( A ) Whole - cell lysates from control and patient ( case index , CI ) fibroblasts were assessed by 3 - 12 % native - PAGE gradient gels with proteasome bands ( 30S , 26S and 20S complexes ) visualized by their ability to cleave the Suc - LLVY - AMC fluorogenic peptide .

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(A) Whole-cell lysates from control and patient (case index, CI) fibroblasts were assessed by 3-12% native-PAGE gradient gels with proteasome bands (30S, 26S and 20S complexes) visualized by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide.


( B ) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin - like activity by incubating them with 0 . 1 mM of the Suc - LLVY - AMC substrate at 37 ° C over a 180 - h period of time in quadruplicates on a 96 - well plate . Indicated on the y - axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide . Bars show the mean of 4 independent experiments + / - SD .

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(B) Ten micrograms of control and patient cell lysates were tested for their chymotrypsin-like activity by incubating them with 0.1 mM of the Suc-LLVY-AMC substrate at 37°C over a 180-h period of time in quadruplicates on a 96-well plate. Indicated on the y-axis are the raw fluorescence values measured by a microplate reader and reflecting the AMC cleavage from the peptide. Bars show the mean of 4 independent experiments +/- SD.


( C ) Proteasome complexes from control and patient fibroblasts separated by native - PAGE were subjected to western - blotting using antibodies specific for α6 , Rpt5 ( PSMC3 ) and PA28 - α , as indicated .

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(C) Proteasome complexes from control and patient fibroblasts separated by native-PAGE were subjected to western-blotting using antibodies specific for α6, Rpt5 (PSMC3) and PA28-α, as indicated.


( D ) Proteins extracted from control and CI PSMC3 were separated by 10 or 12 . 5 % SDS - PAGE prior to western - blotting using primary antibodies directed against ubiquitin and several proteasome subunits and / or components including α6 , β1 , β ; β5 , β5i , Rpt2 ( PSMC1 ) , Rpt5 ( PSMC3 ) , Rpt3 ( PSMC4 ) , Rpt4 ( PSMC6 ) and PA28 - α , as indicated . For the PSMC3 staining , two exposure times are shown . Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant . Equal protein loading between samples was ensured by probing the membrane with an anti - α - Tubulin antibody .

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(D) Proteins extracted from control and CI PSMC3 were separated by 10 or 12.5% SDS-PAGE prior to western-blotting using primary antibodies directed against ubiquitin and several proteasome subunits and/or components including α6, β1, β; β5, β5i, Rpt2 (PSMC1), Rpt5 (PSMC3), Rpt3 (PSMC4), Rpt4 (PSMC6) and PA28-α, as indicated. For the PSMC3 staining, two exposure times are shown. Arrow indicates an additional PSMC3 species corresponding to the expected size of the truncated PSMC3 variant. Equal protein loading between samples was ensured by probing the membrane with an anti-α-Tubulin antibody.


Control and patient ( index case , IC PSMC3 ) fibroblasts were exposed to a 16 - h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated ( as a negative control ) . Following treatment , cells were collected and subjected to RIPA - mediated protein extraction prior to SDS - PAGE and subsequent western - blotting using antibodies specific for ubiquitin , TCF11 / Nrf1 , Rpt1 ( PSMC2 ) , Rpt3 ( PSMC4 ) , Rpt5 ( PSMC3 ) , Rpt6 ( PSMC5 ) , β1 , β2 , β5 , β5i , β1i , PA28 - α and α - Tubulin ( loading control ) as indicated . For the TCF11 / Nrf1 staining , two exposure times are shown .

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Control and patient (index case, IC PSMC3) fibroblasts were exposed to a 16-h treatment with 30 nM of the proteasome inhibitor carfilzomib or left untreated (as a negative control). Following treatment, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, β1i, PA28-α and α-Tubulin (loading control) as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.


( A ) Control and / or patient ( index case , IC PSMC3 ) fibroblasts were subjected to a 24 - h transfection with pcDNA3 . 1 / empty vector ( mock ) or pcDNA3 . 1 / PSMC3 prior to RIPA - mediated protein extraction and subsequent western - blotting using antibodies specific for ubiquitin , PSMC3 ( i . e . Rpt5 ) and α - Tubulin ( loading control ) .

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(A) Control and/or patient (index case, IC PSMC3) fibroblasts were subjected to a 24-h transfection with pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 prior to RIPA-mediated protein extraction and subsequent western-blotting using antibodies specific for ubiquitin, PSMC3 (i.e. Rpt5) and α-Tubulin (loading control).


( B ) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3 . 1 / empty vector ( mock ) or pcDNA3 . 1 / PSMC3 , as indicated . The y - axis represents the percent changes in densitometry measurements ( of pixel intensities using Image J ) which are set as 100 % for cells transfected with the pcDNA3 . 1 / empty vector ( mock ) at 24 post - transfection ( n = 4 , * : p < 0 . 05 , t - test ) . Bars show the mean + / - SEM .

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(B) Densitometry analysis showing the relative ubiquitin contents detected by western blotting in patient fibroblasts exposed to either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3, as indicated. The y-axis represents the percent changes in densitometry measurements (of pixel intensities using Image J) which are set as 100 % for cells transfected with the pcDNA3.1/empty vector (mock) at 24 post-transfection (n=4, *: p<0.05, t-test). Bars show the mean +/- SEM.


( C ) Patient ( index case , IC PSMC3 ) fibroblasts transfected with either pcDNA3 . 1 / empty vector ( mock ) or pcDNA3 . 1 / PSMC3 were exposed to a 30 - nM treatment of carfilzomib or left untreated ( as a negative control ) . After 16 hours , cells were collected and subjected to RIPA - mediated protein extraction prior to SDS - PAGE and subsequent western - blotting using antibodies specific for ubiquitin , TCF11 / Nrf1 , Rpt1 ( PSMC2 ) , Rpt3 ( PSMC4 ) , Rpt5 ( PSMC3 ) , Rpt6 ( PSMC5 ) , β1 , β2 , β5 , β5i , PA28 - α and α - Tubulin ( loading control ) , as indicated . For the TCF11 / Nrf1 staining , two exposure times are shown .

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

(C) Patient (index case, IC PSMC3) fibroblasts transfected with either pcDNA3.1/empty vector (mock) or pcDNA3.1/PSMC3 were exposed to a 30-nM treatment of carfilzomib or left untreated (as a negative control). After 16 hours, cells were collected and subjected to RIPA-mediated protein extraction prior to SDS-PAGE and subsequent western-blotting using antibodies specific for ubiquitin, TCF11/Nrf1, Rpt1 (PSMC2), Rpt3 (PSMC4), Rpt5 (PSMC3), Rpt6 (PSMC5), β1, β2, β5, β5i, PA28-α and α-Tubulin (loading control), as indicated. For the TCF11/Nrf1 staining, two exposure times are shown.


