( e ) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt / GSK - 3β / mTOR / P70S6K signalling and LC3 expression in OGD / R . Full - size blots are shown in Supplementary Fig . 14 .

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(e) Western blots showing the effect of siRNA for CLIC4 on phosphorylation of Akt/GSK-3β/mTOR/P70S6K signalling and LC3 expression in OGD/R. Full-size blots are shown in Supplementary Fig. 14.


( f ) Representative western blots and bar graph showing the expression of LC3 - I and LC3 - II on steady - state autophagy and autophagic flux under OGD / R when CLIC4 was knocked down ( data are presented as mean ± s . e . m . ; n = 6 ; * P0 . 05 , * * P0 . 01 ; , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) . Full - size blots are shown in Supplementary Fig . 14 .

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(f) Representative western blots and bar graph showing the expression of LC3-I and LC3-II on steady-state autophagy and autophagic flux under OGD/R when CLIC4 was knocked down (data are presented as mean±s.e.m.; n=6; *P0.05, **P0.01; , with ANOVAs followed by the Bonferroni/Dunn post hoc test). Full-size blots are shown in Supplementary Fig. 14.


( g ) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD / R ( data are presented as mean ± s . e . m . ; n = 8 per condition ; * * * P0 . 001 , with ANOVAs followed by the Bonferroni / Dunn post hoc test ) .

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(g) Cell viability was assessed by MTT assay after siRNA for CLIC4 under OGD/R (data are presented as mean±s.e.m.; n=8 per condition; ***P0.001, with ANOVAs followed by the Bonferroni/Dunn post hoc test).


( C ) Graph showing log10 CFU / gland at 4 days post - inoculation with the indicated strains ( n ≥ 5 ) . Each circle represents the values obtained in individual animals , whereas mean ± s . d . is represented with lines inside each group . No statistical differences were found between infection groups using a one - sided ANOVA followed by the post - hoc Fisher ' s PLSD test .

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(C) Graph showing log10 CFU/gland at 4 days post-inoculation with the indicated strains (n≥5). Each circle represents the values obtained in individual animals, whereas mean ± s.d. is represented with lines inside each group. No statistical differences were found between infection groups using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test.


Plots showing the RT - qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands . Results are expressed as the mean ± s . d ( n = 10 for PBS , n = 5 for WT and n = 4 for CV2 ) of the 2 - ΔΔCt relative expression values of the indicated interleukins ; the value from each individual animal was calculated from three technical replicates . The values obtained in the PBS group were used as control for normalization of gene expression ( = 1 ) . Statistical analysis was performed using a one - sided ANOVA followed by the post - hoc Fisher ' s PLSD test : ( * P ≤ 0 . 05 ; * * P ≤ 0 . 01 ; * * * P ≤ 0 . 005 ) .

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Plots showing the RT-qPCR analysis conducted to quantify relative expression of the indicated interleukins coding genes in the mammary glands. Results are expressed as the mean ± s.d (n=10 for PBS, n=5 for WT and n=4 for CV2) of the 2-ΔΔCt relative expression values of the indicated interleukins; the value from each individual animal was calculated from three technical replicates. The values obtained in the PBS group were used as control for normalization of gene expression (=1). Statistical analysis was performed using a one-sided ANOVA followed by the post-hoc Fisher's PLSD test: (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005).


( B ) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals . Culture supernatants ( n = 1 ) were collected at 0 h , 5 h or 24 h post - inoculation as indicated . WT strain and medium ( Hayflick ) were added as controls .

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(B) Comparison of the turbidimetric assay results obtained with mpn142 and mpn142Opt secretion signals. Culture supernatants (n=1) were collected at 0 h, 5 h or 24 h post-inoculation as indicated. WT strain and medium (Hayflick) were added as controls.


( A , B ) Mature S . aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions , or culture supernatants of the CV2 or the CV2 - DispB strains ( A ) or the WT or the WT - DispB strain ( B ) . Biofilm presence was assessed by crystal violet staining and included a negative staining control ( i . e . , crystal violet without biofilm ) . The results are expressed as mean ± s . d . of OD 595 nm absorbance values obtained from three different biological replicates ( n = 3 ) and statistically compared by one - sided ANOVA followed by the post - hoc Fisher ' s PLSD test : * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 ; * * * * P ≤ 0 .

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(A, B) Mature S. aureus biofilms were allowed to develop for 24 h in polystyrene plates and then treated for the indicated time intervals with cell suspensions, or culture supernatants of the CV2 or the CV2-DispB strains (A) or the WT or the WT-DispB strain (B). Biofilm presence was assessed by crystal violet staining and included a negative staining control (i.e., crystal violet without biofilm). The results are expressed as mean ± s.d. of OD 595 nm absorbance values obtained from three different biological replicates (n=3) and statistically compared by one-sided ANOVA followed by the post-hoc Fisher's PLSD test: *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005; ****P ≤ 0.00005


