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

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


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

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


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

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


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

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


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

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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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


( F & G ) 293T ACE2 OE / TRIM21 KO cells reconstituted with EV or TRIM21 , electroporated with IgG or anti - N antibodies , and infected with SARS - CoV - 2 . Viral replication was then determined by RT - qPCR ( F ) or plaque assay ( G ) . Electroporation of anti - N antibodies significantly inhibits SARS - CoV - 2 replication only in TRIM21 - reconstituted cells ( * = p < 0 . 05 ) . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM . Statistical comparisons were performed using two - way ANOVA .

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(F&G) 293T ACE2 OE/TRIM21 KO cells reconstituted with EV or TRIM21, electroporated with IgG or anti-N antibodies, and infected with SARS-CoV-2. Viral replication was then determined by RT-qPCR (F) or plaque assay (G). Electroporation of anti-N antibodies significantly inhibits SARS-CoV-2 replication only in TRIM21-reconstituted cells (* = p < 0.05). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using two-way ANOVA.


( A ) Capillary - based protein detection ( Jess ) of antibodies against SARS - CoV - 2 antigens in convalescent sera .

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(A) Capillary-based protein detection (Jess) of antibodies against SARS-CoV-2 antigens in convalescent sera.


( B ) Quantification of antigen - specific antibodies in convalescent sera . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM .

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(B) Quantification of antigen-specific antibodies in convalescent sera. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM.


( C ) 293T cells OE ACE2 infected with SARS - CoV - 2 in the presence of electroporated convalescent sera . There are statistically significant differences in the ability of serum from different individuals to inhibit viral replication intracellularly ( * * = p < 0 . 005 , * = p < 0 . 05 ) . ( D ) 293T ACE2 OE / TRIM21 KOs reconstituted with either empty vector ( EV ) or TRIM21 - expressing vector ( TRIM21 ) infected with SARS - CoV - 2 in the presence of electroporated convalescent sera . Intracellular neutralization of viral replication is only observed in cells reconstituted with TRIM21 ( * * = p < 0 . 005 ) . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM . Statistical comparisons were performed using a one - way ( C ) or two - way ( D ) ANOVA .

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(C) 293T cells OE ACE2 infected with SARS-CoV-2 in the presence of electroporated convalescent sera. There are statistically significant differences in the ability of serum from different individuals to inhibit viral replication intracellularly (** = p< 0.005, * = p< 0.05). (D) 293T ACE2 OE/TRIM21 KOs reconstituted with either empty vector (EV) or TRIM21-expressing vector (TRIM21) infected with SARS-CoV-2 in the presence of electroporated convalescent sera. Intracellular neutralization of viral replication is only observed in cells reconstituted with TRIM21 (** = p< 0.005). Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM. Statistical comparisons were performed using a one-way (C) or two-way (D) ANOVA.


( C ) Correlation between N - antibody titre , as calculated by Jess , and intracellular neutralization ( fold decrease in SARS - CoV - 2 replication ) , as measured by EDNA , in convalescent sera from multiple individuals . ( D ) Intracellular neutralization of SARS - CoV - 2 by titrated serum from two strongly N - antibody positive individuals . ( Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM .

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(C) Correlation between N-antibody titre, as calculated by Jess, and intracellular neutralization (fold decrease in SARS-CoV-2 replication), as measured by EDNA, in convalescent sera from multiple individuals. (D) Intracellular neutralization of SARS-CoV-2 by titrated serum from two strongly N-antibody positive individuals. ( Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM.


( E ) ELISpot measuring IFNγ production in T cells from SARS - CoV - 2 convalescents upon stimulation with a peptide library covering N - protein . Data information : All data represents at least three independent replicates . Error bars depict the mean + / - SEM .

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(E) ELISpot measuring IFNγ production in T cells from SARS-CoV-2 convalescents upon stimulation with a peptide library covering N-protein. Data information: All data represents at least three independent replicates. Error bars depict the mean +/- SEM.


