Figure 8Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; 100µM cycloheximide Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . ( B ) 200ng / mL Sema3A was bath applied with or without 100µM cycloheximide ( CHX , a translational blocker ) . Mutating TUBB3 3 ' UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons . Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . , isolated axons ( C ) were used for FRAP analysis . Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A ; 2µM co - MO or MOs - 3p . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . isolated transfected axons ( D ) were used for FRAP analysis . Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . In vivo ( E ) , whole embryos with exposed brain were used . The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution . ( E ) The red area on the brain schematic indicate Sema3A expressing territories . Numbers of axons analyzed are reported between brackets . Quantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP analysis . APP , Amyloid beta precursor proteinQuantification ( in percentage ) of the axonal fluorescence recovery after photobleaching ( FRAP ) of different Venus - 3 ' UTR constructs ex vivo and in vivo . Concentrations used : 200ng / mL Sema3A Growth cones expressing Venus alone ( no 3 ' UTR ) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent , non - bleached regions to the bleached growth cone ( Wong et al , 2017 ) . Ex vivo , whole explants were used for FRAP analysis . THBS1 , Thrombospondin 1H , I . Representative growth cones depicting Venus fluorescence intensity as a heatmap . Dashed white lines delineate the growth cones . Abbreviations : wt , wild type ; mut , miR - 181 - 5p responsive elements mutated ; CHX , cycloheximide ; TUBB3 , tubulin beta 3 class III . Scale bars : 10µm .

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

Figure 8Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 100µM cycloheximide Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). (B) 200ng/mL Sema3A was bath applied with or without 100µM cycloheximide (CHX, a translational blocker). Mutating TUBB3 3'UTR did not affect fluorescence recovery in basal conditions compared to WT indicating that mature miRNAs do not regulate the constitutive TUBB3 expression in distal axons.Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). , isolated axons (C) were used for FRAP analysis.Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A; 2µM co-MO or MOs-3p. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). isolated transfected axons (D) were used for FRAP analysis.Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). In vivo (E), whole embryos with exposed brain were used. The electroporated eye was removed prior to mounting the embryo to eliminate somatic contribution. (E) The red area on the brain schematic indicate Sema3A expressing territories. Numbers of axons analyzed are reported between brackets.Quantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. APP, Amyloid beta precursor proteinQuantification (in percentage) of the axonal fluorescence recovery after photobleaching (FRAP) of different Venus-3'UTR constructs ex vivo and in vivo. Concentrations used: 200ng/mL Sema3A Growth cones expressing Venus alone (no 3'UTR) displayed a minimal amount of recovery after photobleaching corresponding to Venus diffusion from adjacent, non-bleached regions to the bleached growth cone (Wong et al, 2017). Ex vivo, whole explants were used for FRAP analysis. THBS1, Thrombospondin 1H,I. Representative growth cones depicting Venus fluorescence intensity as a heatmap. Dashed white lines delineate the growth cones. Abbreviations: wt, wild type; mut, miR-181-5p responsive elements mutated; CHX, cycloheximide; TUBB3, tubulin beta 3 class III. Scale bars: 10µm.


Figure 9B . Schematic representation of the experimental paradigm . Representative images of local translation events of TUBB3 in the growth cone . Dashed white line delineates the growth cone . Each white arrow points to a puro - PLA puncta . Abbreviations : TUBB3 , tubulin beta 3 class III ; Puro , puromycin . Scale bars : 5 μm . Quantification of number of puncta per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 30 ( PBS ) , 36 ( Sema3A ) . n = 2 independent experiments . Values are median with interquartile range . D . Schematic representation of the experimental paradigm . Concentrations used : 2 μM co - MO ; 2 μM MOs - 3p ; 200ng / mL Sema3A . Representative images of local translation events of TUBB3 in the growth cone of transfected axons . Dashed white line delineates the growth cone . White arrows represent number of puro - PLA puncta per growth cone . Abbreviations : co - MO , control morpholino . Scale bars : 5 μm . E . Quantification of number of puncta per growth cone . Each data point corresponds to one growth cone . Total number of growth cones analyzed : 32 ( co - MO + PBS ) , 45 ( co - MO + Sema3A ) , 27 ( MOs - 3p + Sema3A ) . n = 2 independent experiments . Values are median with interquartile range . Abbreviations : co - MO , control morpholino ; ns , not significant . G . Percentage of collapsed growth cones in axons transfected with co - MO , MOs - 3p + co - MO and MOs - 3p + MOs - TUBB3 . Each data point corresponds to one independent experiment . Total number of growth cones counted : 152 ( co - MO ) , 155 ( MOs - 3p + coMO ) , 179 ( MOs - 3p + MOs - TUBB3 ) . n = 3 independent experiments . Values are mean ± SEM . Abbreviations : ns , not significant ; co - MO , control morpholino ; TUBB3 , tubulin beta 3 class III .