( C , D ) Plots showing results obtained from the in vitro or ex vivo dispersion assays with the indicated strains and from the staining control . Each circle represents the OD 595 nm values obtained for individual catheters ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Results from one - sided ANOVA followed by Fisher ' s PLSD test are shown for treatments statistically different from those based on strains not secreting dispersin B ( i . e . , the WT or CV2 strains ) . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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(C, D) Plots showing results obtained from the in vitro or ex vivo dispersion assays with the indicated strains and from the staining control. Each circle represents the OD 595 nm values obtained for individual catheters (n≥4), whereas mean ± s.d. is represented with lines inside each group. Results from one-sided ANOVA followed by Fisher's PLSD test are shown for treatments statistically different from those based on strains not secreting dispersin B (i.e., the WT or CV2 strains). *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


( C , D ) Plots showing the SUV60 variation ( % ) between day 1 and day 4 of the different treatments . Each circle represents the SUV60 variation obtained for individual animals ( n ≥ 4 ) , whereas mean ± s . d . is represented with lines inside each group . Data below the dotted lines indicate that the SUV 60 values decreased at D4 post - treatment . Results from one - sided ANOVA followed by Fisher ' s PLSD test are shown . * P ≤ 0 . 05 ; * * P ≤ 0 . 005 ; * * * P ≤ 0 . 0005 .

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(C, D) Plots showing the SUV60 variation (%) between day 1 and day 4 of the different treatments. Each circle represents the SUV60 variation obtained for individual animals (n≥4), whereas mean ± s.d. is represented with lines inside each group. Data below the dotted lines indicate that the SUV 60 values decreased at D4 post-treatment. Results from one-sided ANOVA followed by Fisher's PLSD test are shown. *P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0005.


( A ) Yeast clones harbouring empty bait vector ( pYTH9 , mock ) or those encoding ScAtg8 , LC3B , Atg12 and SUMO1 were transformed with empty prey vector ( pACT2 , mock ) or those encoding fragments of ATG4A , ATG4B , NBR1 , p62 and full - length Nix . Interaction was assessed by yeast growth on the SD ‐ W / ‐ L / ‐ H medium with a β ‐ gal assay .

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

(A) Yeast clones harbouring empty bait vector (pYTH9, mock) or those encoding ScAtg8, LC3B, Atg12 and SUMO1 were transformed with empty prey vector (pACT2, mock) or those encoding fragments of ATG4A, ATG4B, NBR1, p62 and full-length Nix. Interaction was assessed by yeast growth on the SD‐W/‐L/‐H medium with a β‐gal assay.


( B ) GST pulldown assays using cell extracts of Flag ‐ Nix ‐ expressing COS7 cells and immobilized GST or the indicated GST fusions . Coprecipitated Nix was detected with Flag antibodies .

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(B) GST pulldown assays using cell extracts of Flag‐Nix‐expressing COS7 cells and immobilized GST or the indicated GST fusions. Coprecipitated Nix was detected with Flag antibodies.


( C ) Purified GST ‐ tagged NixΔTM was cleaved from the GST moiety by thrombin , purified and used for precipitation by GST fusion proteins . Precipitated proteins were analysed by western blotting ( WB ) with Nix antibodies . Ponceau S staining was used to visualize GST ‐ fusion proteins .

(C) Purified GST‐tagged NixΔTM was cleaved from the GST moiety by thrombin, purified and used for precipitation by GST fusion proteins. Precipitated proteins were analysed by western blotting (WB) with Nix antibodies. Ponceau S staining was used to visualize GST‐fusion proteins.


( D ) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated ( IP ) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies . β ‐ gal , β ‐ galactosidase ; BafA1 , bafilomycin ‐ A1 ; CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; GST , glutathione ‐ S ‐ transferase ; IgG , immunoglobulin G ; NixΔTM , recombinant Nix lacking a TM ; SUMO1 , small ubiquitin ‐ like modifier 1 ; TCL , total cell lysate ; TM , transmembrane domain .

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

(D) HeLa cells were treated as indicated and endogenous proteins were immunoprecipitated (IP) from cell extracts using Nix antibody and analysed by using Nix and LC3 antibodies. β‐gal, β‐galactosidase; BafA1, bafilomycin‐A1; CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; GST, glutathione‐S‐transferase; IgG, immunoglobulin G; NixΔTM, recombinant Nix lacking a TM; SUMO1, small ubiquitin‐like modifier 1; TCL, total cell lysate; TM, transmembrane domain.


( C ) Lysates of COS7 cells transfected with Flag ‐ wt ‐ Nix or the indicated mutants were incubated with immobilized GST ‐ LC3A . The coprecipitated proteins were detected by western blotting ( WB ) with Flag antibodies . BNIP3 , Bcl2 / E1B 19kDa ‐ interacting protein 3 ‐ like protein ; Dr , Danio reio ; GST , glutathione ‐ S ‐ transferase ; Hs , Homo sapiens ; LIR , LC3 ‐ interacting region ; Mm , Mus musculus ; wt , wild type ; Xl , Xenopus laevis .

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(C) Lysates of COS7 cells transfected with Flag‐wt‐Nix or the indicated mutants were incubated with immobilized GST‐LC3A. The coprecipitated proteins were detected by western blotting (WB) with Flag antibodies. BNIP3, Bcl2/E1B 19kDa‐interacting protein 3‐like protein; Dr, Danio reio; GST, glutathione‐S‐transferase; Hs, Homo sapiens; LIR, LC3‐interacting region; Mm, Mus musculus; wt, wild type; Xl, Xenopus laevis.