( B - B ' ) . Cataract detection revealed abnormal lens reflection in psmc3 morpholino ( MO ) - mediated knockdown but not in controls ( uninj , ctrl - mo ) . Similarly , abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 ( sgRNA2 ) . Co - injection of wt psmc3 mRNA with either psmc3 - mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection . Scale bar = 50 µm . ( B ' ) Quantification of embryos with abnormal lens reflection .

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(B-B'). Cataract detection revealed abnormal lens reflection in psmc3 morpholino (MO)-mediated knockdown but not in controls (uninj, ctrl-mo). Similarly, abnormal lens reflection was also observed in embryos injected with sgRNA + Cas9 but not in sgRNA injected embryos without Cas9 (sgRNA2). Co-injection of wt psmc3 mRNA with either psmc3-mo or sgRNA2 + Cas9 reduced the number of embryos presenting abnormal lens reflection. Scale bar = 50 µm. (B') Quantification of embryos with abnormal lens reflection.


( D - D ' ) bright field images of inner ear development ( lateral position ) . ( D ) Epithelial projections were fused and formed canal pillars in 4 - day - old uninjected and control injected fish ( ctrl - mo , sgRNA2 ) but not in morphants ( mo ) and crispants ( sgRNA2 + Cas9 ) . Co - injection of wt psmc3 mRNA with psmc3 - mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype . Black asterisks indicate fused pillars . Red arrowheads mark unfused projections . Scale bar = 100 µm . ( D ' ) Quantification of embryos with abnormal projection outgrowth .

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(D-D') bright field images of inner ear development (lateral position). (D) Epithelial projections were fused and formed canal pillars in 4-day-old uninjected and control injected fish (ctrl-mo, sgRNA2) but not in morphants (mo) and crispants (sgRNA2+Cas9). Co-injection of wt psmc3 mRNA with psmc3-mo or sgRNA + Cas9 reduced the number of embryos presenting abnormal ear phenotype. Black asterisks indicate fused pillars. Red arrowheads mark unfused projections. Scale bar = 100 µm. (D') Quantification of embryos with abnormal projection outgrowth.


( E - E ' ) An anti - acetylated tubulin antibody ( green ) staining revealed an abnormal amount of kinocilia in psmc3 crispants ( sgRNA2 + Cas9 ) compared to uninjected and control injected embryos ( sgRNA2 ) . Nuclei are stained in blue with DAPI . Representative images show kinocilia of the lateral cristae . Scale bar = 20 µm . ( E ' ) Quantification of embryos with an abnormal amount of kinocilia .

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(E-E') An anti-acetylated tubulin antibody (green) staining revealed an abnormal amount of kinocilia in psmc3 crispants (sgRNA2+Cas9) compared to uninjected and control injected embryos (sgRNA2). Nuclei are stained in blue with DAPI. Representative images show kinocilia of the lateral cristae. Scale bar = 20 µm. (E') Quantification of embryos with an abnormal amount of kinocilia.


( B ) Phenotyping of the same mice , presented in the same order , inoculated i . n . with 0 . 3 × 105 TCID50 of SARS - CoV - 2 at the age of 5 - 7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E - specific qRT - PCR . Red lines indicate the qRT - PCR limits of detection .

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(B) Phenotyping of the same mice, presented in the same order, inoculated i.n. with 0.3 × 105 TCID50 of SARS-CoV-2 at the age of 5-7 wks and viral RNA content was determination in the indicated organs at 3 dpi by conventional E-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection.


( C , D ) Quantification ( C ) and images ( D ) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B , presented in the same order .

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(C, D) Quantification (C) and images (D) of hACE2 protein expression level by Western blot from the lungs of the same mice as in panels A and B, presented in the same order.


( B - E ) Examples of lesions observed in the lungs of infected mice at higher magnification . The two top panels depict mild ( B ) ( scale bar : 100 μm ) and mild to moderate ( C ) interstitium thickening accompanied by dense inflammatory infiltrates predominantly localized in the vicinity of bronchioles ( yellow stars ) and also present around blood vessels ( green stars ) ( scale bar : 200 μm ) . ( D ) Alveoli filled with a proteinaceous exudate containing a few cells ( green arrows ) ( scale bar : 50 μm ) . ( E ) Discrete degenerative lesions of the bronchiolar epithelium , such as perinuclear clear spaces ( blue arrows ) , hyper - eosinophilic cells with condensed nuclei ( orange arrow ) and some intraluminal fibrinous and cell debris ( black arrow ) in an overall well - preserved epithelium ( scale bar : 20 μm ) .

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(B-E) Examples of lesions observed in the lungs of infected mice at higher magnification. The two top panels depict mild (B) (scale bar: 100 μm) and mild to moderate (C) interstitium thickening accompanied by dense inflammatory infiltrates predominantly localized in the vicinity of bronchioles (yellow stars) and also present around blood vessels (green stars) (scale bar: 200 μm). (D) Alveoli filled with a proteinaceous exudate containing a few cells (green arrows) (scale bar: 50 μm). (E) Discrete degenerative lesions of the bronchiolar epithelium, such as perinuclear clear spaces (blue arrows), hyper-eosinophilic cells with condensed nuclei (orange arrow) and some intraluminal fibrinous and cell debris (black arrow) in an overall well-preserved epithelium (scale bar: 20 μm).


( G ) Representative NCoV - 2 - specific IHC image of the brain in NI control or SARS - CoV - 2 - infected mice at 3 dpi . Scale bar : 500 μm . ( H ) Closer view of a NCoV - 2 positive area from an infected mouse . Scale bar : 500 μm .

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(G) Representative NCoV-2-specific IHC image of the brain in NI control or SARS-CoV-2-infected mice at 3 dpi. Scale bar: 500 μm. (H) Closer view of a NCoV-2 positive area from an infected mouse. Scale bar: 500 μm.


( A ) Comparative permissiveness of various organs from B6 . K18 - hACE2IP - THV and B6 . K18 - ACE22prlmn / Jax transgenic mice ( n = 5 - 6 / group ) to SARS - CoV - 2 replication , as determined at 3 dpi by conventional E - specific or sub - genomic Esg - specific qRT - PCR . Red lines indicate the qRT - PCR limits of detection . ( B ) Comparative quantitation of hACE - 2 mRNA in the lungs and brain of B6 . K18 - hACE2IP - THV and B6 . K18 - ACE22prlmn / Jax transgenic mice ( n = 5 - 6 / group ) . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , ns = not significant ) .

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(A) Comparative permissiveness of various organs from B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group) to SARS-CoV-2 replication, as determined at 3 dpi by conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR limits of detection. (B) Comparative quantitation of hACE-2 mRNA in the lungs and brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax transgenic mice (n = 5-6/group). Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ns = not significant).


( C ) Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression in the lungs or brain of B6 . K18 - hACE2IP - THV and B6 . K18 - ACE22prlmn / Jax mice at 3 dpi ( n = 5 - 6 / group ) . Data were normalized versus untreated controls .