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

Figure 9B. Schematic representation of the experimental paradigm. Representative images of local translation events of TUBB3 in the growth cone. Dashed white line delineates the growth cone. Each white arrow points to a puro-PLA puncta. Abbreviations: TUBB3, tubulin beta 3 class III; Puro, puromycin. Scale bars: 5 μm. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 30 (PBS), 36 (Sema3A). n= 2 independent experiments. Values are median with interquartile range.D. Schematic representation of the experimental paradigm. Concentrations used: 2 μM co-MO; 2 μM MOs-3p; 200ng/mL Sema3A. Representative images of local translation events of TUBB3 in the growth cone of transfected axons. Dashed white line delineates the growth cone. White arrows represent number of puro-PLA puncta per growth cone. Abbreviations: co-MO, control morpholino. Scale bars: 5 μm. E. Quantification of number of puncta per growth cone. Each data point corresponds to one growth cone. Total number of growth cones analyzed: 32 (co-MO + PBS), 45 (co-MO + Sema3A), 27 (MOs-3p + Sema3A). n=2 independent experiments. Values are median with interquartile range. Abbreviations: co-MO, control morpholino; ns, not significant.G. Percentage of collapsed growth cones in axons transfected with co-MO, MOs-3p + co-MO and MOs-3p + MOs-TUBB3. Each data point corresponds to one independent experiment. Total number of growth cones counted: 152 (co-MO), 155 (MOs-3p + coMO), 179 (MOs-3p + MOs-TUBB3). n= 3 independent experiments. Values are mean ± SEM. Abbreviations: ns, not significant; co-MO, control morpholino; TUBB3, tubulin beta 3 class III.


Figure 1Left graph reports the tumor volume in NSG mice injected with control ( shCTR ) and antimiR - 9 FaDu cells followed for up to 35 days ( n = 5 mice / group ) . Data represent the mean ( ± SD ) and unpaired t - test was used to verify the statistical significance at each time point . On the right , typical images of explanted tumors formed by control ( shCTR ) and antimiR - 9 FaDu cells at necropsy . Typical images of Hematoxylin & Eosin ( H & E - upper images ) and Ki67 expression ( Ki67 - bottom images ) evaluated by immunohistochemistry ( IHC ) in tumors explanted from mice treated as in H . 4X and 20X magnification are shown . Right graph reports the percentage of Ki67 positive cells . Each dot represents one tumor . Data are expressed as mean ( ± SD ) and unpaired t - test was used to verify the statistical significance .

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Figure 1Left graph reports the tumor volume in NSG mice injected with control (shCTR) and antimiR-9 FaDu cells followed for up to 35 days (n=5 mice/group). Data represent the mean (±SD) and unpaired t-test was used to verify the statistical significance at each time point. On the right, typical images of explanted tumors formed by control (shCTR) and antimiR-9 FaDu cells at necropsy.Typical images of Hematoxylin&Eosin (H&E - upper images) and Ki67 expression (Ki67 - bottom images) evaluated by immunohistochemistry (IHC) in tumors explanted from mice treated as in H. 4X and 20X magnification are shown. Right graph reports the percentage of Ki67 positive cells. Each dot represents one tumor. Data are expressed as mean (±SD) and unpaired t-test was used to verify the statistical significance.


Figure 2qRT - PCR analyses of normalized miR - 9 expression , expressed as fold over the untreated condition , in control ( shCTR ) and antimiR - 9 FaDu cells treated with Cetuximab ( CTX ) for up to four hours . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and two - way ANOVA test was used to verify the statistical significance . Graph reporting the cell viability of control ( shCTR ) and antimiR - 9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay . Data represent the mean ( ± SD ) of two independent experiments performed in sextuplicate , and unpaired t - test was used to verify the statistical significance per each dose .