Nix recruits EGFP ‐ GABARAP ‐ L1 to stressed mitochondria in reconstituted Nix − / − MEFs . Nix − / − MEFs cotransfected with EGFP ‐ GABARAP ‐ L1 and wt ‐ Nix ( A ) , or Nix ‐ W35A ( B ) , were incubated with and without mitochondrial poison CCCP for 3 h . Cells were then fixed and analysed for colocalization between EGFP ‐ GABARAP ‐ L1 and Nix ( stained with Flag antibodies ) . The final panel in each row shows colocalization between Flag ‐ Nix ‐ Cy3 and GABARAP ‐ L1 using the ' colocalization ' highlighter plug ‐ in for ImageJ software ( NIH , Bethesda , MA , USA ) .

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Nix recruits EGFP‐GABARAP‐L1 to stressed mitochondria in reconstituted Nix−/− MEFs. Nix−/− MEFs cotransfected with EGFP‐GABARAP‐L1 and wt‐Nix (A), or Nix‐W35A (B), were incubated with and without mitochondrial poison CCCP for 3 h. Cells were then fixed and analysed for colocalization between EGFP‐GABARAP‐L1 and Nix (stained with Flag antibodies). The final panel in each row shows colocalization between Flag‐Nix‐Cy3 and GABARAP‐L1 using the 'colocalization' highlighter plug‐in for ImageJ software (NIH, Bethesda, MA, USA).


( C ) Quantification was achieved by counting the number of complete colocalizations , EGFP / Cy3 per cell for approximately 30 cells per condition . Results are expressed as a percentage of colocalization ( EGFP : Cy3 ) in control and CCCP ‐ treated cells . Error bars in ( C ) show the standard deviation ( s . d . ) of three independent experiments . Statistical analysis was performed using the Mann - Whitney t ‐ test . CCCP , carbonyl cyanide m ‐ chlorophenyl hidrazone ; DMSO , dimethyl sulfoxide ; EGFP , enhanced green fluorescent protein ; MEF , mouse embryonic fibroblast ; wt , wild type .

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

(C) Quantification was achieved by counting the number of complete colocalizations, EGFP/Cy3 per cell for approximately 30 cells per condition. Results are expressed as a percentage of colocalization (EGFP:Cy3) in control and CCCP‐treated cells. Error bars in (C) show the standard deviation (s.d.) of three independent experiments. Statistical analysis was performed using the Mann-Whitney t‐test. CCCP, carbonyl cyanide m‐chlorophenyl hidrazone; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescent protein; MEF, mouse embryonic fibroblast; wt, wild type.


( A ) Flow cytometry of reticulocyte ‐ enriched blood , cultured in vitro for 3 days , stained with MTR . The blood was taken from mice transplanted with Nix − / − bone marrow and reconstituted with wt ‐ Nix and Nix ‐ W35A viruses . Reticulocytes were induced by phenylhydrazine treatment . The viruses also express GFP .

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(A) Flow cytometry of reticulocyte‐enriched blood, cultured in vitro for 3 days, stained with MTR. The blood was taken from mice transplanted with Nix−/− bone marrow and reconstituted with wt‐Nix and Nix‐W35A viruses. Reticulocytes were induced by phenylhydrazine treatment. The viruses also express GFP.


( B ) Mitochondrial clearance in the GFP ‐ positive fraction of the reticulocyte ‐ enriched blood , as described in panel ( A ) . Mice were reconstituted with wt ‐ Nix , LIR ‐ W35A or empty vector viruses . Data from Nix − / − and Nix + / + mice are shown for comparison .

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(B) Mitochondrial clearance in the GFP‐positive fraction of the reticulocyte‐enriched blood, as described in panel (A). Mice were reconstituted with wt‐Nix, LIR‐W35A or empty vector viruses. Data from Nix−/− and Nix+/+ mice are shown for comparison.


( C ) Anti ‐ Flag , anti ‐ GFP and anti ‐ β ‐ actin western blots of fetal liver cells transduced with Flag ‐ Nix and Flag ‐ W35A ‐ Nix viruses , normalized to total protein . GFP , green fluorescent protein ; LIR , LC3 ‐ interacting region ; MTR , MitoTracker Red ; wt , wild type .

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(C) Anti‐Flag, anti‐GFP and anti‐β‐actin western blots of fetal liver cells transduced with Flag‐Nix and Flag‐W35A‐Nix viruses, normalized to total protein. GFP, green fluorescent protein; LIR, LC3‐interacting region; MTR, MitoTracker Red; wt, wild type.


( A ) Western Blot for pUb on HeLa control and FBXO7 - / - cells after treatment with AO for indicated times .

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(A) Western Blot for pUb on HeLa control and FBXO7-/- cells after treatment with AO for indicated times.


( B ) Quantification of pUb signal relative to loading . Western Blots run in triplicates from three independent clones . Error bars depict S . D . . Data from three biological replicates . One - way ANOVA with multiple comparisons . p ( * * ) = 0 . 0011 ; p ( * * * * ) < 0 . 0001 .