(C) Heatmaps represent log2 fold change in cytokine and chemokine mRNA expression in the lungs or brain of B6.K18-hACE2IP-THV and B6.K18-ACE22prlmn/Jax mice at 3 dpi (n = 5-6/group). Data were normalized versus untreated controls.


( C ) Viral RNA content as determined in various organs at 3 dpi ( n = 6 / group ) by use of conventional E - specific or sub - genomic Esg - specific qRT - PCR . Red lines indicate the qRT - PCR detection limits . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(C) Viral RNA content as determined in various organs at 3 dpi (n = 6/group) by use of conventional E-specific or sub-genomic Esg-specific qRT-PCR. Red lines indicate the qRT-PCR detection limits. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).


( D ) Percentages of NK cells or neutrophils in the lungs of LV : : S - or sham - vaccinated and SARS - CoV - 2 - challenged B6 . K18 - hACE2IP - THV transgenic mice at 3 dpi ( n = 6 / group ) . Percentages were calculated versus total lung live CD45 + cells . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(D) Percentages of NK cells or neutrophils in the lungs of LV::S- or sham-vaccinated and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Percentages were calculated versus total lung live CD45+ cells. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).


( E ) Relative log2 fold change in cytokine and chemokine mRNA expression in the brain of LV : : S - or sham - immunized and SARS - CoV - 2 - challenged B6 . K18 - hACE2IP - THV transgenic mice at 3 dpi ( n = 6 / group ) . Means ± SD are shown . Data were normalized versus untreated controls . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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(E) Relative log2 fold change in cytokine and chemokine mRNA expression in the brain of LV::S- or sham-immunized and SARS-CoV-2-challenged B6.K18-hACE2IP-THV transgenic mice at 3 dpi (n = 6/group). Means ± SD are shown. Data were normalized versus untreated controls. Data information: Statistical significance was evaluated by Mann-Whitney test (* = p<0.05, ** = p<0.01).


B6 . K18 - hACE2IP - THV mice were primed ( i . m . ) at wk 0 and boosted ( i . n . ) at wk 5 ( n = 5 ) with non - integrative LV : : S . Control mice were injected with an empty LV ( sham ) . ( B , C ) Cytometric strategy to detect lung CD8 + T central memory ( Tcm , CD44 + CD62L + CD69 - ) , T effector memory ( Tem , CD44 + CD62L - CD69 - ) and T resident memory ( Trm , CD44 + CD62L - CD69 + CD103 + ) and ( C ) percentages of these subsets among CD8 + T - cells in LV : : S - vaccinated ( n = 9 ) or sham ( n = 5 ) mice . Data information : Statistical significance of the difference between the two groups was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham). (B, C) Cytometric strategy to detect lung CD8+ T central memory (Tcm, CD44+CD62L+CD69-), T effector memory (Tem, CD44+CD62L-CD69-) and T resident memory (Trm, CD44+CD62L-CD69+CD103+) and (C) percentages of these subsets among CD8+ T-cells in LV::S-vaccinated (n = 9) or sham (n = 5) mice. Data information: Statistical significance of the difference between the two groups was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01).


B6 . K18 - hACE2IP - THV mice were primed ( i . m . ) at wk 0 and boosted ( i . n . ) at wk 5 ( n = 5 ) with non - integrative LV : : S . Control mice were injected with an empty LV ( sham ) . ( D ) Cytometric strategy to detect SCoV - 2 - specific CD8 + T cells by use of the H - 2Db - SCoV - 2 : 538 - 546 dextramer in the lungs of LV : : S or sham - vaccinated mice . Inside CD8 + dextramer + T - cell subset , Tcm , Tem and Trm have been distinguished .

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B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham). (D) Cytometric strategy to detect SCoV-2-specific CD8+ T cells by use of the H-2Db-SCoV-2:538-546 dextramer in the lungs of LV::S or sham-vaccinated mice. Inside CD8+ dextramer+ T-cell subset, Tcm, Tem and Trm have been distinguished.


B6 . K18 - hACE2IP - THV mice were primed ( i . m . ) at wk 0 and boosted ( i . n . ) at wk 5 ( n = 5 ) with non - integrative LV : : S . Control mice were injected with an empty LV ( sham ) . ( F , G ) Anti - SCoV - 2 IgG or IgA titers ( F ) and neutralizing activity ( EC50 ) ( G ) in the sera or lung homogenates at 3 dpi . Samples from individual mice ( n = 4 / group ) were studied . Data information : Statistical significance of the difference between the two groups was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 ) .

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B6.K18-hACE2IP-THV mice were primed (i.m.) at wk 0 and boosted (i.n.) at wk 5 (n = 5) with non-integrative LV::S. Control mice were injected with an empty LV (sham). (F, G) Anti-SCoV-2 IgG or IgA titers (F) and neutralizing activity (EC50) (G) in the sera or lung homogenates at 3 dpi. Samples from individual mice (n = 4/group) were studied. Data information: Statistical significance of the difference between the two groups was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01).


( A ) B6 . K18 - hACE2IP - THV mice were immunized with LV : : S via i . m . or i . n . at wk 0 and boosted via i . m . or i . n . at wk 5 ( n = 5 / group ) with non - integrative LV : : S . Control mice were injected i . m . i . n . with an empty LV ( sham ) . Viral RNA contents were determined by conventional E - specific RT - PCR at 3 dpi , in the brain , lung and nasal washes . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 , ns = not significant ) .

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(A) B6.K18-hACE2IP-THV mice were immunized with LV::S via i.m. or i.n. at wk 0 and boosted via i.m. or i.n. at wk 5 (n = 5/group) with non-integrative LV::S. Control mice were injected i.m. i.n. with an empty LV (sham). Viral RNA contents were determined by conventional E-specific RT-PCR at 3 dpi, in the brain, lung and nasal washes. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p <0.01, ****= p < 0.0001, ns = not significant).


( B ) Lung histology in B6 . K18 - hACE2IP - THV mice , vaccinated with LV : : S after SARS - CoV - 2 inoculation . H & E ( rows 1 and 3 ) ( scale bar : 500 μm ) and NCoV - 2 - specific IHC ( rows 2 and 4 ) ( scale bar : 50 μm ) staining of whole lung sections ( scale bar : 50 μm ) from the primed ( i . m . ) , boosted ( i . n . ) and challenged B6 . K18 - hACE2IP - THV mice compared with their sham controls . H & E and NCoV - 2 - specific IHC were performed on contiguous sections . The IHC fields correspond to the rectangles in the corresponding H & E images above them . A representative NCoV - 2 - specific IHC on a lung section from a non - infected ( NI ) mouse is also shown .

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(B) Lung histology in B6.K18-hACE2IP-THV mice, vaccinated with LV::S after SARS-CoV-2 inoculation. H&E (rows 1 and 3) (scale bar: 500 μm) and NCoV-2-specific IHC (rows 2 and 4) (scale bar: 50 μm) staining of whole lung sections (scale bar: 50 μm) from the primed (i.m.), boosted (i.n.) and challenged B6.K18-hACE2IP-THV mice compared with their sham controls. H&E and NCoV-2-specific IHC were performed on contiguous sections. The IHC fields correspond to the rectangles in the corresponding H&E images above them. A representative NCoV-2-specific IHC on a lung section from a non-infected (NI) mouse is also shown.