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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

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


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

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


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

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


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

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

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


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

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

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


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

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

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


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

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

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


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

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


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

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

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


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

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

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


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

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

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


( A ) Electron micrographs of MEFs . Scale bar ; 1 μm . High - magnification images depicting representative electron - dense materials are shown . Scale bar ; 0 . 2 μm .

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

(A) Electron micrographs of MEFs . Scale bar; 1 μm. High-magnification images depicting representative electron-dense materials are shown. Scale bar; 0.2 μm.


( B ) Citrate synthase activity ( absorbance / min ) was measured in MEFs . Error bars represent ± SD from eight independent experiments . Data information : For graph , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Citrate synthase activity (absorbance/min) was measured in MEFs . Error bars represent ±SD from eight independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( C , D ) Oxygen consumption rates ( OCRs ) of MEFs were measured on an XF24 Analyzer . ( C ) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin ( 2 μg / mL ) ( Oligo ) , FCCP ( 2 μM ) , and rotenone ( 1 μM ) plus actinomycin ( 2 μM ) ( R & A ) . ( D ) OCR ( pmol O2 / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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

(C, D) Oxygen consumption rates (OCRs) of MEFs were measured on an XF24 Analyzer. (C) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin (2 μg/mL) (Oligo), FCCP (2 μM), and rotenone (1 μM) plus actinomycin (2 μM) (R & A). (D) OCR (pmol O2/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( E ) ATP production ( pmol / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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

(E) ATP production (pmol/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( F ) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype . MEFs were treated with tunicamycin ( 5 μg / ml ) or vehicle control in the presence or absence of bafilomycin A1 ( 400 nM ) , and endogenous LC3 and p62 levels were measured by immunoblotting . Data represent the ratios of LC3 - II to LC3 - I and p62 to actin in the absence of bafilomycin , which were normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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

(F) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype. MEFs were treated with tunicamycin (5 μg/ml) or vehicle control in the presence or absence of bafilomycin A1 (400 nM), and endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin in the absence of bafilomycin, which were normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( G ) Survival rate of MEFs treated with tunicamycin ( 5 μg / ml ) , thapsigargin ( 1 μM ) , or hydrogen peroxide ( 100 μM ) . Error bars represent ± SD from four independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

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

(G) Survival rate of MEFs treated with tunicamycin (5 μg/ml), thapsigargin (1 μM), or hydrogen peroxide (100 μM). Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( A ) Absolute [ Ca2 + ] m changes in MEFs of indicated genotype in response to bradykinin ( 2 . 5 μM ) . ( B ) Basal and peak values of Ca2 + transients ( μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (A) Absolute [Ca2+]m changes in MEFs of indicated genotype in response to bradykinin (2.5 μM). (B) Basal and peak values of Ca2+ transients (μM). Error bars represent ±SD from six independent experiments. Data information: For graph P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( C ) Peak values of Ca2 + transients in MEFs transfected with IP3R or shRNA against IP3R , or treated with 2 - AP ( 20 mM ) . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (C) Peak values of Ca2+ transients in MEFs transfected with IP3R or shRNA against IP3R, or treated with 2-AP (20 mM). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( D ) Peak values of Ca2 + transients in MEFs transfected with VDAC1 or shRNA against VDAC1 . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (D) Peak values of Ca2+ transients in MEFs transfected with VDAC1 or shRNA against VDAC1. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( E ) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm ) . Scale bar : 100 nm . Data represent percentage of ERmitochondrial contacts per mitochondria perimeter . Error bars represent ± SD from 40 images MEFs Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(E) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm). Scale bar: 100 nm. Data represent percentage of ERmitochondrial contacts per mitochondria perimeter. Error bars represent ±SD from 40 images MEFs Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( F ) In situ PLA images using anti - IP3R and anti - VDAC1 antibodies . Scale bar : 20 μm . Data represent fluorescent in MEFs normalized LRRK2 + / + MEFs . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(F) In situ PLA images using anti-IP3R and anti-VDAC1 antibodies. Scale bar: 20 μm. Data represent fluorescent in MEFs normalized LRRK2+/+ MEFs. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( G ) Peak values of Ca2 + transients in MEFs transfected with synthetic tethering protein ( TOM - mRFP - ER ) to induce artificial tethering of the ER and mitochondria . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(G) Peak values of Ca2+ transients in MEFs transfected with synthetic tethering protein (TOM-mRFP-ER) to induce artificial tethering of the ER and mitochondria. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Representative immunoblots of MAM components in MEFs of indicated genotype . MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right .