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(B) Quantification of pUb signal relative to loading. Western Blots run in triplicates from three independent clones. Error bars depict S.D.. Data from three biological replicates. One-way ANOVA with multiple comparisons. p(**) = 0.0011; p(****)<0.0001.


( D ) Western Blot for pUb on extracts from day 12 iNeuron control and FBXO7 - / - cells after treatment with BafA or AO for indicated times .

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(D) Western Blot for pUb on extracts from day 12 iNeuron control and FBXO7-/- cells after treatment with BafA or AO for indicated times.


( A ) 3D - SIM images of HeLa control , PINK1 - / - and FBXO7 - / - cell lines after AO - induced mitophagy . Cells were stained for nuclear DNA ( DAPI ) , mitochondria ( HSP60 ) and pUb . Zoom - ins of regions of interested are enlarged in the middle panel . 3D - surface renderings of insets are shown on the right . Scale bar = 5 µm or 1 µm .

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(A) 3D-SIM images of HeLa control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interested are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.


( B , C ) Evaluation of 3D - SIM images from HeLa datasets . The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy - induction are plotted . Error bars depict S . D . from 8 - 14 measured cells per condition . Two - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 .

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(B,C) Evaluation of 3D-SIM images from HeLa datasets. The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy-induction are plotted. Error bars depict S.D. from 8-14 measured cells per condition. Two-way ANOVA with multiple comparisons; p(****)<0.0001.


( D ) 3D - SIM images of iN day 12 control , PINK1 - / - and FBXO7 - / - cell lines after AO - induced mitophagy . Cells were stained for nuclear DNA ( DAPI ) , mitochondria ( HSP60 ) and pUb . Zoom - ins of regions of interest are enlarged in the middle panel . 3D - surface renderings of insets are shown on the right . Scale bar = 5 µm or 1 µm .

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(D) 3D-SIM images of iN day 12 control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (DAPI), mitochondria (HSP60) and pUb. Zoom-ins of regions of interest are enlarged in the middle panel. 3D-surface renderings of insets are shown on the right. Scale bar = 5 µm or 1 µm.


( E , F ) Evaluation of 3D - SIM images from iNeuron datasets . The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy - induction are plotted . Error bars depict S . D . from 7 - 14 measured cells per condition . One - way ANOVA with multiple comparisons , p ( * * * * ) < 0 . 0001 .

(E,F) Evaluation of 3D-SIM images from iNeuron datasets. The changes in pUb volume and minimal distances between mitochondria and pUb after mitophagy-induction are plotted. Error bars depict S.D. from 7-14 measured cells per condition. One-way ANOVA with multiple comparisons, p(****)<0.0001.


( G ) Confocal images of iNeuron d12 Control , PINK1 - / - and FBXO7 - / - cell lines after AO - induced mitophagy . Cells were stained for nuclear DNA ( Hoechst33342 ) , mitochondria ( HSP60 ) and pUb . Scale bar = 10 µm and 5 µm . ( H ) Evaluation of pUb volume after mitophagy induction . Error bars depict S . D . from three biological replicates . One - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 .

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(G) Confocal images of iNeuron d12 Control, PINK1-/- and FBXO7-/- cell lines after AO-induced mitophagy. Cells were stained for nuclear DNA (Hoechst33342), mitochondria (HSP60) and pUb. Scale bar = 10 µm and 5 µm. (H) Evaluation of pUb volume after mitophagy induction. Error bars depict S.D. from three biological replicates. One-way ANOVA with multiple comparisons; p(****)<0.0001.


( B ) Mean Acidic : Neutral mtKeima per - cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO ( Antimycin A ( 5 µM ) and Oligomycin ( 10 µM ) ) or three hours with 25 nM BafilomycinA ( BafA ) . Error bars depict S . D . from biological triplicate measurements from three independent clones . Two - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 . 0001 .

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(B) Mean Acidic:Neutral mtKeima per-cell ratios measured by flow cytometry for HeLa cells expressing Parkin ndicating the number of hours treated with AO (Antimycin A (5 µM) and Oligomycin (10 µM)) or three hours with 25 nM BafilomycinA (BafA). Error bars depict S.D. from biological triplicate measurements from three independent clones. Two-way ANOVA with multiple comparisons; p(****)<0.0001.


( C ) Bar graph showing the number of mtDNA puncta / cell with or without treatment with AO ( 6 h ) in HeLa control or FBXO7 - / - cells . Error bars depict S . D . from triplicate technical replicates from two independent clones . One - way ANOVA with multiple comparisons ; p ( * * * * ) < 0 .

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(C) Bar graph showing the number of mtDNA puncta/cell with or without treatment with AO (6 h) in HeLa control or FBXO7-/- cells. Error bars depict S.D. from triplicate technical replicates from two independent clones. One-way ANOVA with multiple comparisons; p(****)<0.0001


( D ) Indicated hESCs expressing mtKeima were differentiated to iNeurons ( day 12 ) and were subjected to image analysis . Scale bar , 5 μm . ( E ) The number of red - shifted Keima foci per cell is plotted , where each dot represents the average foci - number per image stack , originating from 7 - 12 image stacks of iNeuron differentiations . Error bars represent S . D . . One - way ANOVA with multiple comparisons ; p ( * * * ) = 0 . 0001 , p ( * * * * ) < 0 . 0001 .