( A ) Example of CD3 - positive cells in an olfactory bulb from an LV : : S i . m . - i . n . vaccinated and protected mice and representative results from this group versus sham - vaccinated and unprotected mice at 3 dpi ( n = 7 - 9 / group ) . Scale bar : 50 μm . Statistical significance was evaluated by Mann - Whitney test ( * * = p < 0 . 01 ) .

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(A) Example of CD3-positive cells in an olfactory bulb from an LV::S i.m.-i.n. vaccinated and protected mice and representative results from this group versus sham-vaccinated and unprotected mice at 3 dpi (n = 7-9/group). Scale bar: 50 μm. Statistical significance was evaluated by Mann-Whitney test (**= p <0.01).


Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups .

Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups.


Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups . ( C , D ) Innate immune cells in the olfactory bulbs ( C ) or brain ( D ) . The CD11b + Ly6C + Ly6G + population in the olfactory bulbs are neutrophils and the CD11b + Ly6C + Ly6G - cells of the brain are inflammatory monocytes .

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Cytometric analysis of cells extracted from pooled olfactory bulbs from the same groups. (C, D) Innate immune cells in the olfactory bulbs (C) or brain (D). The CD11b+ Ly6C+ Ly6G+ population in the olfactory bulbs are neutrophils and the CD11b+ Ly6C+ Ly6G- cells of the brain are inflammatory monocytes.


( E ) Brain H & E histology at 3 dpi . The top right and both bottom panels show examples , in two different mice , of leukocyte clusters ( arrows ) alongside the ventricular wall . No such clusters were detected in the LV : : S i . m . - i . n . vaccinated mice ( top left panel ) . Scale bar : 200 μm . The close up view ( bottom right panel ) highlights the thickened , disorganized ependymal lining , compared to the normal ependymal cells and cilia of an LV : : S i . m . - i . n . vaccinated mouse ( top left panel ) . Scale bar : 50 μm .

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(E) Brain H&E histology at 3 dpi. The top right and both bottom panels show examples, in two different mice, of leukocyte clusters (arrows) alongside the ventricular wall. No such clusters were detected in the LV::S i.m.-i.n. vaccinated mice (top left panel). Scale bar: 200 μm. The close up view (bottom right panel) highlights the thickened, disorganized ependymal lining, compared to the normal ependymal cells and cilia of an LV::S i.m.-i.n. vaccinated mouse (top left panel). Scale bar: 50 μm.


( B ) Brain or lung viral RNA contents , determined by conventional E - specific or sub - genomic Esg - specific qRT - PCR at 3 dpi . Two mice out of the 5 sham - vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 ) .

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(B) Brain or lung viral RNA contents, determined by conventional E-specific or sub-genomic Esg-specific qRT-PCR at 3 dpi. Two mice out of the 5 sham-vaccinated mice did not have detectable viral RNA in the lungs despite high viral RNA content in the brain and hACE2 mRNA expression levels comparable to that of the other mice in the same group. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).


( E ) Wild type or µMT KO mice ( n = 5 - 9 / group ) were injected by LV : : S or sham following the time line shown in ( A ) , then pretreated with Ad5 : : hACE2 4 days before challenge with the ancestral SARS - CoV - 2 strain . Lung viral RNA contents were determined at 3 dpi . Data information : Statistical significance was evaluated by Mann - Whitney test ( * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 ) .

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(E) Wild type or µMT KO mice (n = 5-9/group) were injected by LV::S or sham following the time line shown in (A), then pretreated with Ad5::hACE2 4 days before challenge with the ancestral SARS-CoV-2 strain. Lung viral RNA contents were determined at 3 dpi. Data information: Statistical significance was evaluated by Mann-Whitney test (*= p < 0.05, **= p < 0.01, ****= p < 0.0001).


A - C Confocal images of primary hepatocytes grown under indicated conditions and stained with anti - CLUH antibody and Hadha ( A ) , Pcx ( B ) and Actb ( C ) mRNA in situ hybridization . Right panels show 5x magnified boxed areas . Scale bar , 10 μm . D - F Manders ' colocalization coefficient between Hadha ( D ) Pcx ( E ) and Actb ( F ) mRNA molecules and CLUH signal ( n ≥ 50 cells isolated from 3 - 6 mice ) . Data information : In ( D - F ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) .

A-C Confocal images of primary hepatocytes grown under indicated conditions and stained with anti-CLUH antibody and Hadha (A), Pcx (B) and Actb (C) mRNA in situ hybridization. Right panels show 5x magnified boxed areas. Scale bar, 10 μm. D-F Manders' colocalization coefficient between Hadha (D) Pcx (E) and Actb (F) mRNA molecules and CLUH signal (n ≥ 50 cells isolated from 3-6 mice). Data information: In (D-F), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test).


G , H Confocal images of primary hepatocytes grown in HBSS and stained with anti - CLUH and anti - TIA - 1 ( G ) or anti - G3BP1 ( H ) antibodies . Right panels show 5x magnified boxed areas . Scale bar , 10 μm .

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G, H Confocal images of primary hepatocytes grown in HBSS and stained with anti-CLUH and anti-TIA-1 (G) or anti-G3BP1 (H) antibodies. Right panels show 5x magnified boxed areas. Scale bar, 10 μm.


A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti - puromycin antibody . Scale bar , 10 μm . B Quantification of fluorescence intensity per cell of experiment shown in ( A ) . AU , arbitrary units . ( n = 90 - 110 cells per treatment isolated from 4 mice ) . Data information data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values .

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A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti-puromycin antibody. Scale bar, 10 μm. B Quantification of fluorescence intensity per cell of experiment shown in (A). AU, arbitrary units. (n= 90-110 cells per treatment isolated from 4 mice). Data information data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values.


C Quantification of the number of puromycin granules under indicated conditions ( n ≥ 50 cells isolated from 4 mice ) . Data information : data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 ( Student ' s t - test ) .

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C Quantification of the number of puromycin granules under indicated conditions (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).


D Confocal images of primary hepatocytes stained with anti - puromycin and anti - CLUH antibodies . The cells from which the enlarged areas ( 400 μm2 ) have been magnified are shown in Appendix Figure S1B for each individual channel . Scale bar , 4 μm . Cells analyzed were isolated from 4 different mice with similar results . Arrows point to colocalizing particles .

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D Confocal images of primary hepatocytes stained with anti-puromycin and anti-CLUH antibodies. The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S1B for each individual channel. Scale bar, 4 μm. Cells analyzed were isolated from 4 different mice with similar results. Arrows point to colocalizing particles.


