that both WT and Δsir2 yeast cells show similar free EGFP levels after spermidine-mediated autophagy induction.


( C ) Relative alkaline phosphatase ( ALP ) activity indicative of autophagy . n = 3 .

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(C) Relative alkaline phosphatase (ALP) activity indicative of autophagy. n = 3.


( E ) Quantification of reactive oxygen species . Bars indicate the percentages of cells exhibiting the reactive oxygen species - mediated conversion of dihydroethidine ( DHE ) into ethidium ( Eth ; n = 4 ) . Data represent means ± SEM ; * , P < 0 . 001 as compared with untreated cells of the same genotype . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase . RFU , relative fluorescence unit .

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(E) Quantification of reactive oxygen species. Bars indicate the percentages of cells exhibiting the reactive oxygen species-mediated conversion of dihydroethidine (DHE) into ethidium (Eth; n = 4). Data represent means ± SEM; *, P < 0.001 as compared with untreated cells of the same genotype. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. RFU, relative fluorescence unit.


The life - extending and autophagy - inducing effects of spermidine in C . elegans are not mediated by Sir2 . ( A ) Fluorescence microscopy of C . elegans transgenic embryos expressing a full - length plgg - 1DsRed : : LGG - 1 fusion protein indicative of autophagic activity . Two representative pictures of wild - type ( WT ) and sir - 2 . 1 embryos untreated ( Co , control ) or treated with 0 . 2 - mM spermidine ( Spd ) supplementation of food are shown . ( B ) Quantification of autophagic activity through the measurement of DsRed : : LGG - 1 pixel intensity from images of WT animals shown in A . Data represent means ± SEM ( n = 3 ) with ≥ 25 images processed for each trial .

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The life-extending and autophagy-inducing effects of spermidine in C. elegans are not mediated by Sir2. (A) Fluorescence microscopy of C. elegans transgenic embryos expressing a full-length plgg-1DsRed::LGG-1 fusion protein indicative of autophagic activity. Two representative pictures of wild-type (WT) and sir-2.1 embryos untreated (Co, control) or treated with 0.2-mM spermidine (Spd) supplementation of food are shown. (B) Quantification of autophagic activity through the measurement of DsRed::LGG-1 pixel intensity from images of WT animals shown in A. Data represent means ± SEM (n = 3) with ≥25 images processed for each trial.


C ) Survival of WT C . elegans during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 005 ) . ( D ) Survival of sir - 2 . 1 C . elegans ( ok434 phenotype ) during aging with and without ( control ) supplementation of food ( UV - killed E . coli ) with 0 . 2 - mM spermidine ( n = 110 ; P < 0 . 01 ) . P - values were calculated using the log - rank test as described in Materials and methods .

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C) Survival of WT C. elegans during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.005). (D) Survival of sir-2.1 C. elegans (ok434 phenotype) during aging with and without (control) supplementation of food (UV-killed E. coli) with 0.2-mM spermidine (n = 110; P < 0.01). P-values were calculated using the log-rank test as described in Materials and methods.


Convergent alterations in the phosphoproteome status after resveratrol and / or spermidine treatment . ( A - C ) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , and 100 - µM spermidine ( Spd ) , alone or in combination ( Resv + Spd ) . ( A ) Representative phosphoprotein arrays are shown .

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Convergent alterations in the phosphoproteome status after resveratrol and/or spermidine treatment. (A-C) Human colon carcinoma HCT 116 cells were treated for 2 h with vehicle (Co, control), 100-µM resveratrol (Resv), and 100-µM spermidine (Spd), alone or in combination (Resv + Spd). (A) Representative phosphoprotein arrays are shown.


( B ) Clustering analysis for the effects on protein kinase phosphorylation .

(B) Clustering analysis for the effects on protein kinase phosphorylation.


( C ) Representative immunoblots of selected kinases whose phosphorylation status was unaffected ( PRKAA1 , RPS6KB1 , and acetyl - CoA carboxylase a [ ACACA ] ) or affected by resveratrol or / and spermidine treatment ( PTKB , AKT1 , MAPK8 , and CDKN1B ) , validating phosphoprotein array data .

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(C) Representative immunoblots of selected kinases whose phosphorylation status was unaffected (PRKAA1, RPS6KB1, and acetyl-CoA carboxylase a [ACACA]) or affected by resveratrol or/and spermidine treatment (PTKB, AKT1, MAPK8, and CDKN1B), validating phosphoprotein array data.


( D ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle or the indicated dose of resveratrol or spermidine , alone or in combination , for 2 h . Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(D) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle or the indicated dose of resveratrol or spermidine, alone or in combination, for 2 h. Quantitative data. Bars depict the percentages (means ± SD; n = 3; *, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Convergent acetylproteome modification after resveratrol or spermidine treatment . ( A - C ) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes . Cells were treated with 100 - µM resveratrol ( Resv ) or spermidine ( Spd ) for 2 h , fractioned into cytoplasmic , nuclear , and mitochondrial extracts , processed for acetyl lysine peptide enrichment , and analyzed by MS . ( A ) Hierarchical clustering of drug - specific organellar distributions of all acetylated sites quantified in at least one fraction . Fold changes are calculated relative to untreated cells . Only sites regulated > 1 . 5 - fold were included in statistical analyses . ( B ) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way . n = 560 .