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(D) Indicated hESCs expressing mtKeima were differentiated to iNeurons (day 12) and were subjected to image analysis. Scale bar, 5 μm. (E) The number of red-shifted Keima foci per cell is plotted, where each dot represents the average foci-number per image stack, originating from 7-12 image stacks of iNeuron differentiations. Error bars represent S.D.. One-way ANOVA with multiple comparisons; p(***)=0.0001, p(****)<0.0001.


( F ) mtKeima mitophagy flux readout of indicated iNeuron genotypes . Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry ( > 10 , 000 cells ) . Pooled data from biological replicates is shown , normalized to 6h BafA treated cells .

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

(F) mtKeima mitophagy flux readout of indicated iNeuron genotypes. Cells were treated with AO for either 1 or 6 h and mitophagic flux measured by flow cytometry (>10,000 cells). Pooled data from biological replicates is shown, normalized to 6h BafA treated cells.


( A ) Workflow for analysis of total protein abundance in HeLa cells expressing Parkin with and without depolarization with AO ( 16 h ) . Cell extracts from triplicates were digested with trypsin prior to 18 - plex TMT labelling and analysis by mass spectrometry .

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

(A) Workflow for analysis of total protein abundance in HeLa cells expressing Parkin with and without depolarization with AO (16 h). Cell extracts from triplicates were digested with trypsin prior to 18-plex TMT labelling and analysis by mass spectrometry.


( F ) PINK1 levels in control cells and in two FBXO7 - / - clones were measured by TMT - proteomics in fed cells , from biological triplicates from two independent clones .

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

(F) PINK1 levels in control cells and in two FBXO7-/- clones were measured by TMT-proteomics in fed cells, from biological triplicates from two independent clones.


( B ) Log2 FC for the indicated mitochondrial protein - groups in control or FBXO7 - / - cells at day 0 , 4 or 12 during neurogenesis is shown in violin plots . ( C ) Log2 FC for the indicated cellular organelle proteins in control or FBXO7 - / - cells at day 0 , 4 or 12 during neurogenesis is shown in violin plots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Log2 FC for the indicated mitochondrial protein-groups in control or FBXO7-/- cells at day 0, 4 or 12 during neurogenesis is shown in violin plots. (C) Log2 FC for the indicated cellular organelle proteins in control or FBXO7-/- cells at day 0, 4 or 12 during neurogenesis is shown in violin plots.


( F , G ) Mitochondrial morphology in iNeurons ( iN ) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei , in either fed cells or cells treated with AO ( 3 h ) ( left panel ) . The number of median # of mitochondria / cell , median mitochondrial length , and median mitochondrial circularity is shown . Error bars depict S . D . from triplicates from three differentiations ( 9 - 12 image stacks each ) , as shown in panel I left , center , and right , respectively . Two - way ANOVA with multiple comparisons ; p ( * ) = 0 . 0416 , p ( * * * * ) < 0 . 0001 . Scale bar = 10 µm .

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(F,G) Mitochondrial morphology in iNeurons (iN) was assessed using confocal imaging after staining cells with HSP60 to detect mitochondria and DAPI to identify nuclei, in either fed cells or cells treated with AO (3 h) (left panel). The number of median # of mitochondria/cell, median mitochondrial length, and median mitochondrial circularity is shown. Error bars depict S.D. from triplicates from three differentiations (9-12 image stacks each), as shown in panel I left, center, and right, respectively. Two-way ANOVA with multiple comparisons; p(*)=0.0416, p(****)<0.0001. Scale bar = 10 µm.


( H ) Western Blot analysis on HeLa control and FBXO7 - / - whole cell lysate , probed for the mitochondrial fission adapter MiD51 .

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(H) Western Blot analysis on HeLa control and FBXO7-/- whole cell lysate, probed for the mitochondrial fission adapter MiD51.


( a ) Induction of GFP + dots in differentiated THP - 1 cells stably expressing GFP - LC3 , left untransfected or transfected for 6 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , then exposed to control antibody ( Control Ab ) , antibody to neutralize IL - 1β ( Anti - IL - 1β ) , dimethyl sulfoxide ( DMSO ) or 3 - MA ( 5 mM ) ; results are presented as quantification of GFP - LC3 + dots ( top ) and images of individual cells ( middle ) . Scale bars , 10 μm .

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

(a) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3, left untransfected or transfected for 6 h with poly(dA:dT) (1.5 μg/ml), then exposed to control antibody (Control Ab), antibody to neutralize IL-1β (Anti-IL-1β), dimethyl sulfoxide (DMSO) or 3-MA (5 mM); results are presented as quantification of GFP-LC3+ dots (top) and images of individual cells (middle). Scale bars, 10 μm.


( b ) Expression of AIM2 and beclin - 1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin - 1 ; numbers below lanes indicate band intensity ( ratio of cells transfected with targeting siRNA to those transfected with control siRNA ) .

(b) Expression of AIM2 and beclin-1 in cells transfected with control siRNA or siRNA targeting AIM2 or beclin-1; numbers below lanes indicate band intensity (ratio of cells transfected with targeting siRNA to those transfected with control siRNA).