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(A) Representative immunoblots of MAM components in MEFs of indicated genotype. MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right.


( C ) Peak values of Ca2 + transients in MEFs transfected with deletion constructs of LRRK2 ( G2019S ) . Error bars represent ± SD from six independent experiments . Data information : For graph , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

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(C) Peak values of Ca2+ transients in MEFs transfected with deletion constructs of LRRK2(G2019S). Error bars represent ±SD from six independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( D ) Binding assays to detect interactions between deletion constructs of LRRK2 ( G2019S ) and E3 ubiquitin ligases . HEK293 cells were transfected with deletion constructs of LRRK2 ( G2019S ) and E3 ubiquitin ligases , and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody against the V5 or HA epitope , as indicated the right of each panel .

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(D) Binding assays to detect interactions between deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases. HEK293 cells were transfected with deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases, and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the V5 or HA epitope, as indicated the right of each panel.


Peak values of Ca2 + transients in MEFs transfected with ligase - active MARCH5 ( WT ) , MULAN ( WT ) , and Parkin ( W403A ) [ Parkin ( WA ) ] or dominant - negative forms of MARCH5 ( H43W ) [ MARCH ( HW ) ] , MULAN ( C339A ) [ MULAN ( CA ) ] , Parkin ( C431A ) [ Parkin ( CA ) ] ( E ) Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

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Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


Peak values of Ca2 + transients in MEFs transfected with active USP30 or inactive USP30 ( C77S ) [ USP ( CS ) ] ( F ) . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

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Peak values of Ca2+ transients in MEFs transfected with active USP30 or inactive USP30(C77S) [USP(CS)] (F). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( A ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2 + / + and LRRK2 ( G2019S ) - expressing MEFs treated with vehicle ( Control ) or tunicamycin ( 1 μg / ml ) . Endogenous PERK , MARCH5 , MULAN and Parkin were immunoprecipitated with antibody , and precipitates were immunoblotted with antibody indicated at the right . Data represent the ratio of phosphorylated to total protein levels . Error bars represent ± SD from independent experiments . Data information : For graph A the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(A) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments. Data information: For graph A the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) Immunoprecipitation / immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin ( 5 μg / ml ) . LRRK2 ( G2019S ) MEFs were also pretreated with LRRK2 - IN - 1 ( 1 μM ) . MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody against the Myc epitope , phosphoserine or mitofin2 as indicated to the right of each panel . Mfn2 : mitofusin 2 , Ub : ubiquitin . Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin . Error bars represent ± SD from four independent experiments . Data information : For graph B , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(B) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments. Data information: For graph B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Peak values of Ca2 + transients in MEFs treated with LRRK2 - IN - 1 ( 1 μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph A , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(A) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments. Data information: For graph A , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) ( Upper panel ) Diagram showing full - length PERK Myc at the N - terminus and FLAG at the C - terminus ( M - PERK - F ) . S1P recognition sequence R33SLL is mutated to A33SLL ( M - PERK ( R33A ) - F ) . ( Lower panel ) Immunoblot of PERK and PERK ( R33A ) from transfected MEFs under tunicamycin . MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method . Lysates were immunoprecipitated with antibody against FLAG . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody as indicated to the right .

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(B)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F). (Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.


( C ) In vitro kinase assay using isolated PERK , isolated E3 ubiquitin ligase and [ γ - 32P ] ATP . Reaction mixture was subjected to SDS / PAGE . Blots were with antibody against Myc , FLAG or phosphoserine . [ γ - 32P ] ATP - incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ) .

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(C) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).