E Manders ' colocalization coefficient between puromycin and CLUH from experiment shown in ( D ) ( n ≥ 50 cells isolated from 4 mice ) . Data information : data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . ( E ) * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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E Manders' colocalization coefficient between puromycin and CLUH from experiment shown in (D) (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (E) ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


F Quantification of puromycin granules containing CLUH signal ( n ≥ 80 cells isolated from 4 mice ) . Data information : data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 ( Student ' s t - test ) .

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F Quantification of puromycin granules containing CLUH signal (n ≥ 80 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).


A - C Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for ( A ) Hadha , ( B ) Pcx and ( C ) Hmgcs2 . Scale bar , 4 µm . D - F Fluorescence profile of 100 - pixel line from the merged channel shown in ( A - C ) . The cells from which the enlarged areas ( 400 μm2 ) have been magnified are shown in Appendix Figure S2 for each individual channel . Cells analyzed were isolated from 6 different mice with similar results .

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A-C Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for (A) Hadha, (B) Pcx and (C) Hmgcs2. Scale bar, 4 µm. D-F Fluorescence profile of 100-pixel line from the merged channel shown in (A-C). The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S2 for each individual channel. Cells analyzed were isolated from 6 different mice with similar results.


A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH . Asterisks indicates signal of previous incubation with anti - G3BP1 antibody . Pan - actin was used as loading control .

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A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Asterisks indicates signal of previous incubation with anti-G3BP1 antibody. Pan-actin was used as loading control.


B Confocal images of anti - CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH . Insets show 2 . 6x enlarged area . Scale bar , 10 µm . C Quantification of cells with CLUH - granules from experiments shown in ( B ) ( n = 3 independent experiments , > 50 cells per experiment per condition ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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B Confocal images of anti-CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Insets show 2.6x enlarged area. Scale bar, 10 µm. C Quantification of cells with CLUH-granules from experiments shown in (B) (n=3 independent experiments, >50 cells per experiment per condition). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1 - GFP plasmid and incubated in HBSS medium with or without CHX . Cells were recorded for a maximum of 2 h . Insets show 2 . 5x enlarged areas . Scale bar , 10 µm . E Total number of cells analyzed by life imaging for the indicated experiments shown in ( D ) . " Positive " relates to a cell which formed G3BP1 granules at the end of the recording .

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D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and incubated in HBSS medium with or without CHX. Cells were recorded for a maximum of 2 h. Insets show 2.5x enlarged areas. Scale bar, 10 µm. E Total number of cells analyzed by life imaging for the indicated experiments shown in (D). "Positive" relates to a cell which formed G3BP1 granules at the end of the recording.


F Confocal images of anti - G3BP1 staining in primary hepatocytes derived from Li - CluhWT and Li - CluhKO mice grown in indicated media . Arrows point to G3BP1 - granules . Scale bar , 10 µm . G Quantification of percentage of cells with G3BP1 - positive granules of experiment shown in F . Cells were analyzed in a blind fashion ( n = 3 mice per genotype ; number of cells in total : WT Basal , 449 ; KO Basal , 303 ; WT HBSS , 146 ; KO HBSS , 161 ) . Data information : , data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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F Confocal images of anti-G3BP1 staining in primary hepatocytes derived from Li-CluhWT and Li-CluhKO mice grown in indicated media. Arrows point to G3BP1-granules. Scale bar, 10 µm. G Quantification of percentage of cells with G3BP1-positive granules of experiment shown in F. Cells were analyzed in a blind fashion (n=3 mice per genotype; number of cells in total: WT Basal, 449; KO Basal, 303; WT HBSS, 146; KO HBSS, 161). Data information: , data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


H Western blot of primary WT and KO hepatocytes stained with indicated antibodies . Asterisks indicates unspecific signal . Pan - actin was used as loading control .

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H Western blot of primary WT and KO hepatocytes stained with indicated antibodies. Asterisks indicates unspecific signal. Pan-actin was used as loading control.


I Quantification of western blot ( n = 3 per genotype per condition ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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I Quantification of western blot (n=3 per genotype per condition). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions . ( n = 4 per genotype per condition ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions. (n=4 per genotype per condition). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


A , B Volcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal ( A ) and HBSS ( B ) conditions .

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A, B Volcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal (A) and HBSS (B) conditions.


F Correlation of protein fold changes in KO in respect to WT hepatocytes under basal condition and upon HBSS ( 2 h ) . Only genes previously found in CLUH RIP experiments in HeLa cells ( Gao et al . , 2014 ) are plotted . Inset shows magnification of upper part of the graph .

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F Correlation of protein fold changes in KO in respect to WT hepatocytes under basal condition and upon HBSS (2 h). Only genes previously found in CLUH RIP experiments in HeLa cells (Gao et al., 2014) are plotted. Inset shows magnification of upper part of the graph.


A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies . Pan - actin was used as loading control . B , C Quantification of western blots as shown in ( A ) . Antibody signal was normalized to pan - actin signal and signal of phospho - protein was normalized to signal of total protein ( n = 4 independent experiments ) . Data information : In ( B , C ) , data shows the mean ± SEM . * * , P ≤ 0 . 01 for genotype and * * * , P ≤ 0 . 001 for time ( Two - way ANOVA ) . Error bars show minimum and maximum values .

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A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies. Pan-actin was used as loading control. B, C Quantification of western blots as shown in (A). Antibody signal was normalized to pan-actin signal and signal of phospho-protein was normalized to signal of total protein (n=4 independent experiments). Data information: In (B, C), data shows the mean ± SEM. **, P≤0.01 for genotype and ***, P≤0.001 for time (Two-way ANOVA). Error bars show minimum and maximum values.


D Representative western blot of total protein extracts from livers of starved Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . GAPDH was used as loading control . E , F Quantification of western blots as shown in ( D ) . Antibody signal was normalized to GAPDH signal and signal of phospho - protein was normalized to signal of the total protein ( n = 8 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . ( H , J ) * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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D Representative western blot of total protein extracts from livers of starved Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. E, F Quantification of western blots as shown in (D). Antibody signal was normalized to GAPDH signal and signal of phospho-protein was normalized to signal of the total protein (n=8 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti - cleaved caspase 3 antibody . Scale bar , 25 µm .

G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti-cleaved caspase 3 antibody. Scale bar, 25 µm.


H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin . More than 100 cells per replicate were analyzed in a blind fashion ( n = 3 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . Error bars show minimum and maximum values .

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H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin. More than 100 cells per replicate were analyzed in a blind fashion (n=3 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values.


I IHC staining of cleaved caspase 3 in liver sections of starved Li - CluhWT and Li - CluhKO mice . Arrows point to positive apoptotic foci . Scale bar , 200 µm .

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I IHC staining of cleaved caspase 3 in liver sections of starved Li-CluhWT and Li-CluhKO mice. Arrows point to positive apoptotic foci. Scale bar, 200 µm.


J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed ( n = 5 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM .

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J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed (n=5 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM.