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Convergent acetylproteome modification after resveratrol or spermidine treatment. (A-C) Colon carcinoma HCT 116 cells were cultured for 2 wk in three different SILAC media containing different arginine and lysine isotopes. Cells were treated with 100-µM resveratrol (Resv) or spermidine (Spd) for 2 h, fractioned into cytoplasmic, nuclear, and mitochondrial extracts, processed for acetyl lysine peptide enrichment, and analyzed by MS. (A) Hierarchical clustering of drug-specific organellar distributions of all acetylated sites quantified in at least one fraction. Fold changes are calculated relative to untreated cells. Only sites regulated >1.5-fold were included in statistical analyses. (B) Graphical representation of peptides whose acetylation status was affected in a convergent or divergent way. n = 560.


Significant motifs among sites undergoing acetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Consensus acetylation motifs identified upon resveratrol ( left ) or spermidine ( right ) treatment are depicted using the MotifX algorithm ( Schwartz and Gygi , 2005 ) . Among sites that were hyperacetylated in response to both agents , the K ( F / Y ) motif is significantly enriched when tested against the whole proteome ( P < 0 . 00001 ) . ( C ) When testing against the largest acetylation site dataset from Choudhary et al . ( 2009 ) ( acetylation background dataset [ ABD ] ) , the SxK motif is significant ( P < 0 . 0001 ) for sites whose acetylation increased upon spermidine treatment . No general consensus motifs were found for sites whose acetylation increased in response to both agents .

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Significant motifs among sites undergoing acetylation. (A) Hierarchical clustering of the organellar distributions of sites whose acetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Consensus acetylation motifs identified upon resveratrol (left) or spermidine (right) treatment are depicted using the MotifX algorithm (Schwartz and Gygi, 2005). Among sites that were hyperacetylated in response to both agents, the K(F/Y) motif is significantly enriched when tested against the whole proteome (P < 0.00001). (C) When testing against the largest acetylation site dataset from Choudhary et al. (2009) (acetylation background dataset [ABD]), the SxK motif is significant (P < 0.0001) for sites whose acetylation increased upon spermidine treatment. No general consensus motifs were found for sites whose acetylation increased in response to both agents.


Significant motifs among sites undergoing deacetylation . ( A ) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by > 1 . 5 - fold in response to resveratrol or spermidine , at least in one organellar fraction . ( B ) Among sites that were hypoacetylated in response to both agents , the KP motif is significantly enriched when tested against ABD ( P < 0 . 001 ) . ( C and D ) When tested against the ABD ( C ) or the whole proteome ( D ) , the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment ( P < 0 . 0001 ) . No general consensus motifs were found for sites whose acetylation decreased in response to both agents .

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Significant motifs among sites undergoing deacetylation. (A) Hierarchical clustering of the organellar distributions of sites whose deacetylation was increased by >1.5-fold in response to resveratrol or spermidine, at least in one organellar fraction. (B) Among sites that were hypoacetylated in response to both agents, the KP motif is significantly enriched when tested against ABD (P < 0.001). (C and D) When tested against the ABD (C) or the whole proteome (D), the KP motif is significant for sites undergoing hypoacetylation upon spermidine treatment (P < 0.0001). No general consensus motifs were found for sites whose acetylation decreased in response to both agents.


Autophagy can be efficiently regulated by cytoplasmic ( de ) acetylation reactions . ( A and B ) HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and enucleated to obtain cytoplasts . The cytoplasts were treated with either vehicle ( Co , control ) , 100 - µM resveratrol ( Resv ) , or 100 - µM spermidine ( Spd ) for 4 h . 1 - µM rapamycin ( Rapa ) was used as a positive control . ( A ) Representative images of cytoplasts indicative of autophagic activity . Arrows indicate GFP - LC3 - transfected cytoplasts , whereas insets show nonenucleated transfected cells . ( B ) Quantitative data . Bars show the percentages of cells or cytoplasts showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) .

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Autophagy can be efficiently regulated by cytoplasmic (de)acetylation reactions. (A and B) HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and enucleated to obtain cytoplasts. The cytoplasts were treated with either vehicle (Co, control), 100-µM resveratrol (Resv), or 100-µM spermidine (Spd) for 4 h. 1-µM rapamycin (Rapa) was used as a positive control. (A) Representative images of cytoplasts indicative of autophagic activity. Arrows indicate GFP-LC3-transfected cytoplasts, whereas insets show nonenucleated transfected cells. (B) Quantitative data. Bars show the percentages of cells or cytoplasts showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac).