( D ) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases , HA - tagged Ubl , and His - tagged mitofusin2 in the presence of E1 enzyme , UbcH7 and ATP . E3 ubiquitin ligases were initially phosphorylated PERK or PERK ( K618R ) in vitro . Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination . itofusin 2 with Ni - NTA subjected to SDS / PAGE . Blots were with antibody against HA or mitofusin2 . Mfn2 : mitofusin 2 , Ub : ubiquitin .

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(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.


( E ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2 ( G2019S ) - expressing MEFs transfected with the increasing amounts of LRRK2 - d1 - V5 ( 1 , 5 , 25 μg / 106 cells ) treated with tunicamycin ( 1 μg / ml ) . Endogenous MARCH5 , MULAN and Parkin were immunoprecipitated with antibody , and precipitates were immunoblotted with antibody indicated at the right . Endogenous mitofusin 2 was immunoblotted with anti - mitofusin 2 antibody . Data represent the ratio of phosphorylated to total protein levels of MARCH5 , MULAN and Parkin , and the ratio of mitofusin 2 to actin . Error bars represent ± SD from independent experiments . Data information : For graph E , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

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(E) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments. Data information: For graph E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2 . HEK293 cells were transfected with PERK and either MARCH5 , MULAN or Parkin , LRRK2 , LRRK2 ( D1994A ) or LRRK2 ( S1292D ) . Cell lysates were immunoprecipitated with antibody against Myc or V5 , and precipitates were immunoblotted with antibody indicated the right .

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(A) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2. HEK293 cells were transfected with PERK and either MARCH5, MULAN or Parkin, LRRK2, LRRK2(D1994A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc or V5, and precipitates were immunoblotted with antibody indicated the right.


( B ) In vitro kinase assay for auto - phosphorylated LRRK2 using isolated LRRK2 mutants and [ γ - 32P ] ATP with or without LRRK2 - IN - 1 ( 1 μM ) . Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS / PAGE . Blots were with antibody against V5 or phosphoserine . [ γ - 32P ] ATP - incorporated LRRK2 visualized by autoradiography .

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(B) In vitro kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.


( C ) Binding assays to detect interactions between phosphomimetic LRRK2 ( S1292D ) or phosphorylation - defective LRRK2 ( S1292A ) and E3 ubiquitin ligases . HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope . Precipitated proteins were subjected to SDS / PAGE , for immunoblotwith antibody against V5 or Myc .

(C) Binding assays to detect interactions between phosphomimetic LRRK2(S1292D) or phosphorylation-defective LRRK2(S1292A) and E3 ubiquitin ligases. HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope. Precipitated proteins were subjected to SDS/PAGE, for immunoblotwith antibody against V5 or Myc.


( D ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK - ΔN in the presence of LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . HEK293 cells were transfected with MARCH5 , MULAN , Parkin , and PERK - ΔN LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . Cell lysates were immunoprecipitated with antibody against Myc , and precipitates were immunoblotted with antibody indicated the right .

(D) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK-ΔN in the presence of LRRK2(S1292A) or LRRK2(S1292D). HEK293 cells were transfected with MARCH5, MULAN, Parkin, and PERK-ΔN LRRK2(S1292A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc, and precipitates were immunoblotted with antibody indicated the right.


( A ) Immunoblot of LC3 and p62 . MEFs were transfected with PERK - ΔN or PERK shRNA . Endogenous LC3 and p62 levels were measured by immunoblotting . Data represent the ratios of LC3 - II to LC3 - I and p62 to actin normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph A , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 .

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(A) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05.


( B ) Peak values of Ca2 + transients in MEFs transfected with PERK - ΔN or PERK shRNA . Error bars represent ± SD from six independent experiments .

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(B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.


Left panel , a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan - Cancer dataset . Vertical red line indicates the localization of VPS4B . Right panel , enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples . Deletions are marked in blue , amplified regions are marked in red . Analysis of VPS4B copy number alterations in TCGA Pan - Cancer dataset . Cancer types were sorted according to the mean VPS4B copy number after removing germline values .