( c ) Induction of GFP + dots in differentiated THP - 1 cells stably expressing GFP - LC3 and treated with various siRNAs ( below graph ) , quantified 6 h after transfection with poly ( dA : dT ) ( + ) or no transfection ( - ) .

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

(c) Induction of GFP+ dots in differentiated THP-1 cells stably expressing GFP-LC3 and treated with various siRNAs (below graph), quantified 6 h after transfection with poly(dA:dT) (+) or no transfection (-).


( d ) Immunoblot analysis of the expression of LC3 - I and LC3 - II ( above ) in wild - type ( WT ) and Aim2 − / − BMDMs left untransfected or transfected for 6 h with poly ( dA : dT ) ; numbers below lanes indicate division of the ratio of induced LC3 - I to induced LC3 - II by the ratio of basal LC3 - I to basal LC3 - II ( band intensities ) . Below , immunoblot analysis of IL - 1β in supernatants after overnight culture .

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

(d) Immunoblot analysis of the expression of LC3-I and LC3-II (above) in wild-type (WT) and Aim2−/− BMDMs left untransfected or transfected for 6 h with poly(dA:dT); numbers below lanes indicate division of the ratio of induced LC3-I to induced LC3-II by the ratio of basal LC3-I to basal LC3-II (band intensities). Below, immunoblot analysis of IL-1β in supernatants after overnight culture.


( e ) Immunoblot analysis of LC3 - I and LC3 - II in or wild - type , ASC - deficient ( Pycard - / - ; called ' Asc - / - ' here ) or caspase - 1 - deficient ( Casp1 - / - ) BMDMs primed with LPS ( 20 ng / ml ) and then left untransfected or transfected with poly ( dA : dT ) or left untreated or treated with nigericin for 4 h ; numbers below lanes as in d . Actin serves as a loading control throughout . NS , not significant ; * P 0 . 05 and * * P 0 . 01 ( unpaired t - test ) . Data are from three individual

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

(e) Immunoblot analysis of LC3-I and LC3-II in or wild-type, ASC-deficient (Pycard-/-; called 'Asc-/-' here) or caspase-1-deficient (Casp1-/-) BMDMs primed with LPS (20 ng/ml) and then left untransfected or transfected with poly(dA:dT) or left untreated or treated with nigericin for 4 h; numbers below lanes as in d. Actin serves as a loading control throughout. NS, not significant; *P 0.05 and **P 0.01 (unpaired t-test). Data are from three individual experiments


( a ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells transfected for 3 h with Cy3 - labeled poly ( dA : dT ) ( 1 . 5 μg / ml ) . Yellow dots ( Merge ) indicate colocalization of green ( LC - 3 ) and red ( Cy3 ) signals . Scale bars , 10 μm .

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

(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected for 3 h with Cy3-labeled poly(dA:dT) (1.5 μg/ml). Yellow dots (Merge) indicate colocalization of green (LC-3) and red (Cy3) signals. Scale bars, 10 μm.


( b ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells transfected to express Cy3 - poly ( dA : dT ) , imaged every 15 min starting 1 h after transfection ( numbers in bottom left corners indicate time in min ( m ) ) . Arrow indicates coalescence of the Cy3 and GFP signals ( also in bottom right image ) .

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(b) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells transfected to express Cy3-poly(dA:dT), imaged every 15 min starting 1 h after transfection (numbers in bottom left corners indicate time in min (m)). Arrow indicates coalescence of the Cy3 and GFP signals (also in bottom right image).


( c ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells sham - transfected with no poly ( dA : dT ) ( Control ) or transfected with poly ( dA : dT ) , then fixed and immunostained for ASC ( red ) . Insets ( bottom right ) , 5 × enlargement of areas indicated by arrowheads at left . Scale bars , 10 μm ( top ) or 20 μm ( bottom ) .

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

(c) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells sham-transfected with no poly(dA:dT) (Control) or transfected with poly(dA:dT), then fixed and immunostained for ASC (red). Insets (bottom right), 5× enlargement of areas indicated by arrowheads at left. Scale bars, 10 μm (top) or 20 μm (bottom).


( d ) Confocal microscopy of differentiated THP - 1 cells transfected with poly ( dA : dT ) and immunostained for LAMP - 1 and ASC ; below , z - axis projections . Scale bars , 10 μm .

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(d) Confocal microscopy of differentiated THP-1 cells transfected with poly(dA:dT) and immunostained for LAMP-1 and ASC; below, z-axis projections. Scale bars, 10 μm.


( e ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells treated for 2 h with LPS ( 500 ng / ml ) and ATP ( 3 mM ) , then immunostained for ASC ( red ) : left image in each pair , three - dimensional volume renderings reconstructed from z - stack images ; right image in each pair , surface - segmentation models ( 2 × enlargement ) . Scale bars , 10 μm .

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

(e) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for ASC (red): left image in each pair, three-dimensional volume renderings reconstructed from z-stack images; right image in each pair, surface-segmentation models (2× enlargement). Scale bars, 10 μm.