K . Confocal images of primary hepatocytes grown in indicated media and stained with anti - G3BP1 and anti - mTOR . Scale bar , 10 µm . L Manders ' colocalization coefficient between G3BP1 and mTOR staining ( n > 50 cells isolated from 3 mice ) . Data information : In ( L ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . ( H , J ) * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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K. Confocal images of primary hepatocytes grown in indicated media and stained with anti-G3BP1 and anti-mTOR. Scale bar, 10 µm. L Manders' colocalization coefficient between G3BP1 and mTOR staining (n >50 cells isolated from 3 mice). Data information: In (L), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


A Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO - LC3 - GFP mice . Scale bar , 25 µm . B Quantification of LC3 signal morphology of images shown in ( A ) . Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 4 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) .

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A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Scale bar, 25 µm. B Quantification of LC3 signal morphology of images shown in (A). Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei. 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=4 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).


C Confocal images of primary hepatocytes derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice . Hepatocytes were starved for 2 h in HBSS . Scale bar , 10μm . D - E Quantification of LC3 puncta number ( D ) and area ( E ) per cell ( n ≥ 100 cells isolated from 3 mice ) . Data information : In ( D , E ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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C Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Hepatocytes were starved for 2 h in HBSS. Scale bar, 10μm. D-E Quantification of LC3 puncta number (D) and area (E) per cell (n ≥100 cells isolated from 3 mice). Data information: In (D, E), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


F Western blot of total protein extracts from livers of Li - CluhWT and Li - CluhKO mice probed with LAMP1 . GAPDH was used as loading control . G Quantification of western blot shown in ( F ) . WT : n = 4 ; KO : n = 3 . Data information : In data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) .

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F Western blot of total protein extracts from livers of Li-CluhWT and Li-CluhKO mice probed with LAMP1. GAPDH was used as loading control. G Quantification of western blot shown in (F). WT: n=4; KO: n=3. Data information: In data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).


H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies . Two different exposure times are shown for LC3 . I refers to total LC3 and II indicates lipidated form of LC3 . Asterisk indicates signal of previous incubation . I Quantification of LC3 levels in western blots as shown in ( H ) ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies. Two different exposure times are shown for LC3. I refers to total LC3 and II indicates lipidated form of LC3. Asterisk indicates signal of previous incubation. I Quantification of LC3 levels in western blots as shown in (H) (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


J Quantification of autophagic flux of western blot as shown in ( H ) . The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition . The flux in KO cells was normalized to flux in WT cells . ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) .

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J Quantification of autophagic flux of western blot as shown in (H). The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition. The flux in KO cells was normalized to flux in WT cells. (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).


Quantification of p62 levels in western blots ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

Quantification of p62 levels in western blots (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Quantification of CLUH levels in western blots ( n = 4 independent experiments ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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Quantification of CLUH levels in western blots (n=4 independent experiments). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Western blot analysis of total protein extracts from livers of 8 weeks old Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . GAPDH was used as loading control . B Quantification of western blot shown in ( A ) . WT : n = 4 ; KO : n = 3 . Data information data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) .

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Western blot analysis of total protein extracts from livers of 8 weeks old Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. B Quantification of western blot shown in (A). WT: n=4; KO: n=3. Data information data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).


C Western blot analysis of isolated mitochondria from Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . PonceauS staining was used as a loading control .

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C Western blot analysis of isolated mitochondria from Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. PonceauS staining was used as a loading control.


D Quantification of western blot ( n = 3 mice per genotype ) . Data information : , data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) .

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D Quantification of western blot (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).


E Confocal images of liver cryosections derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice and stained with anti - TOM20 antibody . On the right side , indicated boxes were 3 . 5x magnified . Arrows point to colocalizing spots . Scale bar , 25 μm . F Manders ' colocalization coefficient between TOM20 and LC3 from experiments shown in ( E ) . 5 images were analyzed per animal to get an averaged value ( n = 5 mice per genotype ) . Data information , data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) .

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E Confocal images of liver cryosections derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. On the right side, indicated boxes were 3.5x magnified. Arrows point to colocalizing spots. Scale bar, 25 μm. F Manders' colocalization coefficient between TOM20 and LC3 from experiments shown in (E). 5 images were analyzed per animal to get an averaged value (n=5 mice per genotype). Data information , data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).


G Confocal images of primary hepatocytes derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice and stained with anti - TOM20 antibody . When indicated cells were treated with rapamycin ( 200 nM for 4h ) . Bottom panels show 2 . 3x magnified areas for each channel of indicated boxes . Arrows point to colocalizing spots . Scale bar , 10 μm . H Manders ´ colocalization coefficient from experiment shown in ( G ) ( n ≥ 50 cells isolated from 3 mice per genotype ) . Data information : In ( H ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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G Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. When indicated cells were treated with rapamycin (200 nM for 4h). Bottom panels show 2.3x magnified areas for each channel of indicated boxes. Arrows point to colocalizing spots. Scale bar, 10 μm. H Manders´ colocalization coefficient from experiment shown in (G) (n ≥ 50 cells isolated from 3 mice per genotype). Data information: In (H), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


I Confocal images of WT and Cluh KO MEFs transfected with mCherry - GFP - FIS101 - 152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A / oligomycin . Arrows indicate mitophagosomes . Scale bar , 10μm . J Quantification of percentage of cells with mitophagosomes . Cells were considered positive when red signal was clearly recognizable ( n = 3 - 5 independent experiments , > 100 cells per experiment ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10μm. J Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


K Confocal images of WT and Cluh KO MEFs transfected with mCherry - GFP - FIS101 - 152 , incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A / oligomycin . Arrows indicate mitophagosomes . Scale bar , 10 μm . L Quantification of percentage of cells with mitophagosomes . Cells were considered positive when red signal was clearly recognizable ( n = 3 - 5 independent experiments , > 100 cells per experiment ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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K Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152, incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 μm. L Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


A Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO mice injected with rapamycin or mock solution and stained with anti - TOM20 antibody . Scale bar , 25 µm . B Quantification of morphological parameters of experiments shown in ( A ) . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 3 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . * * , P ≤ 0 . 01 ; * , P ≤ 0 . 05 ( Student ' s t - test ) .

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A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-TOM20 antibody. Scale bar, 25 µm. B Quantification of morphological parameters of experiments shown in (A). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).Data information: data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).


C Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO mice injected with rapamycin or mock solution and stained with anti - LAMP1 antibody . Scale bar , 25 µm . D Quantification of morphological parameters of experiments shown in ( C ) . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 3 mice per genotype ) . Data information : , data are presented as histograms showing the mean ± SEM . * * , P ≤ 0 . 01 ; * , P ≤ 0 . 05 ( Student ' s t - test ) .

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C Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-LAMP1 antibody. Scale bar, 25 µm. D Quantification of morphological parameters of experiments shown in (C). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).


E Confocal images of COS - 7 cells downregulated for the indicated genes and stained with anti - TOM20 antibody . Scale bar , 10 µm .

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E Confocal images of COS-7 cells downregulated for the indicated genes and stained with anti-TOM20 antibody. Scale bar, 10 µm.


F Representative western blot of COS - 7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies . Asterisks indicate unspecific signal or signal of previous incubation .