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Left panel, a scheme of chromosome 18 copy number alterations depicting the distal long arm loss across TCGA Pan-Cancer dataset. Vertical red line indicates the localization of VPS4B. Right panel, enlarged fragment of chromosome 18 showing frequent deletions of VPSB locus in cancer samples. Deletions are marked in blue, amplified regions are marked in red. Analysis of VPS4B copy number alterations in TCGA Pan-Cancer dataset. Cancer types were sorted according to the mean VPS4B copy number after removing germline values.


Scatter plot , analysis of VPS4B mRNA expression ( number of transcripts per million ) plotted against VPS4B copy number from TCGA CRC patient samples ( n = 376 ) ; plot generated using cBioPortal Gao et al , 2013 ( ) . Pie chart , summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma ( TCGA , Provisional )

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Scatter plot, analysis of VPS4B mRNA expression (number of transcripts per million) plotted against VPS4B copy number from TCGA CRC patient samples (n=376); plot generated using cBioPortal Gao et al, 2013(). Pie chart, summary of all types of VPS4B copy number alterations based on the analysis of data from 615 CRC samples deposited in the Colorectal Adenocarcinoma (TCGA, Provisional)


qPCR analysis of VPS4B mRNA abundance in normal colon , adenoma and CRC samples . Adenocarcinoma ( n = 26 ) ; adenoma ( n = 42 ) ; normal colon ( n = 24 ) . Green horizontal bars indicate means and red whiskers indicate SD .

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qPCR analysis of VPS4B mRNA abundance in normal colon, adenoma and CRC samples. Adenocarcinoma (n=26); adenoma (n=42); normal colon (n=24). Green horizontal bars indicate means and red whiskers indicate SD.


Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in ( F ) . 3 + - very intensive staining , 2 + - medium - intensive staining , 1 + - weak staining , 0 - no staining . Scale bar , 50 µm .

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Examples of immunohistochemical staining of VPS4B in normal colon and matched CRC samples as an illustration of the scoring system used for the evaluation presented in (F). 3+ - very intensive staining, 2+ - medium-intensive staining, 1+ - weak staining, 0 - no staining. Scale bar, 50 µm.


Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 1 or # 2 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± SEM . All values were normalized , averaged ( avrg ) viability of siCTRL # 1 and # 2 - transfected cells was set as 100 % , the Kruskal - Wallis test followed by Dunn ' s multiple comparison post hoc test ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 .

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Analysis of viability of HCT116 cells assessed 96 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #1 or #2), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%, the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test; ns - non-significant (p≥0.05), **p<0.01.


Left panel , analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non - targeting ( siCTRL # 1 ) , VPS4A - or / and VPS4B - targeting siRNAs used in various duplex combinations as indicated . Right panel , images of HCT116 clones taken at the day of clonogenic growth assessment . Data are means of 3 independent experiments ± SEM . NT - non - transfected . All values were normalized , clonogenic growth of siCTRL # 1 - transfected cells was set as 1 . One - sample t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * * * * p < 0 . 0001 .

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Left panel, analysis of clonogenic growth of HCT116 cells assessed 15 days after transfection with non-targeting (siCTRL#1), VPS4A- or/and VPS4B-targeting siRNAs used in various duplex combinations as indicated. Right panel, images of HCT116 clones taken at the day of clonogenic growth assessment. Data are means of 3 independent experiments ±SEM. NT - non-transfected. All values were normalized, clonogenic growth of siCTRL#1-transfected cells was set as 1. One-sample t-test; ns - non-significant (p≥0.05), ****p<0.0001.


Viability of RKO and SW480 cells assessed as in ( A ) , growth of DLD - 1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection . Various independent non - targeting ( siCTRL # 1 or # 2 ) and VPS4A - or B - targeting siRNA duplexes were used ( # 1 , # 2 , # 5 or # 1 , # 2 , respectively ) . All values were normalized , averaged ( avrg ) viability of siCTRL # 1 and # 2 - transfected cells was set as 100 % . Data are means of 3 independent experiments ± SEM . The Kruskal - Wallis test followed by Dunn ' s multiple comparison post hoc ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 , * * * p < 0 . 001 .