( f ) Electron microscopy of differentiated THP - 1 cells stimulated with LPS and ATP ( left ) or transfected with poly ( dA : dT ) ( right ) for 2 h and immunostained for ASC , visualized with 15 - nm gold - conjugated protein A . Red arrows indicate ASC immunostaining . Right image in each pair , 4 . 5 × enlargement of outlined area at left . Scale bars , 500 nm . Data are representative of three experiments ( a , c ) or two experiments ( b , d - f ) .

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

(f) Electron microscopy of differentiated THP-1 cells stimulated with LPS and ATP (left) or transfected with poly(dA:dT) (right) for 2 h and immunostained for ASC, visualized with 15-nm gold-conjugated protein A. Red arrows indicate ASC immunostaining. Right image in each pair, 4.5× enlargement of outlined area at left. Scale bars, 500 nm. Data are representative of three experiments (a,c) or two experiments (b,d-f).


( a ) Confocal microscopy of differentiated GFP - LC3 + THP - 1 cells left untransfected ( Control ) or transfected for 2 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , then immunostained for endogenous

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

(a) Confocal microscopy of differentiated GFP-LC3+ THP-1 cells left untransfected (Control) or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), then immunostained for endogenous AIM2


( b ) Confocal microscopy of differentiated THP - 1 cells transfected for 3 h with poly ( dA : dT ) , then immunostained for LAMP - 1 and AIM2 .

O O O O O O O O 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) Confocal microscopy of differentiated THP-1 cells transfected for 3 h with poly(dA:dT), then immunostained for LAMP-1 and AIM2.


( c ) Confocal microscopy of differentiated THP - 1 cells left untreated or treated for 2 h with LPS ( 500 ng / ml ) and ATP ( 3 mM ) , then immunostained for endogenous LC3 and NLRP3 . Scale bars , 10 μm . Data are representative of at least three experiments .

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(c) Confocal microscopy of differentiated THP-1 cells left untreated or treated for 2 h with LPS (500 ng/ml) and ATP (3 mM), then immunostained for endogenous LC3 and NLRP3. Scale bars, 10 μm. Data are representative of at least three experiments.


( a ) Immunoblot analysis of AIM2 , ASC and LC3 - II in lysates of differentiated THP - 1 cells left untransfected or transfected with poly ( dA : dT ) ( 1 . 5 μg / ml ) and left untreated or treated with 3 - MA , rapamycin or amino - acid starvation , then separated into an inflammasome - enriched fraction ( top ) or an autophagosome - enriched fraction ( middle ) . Bottom , immunoblot analysis of total cell lysates .

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

(a) Immunoblot analysis of AIM2, ASC and LC3-II in lysates of differentiated THP-1 cells left untransfected or transfected with poly(dA:dT) (1.5 μg/ml) and left untreated or treated with 3-MA, rapamycin or amino-acid starvation, then separated into an inflammasome-enriched fraction (top) or an autophagosome-enriched fraction (middle). Bottom, immunoblot analysis of total cell lysates.


( b ) Immunoblot analysis of unprocessed and processed caspase - 1 and IL - 1β in lysates and supernatants of differentiated THP - 1 cells treated as in a .

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(b) Immunoblot analysis of unprocessed and processed caspase-1 and IL-1β in lysates and supernatants of differentiated THP-1 cells treated as in a.


( c ) Immunoblot analysis of LC3 - I and LC3 - II ( top ) in lysates of mouse BMDMs primed with LPS ( 20 ng / ml ) and left untreated or treated with alum , then left unexposed or exposed for 5 h to live ( Live TB ) or dead ( Dead TB ) M . tuberculosis ( numbers below lanes as in Fig . 1d ) . Below , immunoblot analysis of IL - 1β in supernatants of LPS - primed mouse BMDMs infected overnight with live M . tuberculosis in the presence or absence of 3 - MA ( 2 mM ) ; treatment with LPS and ATP serves as a positive control . Data are representative of at least two experiments .

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

(c) Immunoblot analysis of LC3-I and LC3-II (top) in lysates of mouse BMDMs primed with LPS (20 ng/ml) and left untreated or treated with alum, then left unexposed or exposed for 5 h to live (Live TB) or dead (Dead TB) M. tuberculosis (numbers below lanes as in Fig. 1d). Below, immunoblot analysis of IL-1β in supernatants of LPS-primed mouse BMDMs infected overnight with live M. tuberculosis in the presence or absence of 3-MA (2 mM); treatment with LPS and ATP serves as a positive control. Data are representative of at least two experiments.


( a ) Immunoblot analysis of ASC immunoprecipitates ( IP ; left ) and total lysates ( right ) of differentiated THP - 1 cells left untransfected or transfected for 2 h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for beclin - 1 and ASC ( below ) , then stripped and reprobed for p62 ( above ) .

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

(a) Immunoblot analysis of ASC immunoprecipitates (IP; left) and total lysates (right) of differentiated THP-1 cells left untransfected or transfected for 2 h with poly(dA:dT) (1.5 μg/ml), probed for beclin-1 and ASC (below), then stripped and reprobed for p62 (above).