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F Representative western blot of COS-7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies. Asterisks indicate unspecific signal or signal of previous incubation.


G Quantification of morphological parameters of experiments ( n > 100 cells from 3 independent experiments ) . Data information : In ( G ) , data presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . ( G ) * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 * , P ≤ 0 . 05 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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G Quantification of morphological parameters of experiments (n>100 cells from 3 independent experiments). Data information: In (G), data presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (G) ***, P≤0.001; **, P≤0.01 *, P≤0.05 (One-way ANOVA, Tukey's multiple comparisons test).


D ) Proline biosynthesis protein levels in breast cancer subtypes ( Log2 ratios of light vs . SILAC standard ) . The upper and lower limits of the box show values from the first to the third quartile , with the horizontal line indicating the median . The whiskers extend 1 . 5 times the interquartile range from the edges of the box . Outlier values were plotted outside the whiskers . Significance was determined using paired Student ' s t - test and p values were corrected using permutation based FDR . * p < 0 . 05 and * * * p < 0 . 001 .

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D) Proline biosynthesis protein levels in breast cancer subtypes (Log2 ratios of light vs. SILAC standard). The upper and lower limits of the box show values from the first to the third quartile, with the horizontal line indicating the median. The whiskers extend 1.5 times the interquartile range from the edges of the box. Outlier values were plotted outside the whiskers. Significance was determined using paired Student's t-test and p values were corrected using permutation based FDR . *p < 0.05 and *** p < 0.001.


E ) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study ( n = 3 ) ; Scale bar : 200 μm .

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E) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study (n=3); Scale bar: 200 μm.


F ) Quantification of IHC shows percentage of cells with 2 + and 3 + PYCR1 staining intensity . Data is expressed as mean ± SD of three patient samples . Paired samples were compared by a Student ' s t - test . * p < 0 . 05 and * * * p < 0 . 001 .

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F) Quantification of IHC shows percentage of cells with 2+ and 3+ PYCR1 staining intensity. Data is expressed as mean ± SD of three patient samples. Paired samples were compared by a Student's t-test. *p < 0.05 and *** p < 0.001.


G ) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data . Cox univariate p value , risk table and hazard ratio with 95 % CI indicated .

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G) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data. Cox univariate p value, risk table and hazard ratio with 95% CI indicated.


A ) Seahorse experiment measuring oxygen consumption rate ( OCR ) in MCF7 cells upon addition of oligomycin , FCCP and a mixture of antimycin and rotenone . Arrows indicate the points of addition of each inhibitor . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Similar results were obtained in three biological experiments . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values were indicated as follows * * p < 0 . 01 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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A) Seahorse experiment measuring oxygen consumption rate (OCR) in MCF7 cells upon addition of oligomycin, FCCP and a mixture of antimycin and rotenone. Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values were indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


B ) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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B) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


C ) 13C5 - Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate , malate and citrate in MCF7 cells . Data are presented as mean ± SD of triplicate samples . Isotopologues are represented as M + n where M indicates the mass and n equals number of 13C

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C) 13C5 -Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate, malate and citrate in MCF7 cells. Data are presented as mean ± SD of triplicate samples. Isotopologues are represented as M+n where M indicates the mass and n equals number of 13C incorporated


D ) Seahorse experiment measuring extracellular acidification rate ( ECAR ) in MCF7 cells upon addition of glucose , oligomycin and 2 - deoxyglucose ( 2DG ) . Arrows indicate the points of addition of each inhibitor . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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D) Seahorse experiment measuring extracellular acidification rate (ECAR) in MCF7 cells upon addition of glucose, oligomycin and 2-deoxyglucose (2DG). Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


E ) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Similar results were obtained in three biological experiments . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * * p < 0 . 01 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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E) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments.Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


F ) Normalized lactate concentration upon PYCR1 KO . Data represents mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


A ) Representative pictures of transwell migration and invasion after PYCR1 knockout . Scale bar : 100 μm . B ) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 .

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A) Representative pictures of transwell migration and invasion after PYCR1 knockout. Scale bar :100 μm. B) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p-values are reported as follows *** p < 0.001.


C ) Representative images of colony formation in soft agar upon PYCR1 KO . Scale bar : 100 μm .

C) Representative images of colony formation in soft agar upon PYCR1 KO. Scale bar: 100 μm.


D ) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours . Data represents mean ± SE of three biological experiments . Samples were compared using paired Student ' s t - test .

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D) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours. Data represents mean ± SE of three biological experiments. Samples were compared using paired Student's t-test.


E - H ) Bar plots show percentage of viable cells after treatment with 0 . 75 mM H2O2 ( E ) , 0 . 06 µM paclitaxel ( F ) , 0 . 15 µM doxorubicin ( G ) , 2 . 5 mM cyclophosphamide ( H ) . Data represent mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 .

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E-H) Bar plots show percentage of viable cells after treatment with 0.75 mM H2O2 (E), 0.06 µM paclitaxel (F), 0.15 µM doxorubicin (G), 2.5 mM cyclophosphamide (H). Data represent mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05.


I ) Tumor volume measurements for 26 days in MCF7 injected NSG mice . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) are shown . Data represents mean ± SE . Pax : Paclitaxel , Dox : Doxorubicin . Groups were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * p < 0 . 05 , * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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I) Tumor volume measurements for 26 days in MCF7 injected NSG mice. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) are shown. Data represents mean ± SE. Pax: Paclitaxel, Dox: Doxorubicin. Groups were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *p < 0.05, ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


J ) Bar plot indicates mean ± SE of tumor volume measurements for day 26 . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

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J) Bar plot indicates mean ± SE of tumor volume measurements for day 26. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


( A ) HeLa cells expressing HA - Parkin were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies .

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(A) HeLa cells expressing HA-Parkin were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.


( B ) HeLa cells stably expressing HA - Parkin or intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments . I , C , and M indicate input , cytosol - rich supernatant , and mitochondria - rich membrane pellet , respectively .

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(B) HeLa cells stably expressing HA-Parkin or intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments. I, C, and M indicate input, cytosol-rich supernatant, and mitochondria-rich membrane pellet, respectively.


( D ) Polarized mitochondria stained with MitoTracker red ( red arrowheads ) were not labeled by Parkin . In contrast , damaged mitochondria marked by Parkin ( green arrowheads ) were not stained with MitoTracker red .

(D) Polarized mitochondria stained with MitoTracker red (red arrowheads) were not labeled by Parkin. In contrast, damaged mitochondria marked by Parkin (green arrowheads) were not stained with MitoTracker red.


( E ) HeLa cells expressing HA - Parkin with various pathogenic mutations were treated with CCCP , followed by immunocytochemistry . ( A , D , and E ) Higher magnification views of the boxed areas are shown in the insets . ( F ) Parkin colocalization with mitochondria was analyzed in > 100 cells per mutation . Example figures indicative of robust colocalization ( counted as 1 ) , weak colocalization ( counted as 0 . 5 ) , and no colocalization ( counted as 0 ) are shown . Error bars represent the mean ± SD values of at least three experiments .