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Viability of RKO and SW480 cells assessed as in (A), growth of DLD-1 cells assessed in BrdU incorporation assay 96 h after siRNA transfection. Various independent non-targeting (siCTRL#1 or #2) and VPS4A- or B-targeting siRNA duplexes were used (#1, #2, #5 or #1, #2, respectively). All values were normalized, averaged (avrg) viability of siCTRL#1 and #2-transfected cells was set as 100%. Data are means of 3 independent experiments ±SEM. The Kruskal-Wallis test followed by Dunn's multiple comparison post hoc; ns - non-significant (p≥0.05), **p<0.01, ***p<0.001.


VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR / Cas9 and RNAi screens deposited in the DepMap portal ( https : / / depmap . org / portal / ) .

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VPS4B copy number and dependency scores of selected cancer cell lines obtained in CRISPR/Cas9 and RNAi screens deposited in the DepMap portal (https://depmap.org/portal/).


VPS4B copy number status estimated across different cell lines using TaqMan assay . The whiskers represent the minimal and maximal copy number in a given triplicate readout . The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control .

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VPS4B copy number status estimated across different cell lines using TaqMan assay. The whiskers represent the minimal and maximal copy number in a given triplicate readout. The BCL2 gene localized on 18q in the close vicinity to VPS4B was analyzed as a control.


VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting . Vinculin and GAPDH were used as loading controls .

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VPS4B and VPS4A protein abundance in selected normal and cancer cell lines analyzed by immunoblotting. Vinculin and GAPDH were used as loading controls.


Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non - targeting siRNA ( two different duplexes used , siCTRL # 1 or # 2 ) or targeting VPS4A ( duplexes # 2 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 4 independent experiments ± SEM . All values were normalized , cell viability of averaged ( avrg ) siCTRL # 1 - and # 2 - transfected cells was set as 100 % . Two - tailed unpaired t - test ; * * * * p < 0 . 0001 .

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Analysis of viability of HOP62 lung cancer cells assessed 144 h after transfection with independent non-targeting siRNA (two different duplexes used, siCTRL#1 or #2) or targeting VPS4A (duplexes #2 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 4 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1- and #2-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Top panel , analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated . Bottom panel , images of HOP62 clones taken at the day of clonogenic growth assessment . Data are means of 4 independent experiments ± SEM . All values were normalized , colony area of averaged ( avrg ) siCTRL # 1 and # 2 - transfected cells was set as 1 . The Mann - Whitney U test ; * * p < 0 . 01 .

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Top panel, analysis of clonogenic growth of HOP62 cells assessed 14 days after transfection with various siRNA duplexes as indicated. Bottom panel, images of HOP62 clones taken at the day of clonogenic growth assessment. Data are means of 4 independent experiments ±SEM. All values were normalized, colony area of averaged (avrg) siCTRL#1 and #2-transfected cells was set as 1. The Mann-Whitney U test; **p<0.01.


Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non - targeting siRNA ( three different duplexes used , siCTRL # 1 , # 2 or # 3 ) or targeting VPS4A ( duplexes # 2 , # 4 or # 5 ) , VPS4B ( duplexes # 1 or # 2 ) or both VPS4 ( various combinations of siVPS4A + siVPS4B duplexes ) . Data are means of 3 independent experiments ± SEM . All values were normalized , cell viability of averaged ( avrg ) siCTRL # 1 - , # 2 - and # 3 - transfected cells was set as 100 % . Two - tailed unpaired t - test ; * * * * p < 0 . 0001 .

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Analysis of viability of SNU410 pancreatic cancer cells assessed 168 h after transfection with independent non-targeting siRNA (three different duplexes used, siCTRL#1, #2 or #3) or targeting VPS4A (duplexes #2, #4 or #5), VPS4B (duplexes #1 or #2) or both VPS4 (various combinations of siVPS4A+siVPS4B duplexes). Data are means of 3 independent experiments ±SEM. All values were normalized, cell viability of averaged (avrg) siCTRL#1-, #2- and #3-transfected cells was set as 100%. Two-tailed unpaired t-test; ****p<0.0001.