( C - E ) HCT 116 cells were cotransfected with a plasmid for the expression of RFP - LC3 together with an empty vector ( pcDNA3 ) or plasmids encoding wild - type ( WT ) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP ( WT GFP - SIRT1 and mtNLS GFP - SIRT1 , respectively ) for 12 or 24 h , fixed , and analyzed by fluorescence microscopy . ( C ) Representative images indicative of 24 - h autophagic activity .

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(C-E) HCT 116 cells were cotransfected with a plasmid for the expression of RFP-LC3 together with an empty vector (pcDNA3) or plasmids encoding wild-type (WT) SIRT1 or a SIRT1 variant with a mutation in the nuclear localization signal fused to GFP (WT GFP-SIRT1 and mtNLS GFP-SIRT1, respectively) for 12 or 24 h, fixed, and analyzed by fluorescence microscopy. (C) Representative images indicative of 24-h autophagic activity.


( D ) Quantitative data . Bars depict the percentages of cells exhibiting the accumulation of RFP - LC3 in puncta ( RFP - LC3vac ) . ( B and D ) Means ± SEM ; n = 3 ; * , P < 0 . 05 .

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(D) Quantitative data. Bars depict the percentages of cells exhibiting the accumulation of RFP-LC3 in puncta (RFP-LC3vac). (B and D) Means ± SEM; n = 3; *, P < 0.05.


( E ) Representative immunoblots for 24 - h endogenous LC3 lipidation . The asterisk stands for a nonspecific band . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots for 24-h endogenous LC3 lipidation. The asterisk stands for a nonspecific band. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo . ( A ) Human colorectal carcinoma HCT 116 cells were transfected with a GFP - LC3 - encoding plasmid , cultured in complete medium for 24 h , and then treated with either vehicle ( Co , control ) or the indicated dose of resveratrol ( Resv ) or spermidine ( Spd ) , alone or in combination , for 2 h . ( top ) Quantitative data . Bars depict the percentages ( means ± SD ; n = 3 ; * * , P < 0 . 05 ) of cells showing the accumulation of GFP - LC3 in puncta ( GFP - LC3vac ) . ( bottom ) Representative immunoblots showing endogenous LC3 lipidation .

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Low doses of resveratrol and spermidine synergistically induce autophagy in vitro and in vivo. (A) Human colorectal carcinoma HCT 116 cells were transfected with a GFP-LC3-encoding plasmid, cultured in complete medium for 24 h, and then treated with either vehicle (Co, control) or the indicated dose of resveratrol (Resv) or spermidine (Spd), alone or in combination, for 2 h. (top) Quantitative data. Bars depict the percentages (means ± SD; n = 3; **, P < 0.05) of cells showing the accumulation of GFP-LC3 in puncta (GFP-LC3vac). (bottom) Representative immunoblots showing endogenous LC3 lipidation.


B ) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 ( BafA1 ) . ( C - E ) Transgenic C57BL / 6 mice expressing a GFP - LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations . 3 h later , mice were killed , and tissues were processed for immunofluorescence microscopy determinations of GFP - LC3vac .

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B) Representative immunoblots showing endogenous LC3 lipidation in the presence of bafilomycin A1 (BafA1). (C-E) Transgenic C57BL/6 mice expressing a GFP-LC3 fusion protein were injected with resveratrol and spermidine at the indicated concentrations. 3 h later, mice were killed, and tissues were processed for immunofluorescence microscopy determinations of GFP-LC3vac.


( D ) Quantitative results . Bars represent the percentages ( means ± SEM ; n = 3 ; * , P < 0 . 05 ; and * * , P < 0 . 01 as compared with the same tissue from untreated animals ) of cells exhibiting GFP - LC3vac .

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(D) Quantitative results. Bars represent the percentages (means ± SEM; n = 3; *, P < 0.05; and **, P < 0.01 as compared with the same tissue from untreated animals) of cells exhibiting GFP-LC3vac.


( E ) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL / 6 mouse tissues . GAPDH , glyceraldehyde 3 - phosphate dehydrogenase .

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(E) Representative immunoblots showing endogenous LC3 lipidation and p62 protein content in WT C57BL/6 mouse tissues. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


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 .

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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 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 .

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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 . 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 .

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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 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 .

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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 puncta are indicated ( white arrows ) . Dashed white 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 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 .

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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 .

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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 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 .

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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 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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 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 .

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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 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 .

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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 ) and CD63 - eGFP - labeled vesicles ( green ) are co - trafficked ( white 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 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 .

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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 ) 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 .

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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 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 .

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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 .

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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 .

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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 .

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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 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 .

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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 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 .

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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 .

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 .

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 .

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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 .

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 .

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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 the experimental protocol . B . Representative Xenopus growth cones stained with anti - Dicer and anti - Ago2 . White dashed lines delineate growth cones . Scale bars : 5µm .

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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 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 .

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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 .

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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.


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 .

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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.


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 .

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 .

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