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(E) HeLa cells expressing HA-Parkin with various pathogenic mutations were treated with CCCP, followed by immunocytochemistry. (A, D, and E) Higher magnification views of the boxed areas are shown in the insets. (F) Parkin colocalization with mitochondria was analyzed in >100 cells per mutation. Example figures indicative of robust colocalization (counted as 1), weak colocalization (counted as 0.5), and no colocalization (counted as 0) are shown. Error bars represent the mean ± SD values of at least three experiments.


( A ) HeLa cells expressing wild - type Parkin or E3 - inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies . When E3 - inactivating mutations were introduced into Parkin , the mitochondrial ubiquitylation signal disappeared .

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(A) HeLa cells expressing wild-type Parkin or E3-inactivating mutations were treated with CCCP and then immunostained with the indicated antibodies. When E3-inactivating mutations were introduced into Parkin, the mitochondrial ubiquitylation signal disappeared.


( B and C ) HeLa cells expressing HA - Parkin ( B ) or expressing both Mt - GFP and HA - Parkin ( C ) were treated with CCCP or DMSO ( control ) and then immunostained with the indicated antibodies .

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(B and C) HeLa cells expressing HA-Parkin (B) or expressing both Mt-GFP and HA-Parkin (C) were treated with CCCP or DMSO (control) and then immunostained with the indicated antibodies.


( D ) Localization of GFP - Parkin to the mitochondria after CCCP treatment . ( A - D ) Higher magnification views of the boxed areas are shown in the insets .

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(D) Localization of GFP-Parkin to the mitochondria after CCCP treatment. (A-D) Higher magnification views of the boxed areas are shown in the insets.


( E ) HeLa cell lysates expressing GFP - Parkin were immunoprecipitated by anti - GFP antibody , followed by immunoblotting with the indicated antibodies .

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(E) HeLa cell lysates expressing GFP-Parkin were immunoprecipitated by anti-GFP antibody, followed by immunoblotting with the indicated antibodies.


( F ) Straight immunoblotting of HA - and GFP - Parkin in the absence or presence of CCCP . Note the slower migrating ladders derived from ubiquitylation ( Ub ) in only the GFP - Parkin with CCCP lane .

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(F) Straight immunoblotting of HA- and GFP-Parkin in the absence or presence of CCCP. Note the slower migrating ladders derived from ubiquitylation (Ub) in only the GFP-Parkin with CCCP lane.


( G ) GFP - Parkin - expressing HeLa cells with various pathogenic mutations ( Fig . 1 C ) were treated with CCCP and subjected to immunoblotting . Asterisks show ubiquitylation of GFP - Parkin . Vertical black lines indicate that intervening lanes have been spliced out . IB , immunoblot ; IP , immunoprecipitation .

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(G) GFP-Parkin-expressing HeLa cells with various pathogenic mutations (Fig. 1 C) were treated with CCCP and subjected to immunoblotting. Asterisks show ubiquitylation of GFP-Parkin. Vertical black lines indicate that intervening lanes have been spliced out. IB, immunoblot; IP, immunoprecipitation.


( A ) The number of HeLa cells with N - terminal - or C - terminal - tagged PINK1 localized to the mitochondria was counted in > 100 cells .

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(A) The number of HeLa cells with N-terminal- or C-terminal-tagged PINK1 localized to the mitochondria was counted in >100 cells.


( B and C ) Exogenous nontagged PINK1 ( B ) or endogenous PINK1 ( C ) in HeLa cells was immunostained with the indicated antibodies . ( D ) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A . ( A and D ) Error bars represent the mean ± SD values of least three experiments .

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(B and C) Exogenous nontagged PINK1 (B) or endogenous PINK1 (C) in HeLa cells was immunostained with the indicated antibodies. (D) The number of HeLa cells with endogenous PINK1 localized to the mitochondria was counted as in A. (A and D) Error bars represent the mean ± SD values of least three experiments.


( E ) Endogenous PINK1 gradually accumulated after CCCP treatment . The first through the fifth lanes show endogenous PINK1 , and the sixth lane shows overexpressed untagged PINK1 . Note that the asterisk indicates a cross - reacting band because it was not affected by overproduction of untagged PINK1 .

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(E) Endogenous PINK1 gradually accumulated after CCCP treatment. The first through the fifth lanes show endogenous PINK1, and the sixth lane shows overexpressed untagged PINK1. Note that the asterisk indicates a cross-reacting band because it was not affected by overproduction of untagged PINK1.


( F ) Subcellular fractionation of endogenous PINK1 . Intact SH - SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments ( same sample as Fig . 1 B ) . I , C , and M indicate input , cytosol - rich supernatant , and the mitochondria - rich membrane pellet , respectively .

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(F) Subcellular fractionation of endogenous PINK1. Intact SH-SY5Y cells were treated with CCCP or DMSO and subjected to fractionation experiments (same sample as Fig. 1 B). I, C, and M indicate input, cytosol-rich supernatant, and the mitochondria-rich membrane pellet, respectively.


( G ) HeLa cells were treated with CCCP and cycloheximide as depicted , followed by immunoblotting with the indicated antibodies . LDH , lactate dehydrogenase . ( F and G ) Asterisks indicate a cross - reacting band .

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(G) HeLa cells were treated with CCCP and cycloheximide as depicted, followed by immunoblotting with the indicated antibodies. LDH, lactate dehydrogenase. (F and G) Asterisks indicate a cross-reacting band.


( H ) N - terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP . The top panel shows control HeLa cells expressing only GFP . ( B , C , and H ) Higher magnification views of the boxed areas are shown in the insets . Bars , 10 µm .

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(H) N-terminal 34 aa of PINK1 recruited GFP to the mitochondria both in the absence and presence of CCCP. The top panel shows control HeLa cells expressing only GFP. (B, C, and H) Higher magnification views of the boxed areas are shown in the insets. Bars, 10 µm.


( A ) PINK1 knockout ( PINK1 − / − ) or control ( PINK1 + / + ) MEFs were transfected with HA - Parkin , treated with CCCP , and subjected to immunocytochemistry with the indicated antibodies . Higher magnification views of the boxed areas are shown in the insets . ( B ) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig . 3 A .

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(A) PINK1 knockout (PINK1−/−) or control (PINK1+/+) MEFs were transfected with HA-Parkin, treated with CCCP, and subjected to immunocytochemistry with the indicated antibodies. Higher magnification views of the boxed areas are shown in the insets. (B) The number of MEFs with Parkin localized to the mitochondria was counted as in Fig. 3 A.


( C and D ) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1 − / − MEFs . MEFs stably expressing GFP - Parkin were treated with CCCP for 4 h and subjected to immunoblotting ( C )

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(C and D) Neither activation of Parkin nor mitochondrial degradation was observed in PINK1−/− MEFs. MEFs stably expressing GFP-Parkin were treated with CCCP for 4 h and subjected to immunoblotting (C)