Left panel , growth of HCT116 VPS4B - / - shVPS4A cells as xenografts in mice in the presence or absence of doxycycline . Day 1 indicates the first day of doxycycline administration . n = 9 for each group , each mouse bearing one tumor , ± SEM . Two - tailed unpaired t - test ; ns - non - significant ( p ≥ 0 . 05 ) , * * p < 0 . 01 . Right panel , scatter plot representing end - point volumes of single xenografts . Bars represent means ± SEM .

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Left panel, growth of HCT116 VPS4B-/- shVPS4A cells as xenografts in mice in the presence or absence of doxycycline. Day 1 indicates the first day of doxycycline administration. n=9 for each group, each mouse bearing one tumor, ±SEM. Two-tailed unpaired t-test; ns - non-significant (p≥0.05), **p<0.01. Right panel, scatter plot representing end-point volumes of single xenografts. Bars represent means ±SEM.


Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline - treated mice ( 6 separate xenograft samples for each group ) . Lysates of HCT116 VPS4B - / - cells transfected with control or VPS4A - targeting siRNA marked VPS4A protein detection . GAPDH served as a loading control .

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Immunoblotting analysis of VPS4A abundance in xenograft samples from untreated and doxycycline-treated mice (6 separate xenograft samples for each group). Lysates of HCT116 VPS4B-/- cells transfected with control or VPS4A-targeting siRNA marked VPS4A protein detection. GAPDH served as a loading control.


Growth of HCT116 VPS4B - / - shCTRL # 1 cells as xenografts in mice in the presence or absence of doxycycline . The arrow indicates the first day of doxycycline ( Dox ) administration . n = 2 mice for each group , each mouse bearing two tumors , ± SEM .

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Growth of HCT116 VPS4B-/- shCTRL#1 cells as xenografts in mice in the presence or absence of doxycycline. The arrow indicates the first day of doxycycline (Dox) administration. n=2 mice for each group, each mouse bearing two tumors, ±SEM.


Heatmap visualizing expression of genes related to inflammatory response ( left panel ) and positive regulation of cell death ( right panel ) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates .

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Heatmap visualizing expression of genes related to inflammatory response (left panel) and positive regulation of cell death (right panel) generated from the GO analysis of biological processes across different transfection conditions with at least 3 biological replicates.


Immunoblotting analysis of the canonical and noncanonical branches of the NF - κB pathway and mediators of caspase - dependent cell death . Lysates of HCT116 VPS4B - / - cells were collected 66 h after transfection with siRNA ( siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Lysates of HCT116 VPS4B + / + and non - transfected HCT116 VPS4B - / - cells were used to monitor the basal pathway activity . Representative blot from 10 experiments is shown . NT - non - transfected ; p - RelA - phospho - RelA ; p - IκBα - phospho - IκBα ; cl - cleaved caspases or PARP - 1 . GAPDH or vinculin served as loading controls .

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Immunoblotting analysis of the canonical and noncanonical branches of the NF-κB pathway and mediators of caspase-dependent cell death. Lysates of HCT116 VPS4B-/- cells were collected 66 h after transfection with siRNA (siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Lysates of HCT116 VPS4B+/+ and non-transfected HCT116 VPS4B-/- cells were used to monitor the basal pathway activity. Representative blot from 10 experiments is shown. NT- non-transfected; p-RelA - phospho-RelA; p-IκBα - phospho-IκBα; cl - cleaved caspases or PARP-1. GAPDH or vinculin served as loading controls.


Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in ( A ) . Data are means of 10 ( phospho - Rel and cleaved caspase 7 ) , 9 ( p52 , caspase 8 and cleaved caspase 9 ) , 7 ( phosphorylated and total IκB ) or 5 ( cleaved caspase 3 ) independent experiments . Error bars are SEM . Statistical significance was assessed using the following tests : one - sample t - test ( caspase 8 , cleaved caspases 9 , 7 and 3 , total and phospho - IκBα ) or Wilcoxon signed rank test ( phospho - RelA and p52 ) ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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Densitometry analysis of the abundance of the indicated proteins based on immunoblot images as shown in (A). Data are means of 10 (phospho-Rel and cleaved caspase 7), 9 (p52, caspase 8 and cleaved caspase 9), 7 (phosphorylated and total IκB) or 5 (cleaved caspase 3) independent experiments. Error bars are SEM. Statistical significance was assessed using the following tests: one-sample t-test (caspase 8, cleaved caspases 9, 7 and 3, total and phospho-IκBα) or Wilcoxon signed rank test (phospho-RelA and p52); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Immunoblot showing induction of the NF - κB pathway ( p52 ) and apoptosis ( cleaved caspase 7 ) in xenograft samples described in Fig 3C . cl - cleaved caspase . Vinculin was used as a loading control .