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Figure 2qRT-PCR analyses of normalized miR-9 expression, expressed as fold over the untreated condition, in control (shCTR) and antimiR-9 FaDu cells treated with Cetuximab (CTX) for up to four hours. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA test was used to verify the statistical significance.Graph reporting the cell viability of control (shCTR) and antimiR-9 FaDu cells treated with increasing concentration of Gefitinib as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and unpaired t-test was used to verify the statistical significance per each dose.


Figure 3qRT - PCR analyses of Sp1 expression in control ( shCTR ) and antimiR - 9 FaDu cells transiently transduced with PGK - miR - 9 or control vector as indicated . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and two - way ANOVA with Sidak ' s multiple comparison test was used to verify the statistical significance . qRT - PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR - 9 or control vector . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and unpaired t - test was used to verify the statistical significance . Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib ( GEFI - left panel ) or Cetuximab ( CTX - right panel ) as indicated and evaluated using the MTS assay . Data represent the mean ( ± SD ) of two independent experiments performed in sextuplicate , and two - way ANOVA with Sidak ' s multiple comparison test was used to verify the statistical significance . Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control ( shCTR ) or antimiR - 9 FaDu cells . Data represent the mean ( ± SD ) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance .

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

Figure 3qRT-PCR analyses of Sp1 expression in control (shCTR) and antimiR-9 FaDu cells transiently transduced with PGK-miR-9 or control vector as indicated. Data represent the mean (±SD) of three independent experiments performed in duplicate and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance. qRT-PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR-9 or control vector. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t-test was used to verify the statistical significance.Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of Gefitinib (GEFI - left panel) or Cetuximab (CTX - right panel) as indicated and evaluated using the MTS assay. Data represent the mean (±SD) of two independent experiments performed in sextuplicate, and two-way ANOVA with Sidak's multiple comparison test was used to verify the statistical significance.Graph reporting the normalized Luciferase activity of Sp1 promoter fragments in control (shCTR) or antimiR-9 FaDu cells. Data represent the mean (±SD) of three independent experiments performed in duplicate and unpaired t test was used to verify the statistical significance.


Figure 4On the left , WB analyses of KLF5 and Sp1 expression in control ( shCTR ) and antimiR - 9 FaDu . Histone H3 expression was used as loading control . On the right , protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control . Data represent the mean ( ± SD ) of three biological replicates and unpaired t test was used to verify the statistical significance . Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells . Actin expression was used as loading control . Lower graph shows the SP1 protein quantification normalized over actin expression . Data represents the mean ( ± SD ) of three independent experiments and unpaired t test was used to verify the statistical significance .

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Figure 4On the left, WB analyses of KLF5 and Sp1 expression in control (shCTR) and antimiR-9 FaDu. Histone H3 expression was used as loading control. On the right, protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control. Data represent the mean (±SD) of three biological replicates and unpaired t test was used to verify the statistical significance.Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells. Actin expression was used as loading control. Lower graph shows the SP1 protein quantification normalized over actin expression. Data represents the mean (±SD) of three independent experiments and unpaired t test was used to verify the statistical significance.


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

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

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


Figure 6Graph 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Graph 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.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.


Figure 1 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.


Figure 2 ( 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 . ( 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 .

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

Figure 2(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.(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.


Figure 3 ( 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 . ( 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 .

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

Figure 3(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.(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.


Figure 4 ( 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 .

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

Figure 4(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.


Figure 1Speed 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 ) .

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

Figure 1Speed 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).


Figure 2PMN 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 . 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 ) . 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 ) .

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

Figure 2PMN 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.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).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).


Figure 3Western 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western 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).


Figure 4PMN 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4PMN 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).


Figure 5Western 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western 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).


Figure 6Western 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Western 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.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).


Figure 7The 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7The 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.


Figure 1 ( 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 . ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(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.(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.(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.


Figure 2MEFs 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 . 01MEFs 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 . 01MEFs 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 . 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 . 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 .

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

Figure 2MEFs 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 < 0.01MEFs 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 < 0.01MEFs 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 < 0.01(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 < 0.01(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 < 0.01


Figure 3 ( 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 . 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 . 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 . 05Peak 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 .

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

Figure 3(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 < 0.05(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.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 < 0.05Peak 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


Figure 4 ( 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 .

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

Figure 4(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 < 0.01


Figure 5 ( 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 . ( 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 . ( 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 .

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

Figure 5(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.(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.(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


Figure 6 ( 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 . ( 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 .

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Figure 6(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.(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.


Figure 7 ( 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 .

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

Figure 7(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.