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Immunoblot showing induction of the NF-κB pathway (p52) and apoptosis (cleaved caspase 7) in xenograft samples described in Fig 3C. cl - cleaved caspase. Vinculin was used as a loading control.


Analysis of the impact of RIPK1 inhibitor ( necrostatin - 1 ) or pan - caspase inhibitor ( Q - VD - Oph ) on cell viability of HCT116 VPS4B - / - cells transfected with siRNA ( non - targeting siCTRL # 1 or different siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell viability was assessed 72 h after siRNA transfection . Necrostatin - 1 ( 50 µM ) , Q - VP - Oph ( 20 µM ) or vehicle were added to the medium 48 h before viability assessment . Data are means of 5 independent experiments ± SEM . All values were normalized , viability of siCTRL - transfected and vehicle - treated cells was set as 100 % . Two - tailed unpaired t - test ; * * p < 0 . 01 .

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Analysis of the impact of RIPK1 inhibitor (necrostatin-1) or pan-caspase inhibitor (Q-VD-Oph) on cell viability of HCT116 VPS4B-/- cells transfected with siRNA (non-targeting siCTRL#1 or different siVPS4A duplexes: #2, #4 or #5). Cell viability was assessed 72 h after siRNA transfection. Necrostatin-1 (50 µM), Q-VP-Oph (20 µM) or vehicle were added to the medium 48 h before viability assessment. Data are means of 5 independent experiments ±SEM. All values were normalized, viability of siCTRL-transfected and vehicle-treated cells was set as 100%. Two-tailed unpaired t-test; **p<0.01.


Measurement of ATP released to the cell medium by HCT116 VPS4B - / - cells non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell culture media were exchanged 16 h after transfection , and fresh media were conditioned for the next 52 - 58 h . For non - transfected cells ( NT ) , the same treatment protocol was used but without the transfection mixture .

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Measurement of ATP released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.


Measurement of HMGB1 released to the cell medium by HCT116 VPS4B - / - cells non - transfected ( NT ) or transfected with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A duplexes : # 2 , # 4 or # 5 ) . Cell culture media were exchanged 16 h after transfection , and fresh media were conditioned for the next 52 - 58 h . For non - transfected cells ( NT ) , the same treatment protocol was used but without the transfection mixture .

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Measurement of HMGB1 released to the cell medium by HCT116 VPS4B-/- cells non-transfected (NT) or transfected with siRNA (non-targeting siCTRL#1 or targeting siVPS4A duplexes: #2, #4 or #5). Cell culture media were exchanged 16 h after transfection, and fresh media were conditioned for the next 52-58 h. For non-transfected cells (NT), the same treatment protocol was used but without the transfection mixture.


Microscopy images presenting cell surface calreticulin ( green ) in HCT116 VPS4B - / - cells 48 h after transfection with siRNA ( non - targeting siCTRL # 1 or targeting siVPS4A # 2 ) . As a positive control for detection of cell surface calreticulin , non - transfected cells ( NT ) were treated with 2 µM mitoxantrone for 24 h . In blue , DAPI staining . Scale bar , 15 µm .

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Microscopy images presenting cell surface calreticulin (green) in HCT116 VPS4B-/- cells 48 h after transfection with siRNA (non-targeting siCTRL#1 or targeting siVPS4A#2). As a positive control for detection of cell surface calreticulin, non-transfected cells (NT) were treated with 2 µM mitoxantrone for 24 h. In blue, DAPI staining. Scale bar, 15 µm.