( b ) Confocal microscopy of differentiated THP - 1 cells treated as in a , immunostained for p62 ( green ) and ASC ( red ) ; arrows indicate colocalized proteins .

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(b) Confocal microscopy of differentiated THP-1 cells treated as in a, immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins.


( c ) Confocal microscopy of differentiated THP - 1 cells treated for 2 h with LPS ( 500 ng / ml ) and ATP ( 3mM ) , immunostained for p62 ( green ) and ASC ( red ) : left two images , merged channel volume and surface rendering of a three - dimensional reconstruction ; right four images , enlargement of images at left ( yellow arrow ) , with arrows indicating removal of part of the signal to reveal the red signal ( ASC ) inside the green - stained area ( p62 ) .

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

(c) Confocal microscopy of differentiated THP-1 cells treated for 2 h with LPS (500 ng/ml) and ATP (3mM), immunostained for p62 (green) and ASC (red): left two images, merged channel volume and surface rendering of a three-dimensional reconstruction; right four images, enlargement of images at left (yellow arrow), with arrows indicating removal of part of the signal to reveal the red signal (ASC) inside the green-stained area (p62).


( d ) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence ( − ) or siRNA specific for beclin - 1 or p62 ; numbers below lanes indicate band intensity ( as in Fig . 1b ) .

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(d) Immunoblot analysis of lysates of cells transfected twice with siRNA with scrambled sequence (−) or siRNA specific for beclin-1 or p62; numbers below lanes indicate band intensity (as in Fig. 1b).


( e ) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP - 1 cells transfected as in d , with poly ( dA : dT ) included in the second transfection ; lysates were prepared 4 h after the final transfection .

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(e) Immunoblot analysis of the inflammasome and autophagosome fractions and total lysates of differentiated THP-1 cells transfected as in d, with poly(dA:dT) included in the second transfection; lysates were prepared 4 h after the final transfection.


( f ) Immunoblot analysis of supernatants and lysates of differentiated THP - 1 cells treated as in e ; supernatants were collected after culture for 6 h in serum - free medium . Scale bars ( b , c ) , 10 μm . Data are representative of at least two experiments .

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(f) Immunoblot analysis of supernatants and lysates of differentiated THP-1 cells treated as in e; supernatants were collected after culture for 6 h in serum-free medium. Scale bars (b,c), 10 μm. Data are representative of at least two experiments.


( a ) Immunoblot analysis of ASC immunoprecipitates of differentiated THP - 1 cells transfected for 2h with poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for ubiquitin ( Ub ) , then stripped and reprobed for K63 - linked ubiquitin ( Ub - K63 ) , then stripped and reprobed for ASC .

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

(a) Immunoblot analysis of ASC immunoprecipitates of differentiated THP-1 cells transfected for 2h with poly(dA:dT) (1.5 μg/ml), probed for ubiquitin (Ub), then stripped and reprobed for K63-linked ubiquitin (Ub-K63), then stripped and reprobed for ASC.


( b ) Immunoassay of differentiated THP - 1 cells treated as in a with protein - dissociation buffer , followed by immunoprecipitation of ASC ( 24 kilodaltons ( kDa ) ) and immunoblot analysis .

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(b) Immunoassay of differentiated THP-1 cells treated as in a with protein-dissociation buffer, followed by immunoprecipitation of ASC (24 kilodaltons (kDa)) and immunoblot analysis.


( c ) Confocal microscopy of differentiated THP - 1 cells transfected for 2 h with poly ( dA : dT ) , immunostained for ASC ( red ) and polyubiquitin ( green ) . PolyUb , polyubiquitination . Scale bars , 10 μm . Brackets ( right margins in a , b ) indicate smear of ubiquitinated proteins . Data are representative of three ( a , c ) or two ( b ) experiments .

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

(c) Confocal microscopy of differentiated THP-1 cells transfected for 2 h with poly(dA:dT), immunostained for ASC (red) and polyubiquitin (green). PolyUb, polyubiquitination. Scale bars, 10 μm. Brackets (right margins in a,b) indicate smear of ubiquitinated proteins. Data are representative of three (a,c) or two (b) experiments.


( a ) Immunoblot analysis of lysates of human monocytes - macrophages primed with LPS ( 10 ng ) and then transfected with poly ( dA : dT ) ( 1 . 5 μg / ml ) or treated with nigericin ( 4 μM ) for 6 h ( numbers below lanes as in Fig . 1d ) .

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(a) Immunoblot analysis of lysates of human monocytes-macrophages primed with LPS (10 ng) and then transfected with poly(dA:dT) (1.5 μg/ml) or treated with nigericin (4 μM) for 6 h (numbers below lanes as in Fig. 1d).


( b ) Immunoblot analysis of processed caspase - 1 and IL - 1β ( top ) and enzyme - linked immunosorbent assay of IL - 1β ( middle ) in supernatants of monocytes - macrophages treated for 6 h with various combinations ( bottom ) of poly ( dA : dT ) ( b ) or nigericin ( c ) , plus 3 - MA and amino - acid starvation . * P 0 . 01 ( unpaired t test ) .

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(b) Immunoblot analysis of processed caspase-1 and IL-1β (top) and enzyme-linked immunosorbent assay of IL-1β (middle) in supernatants of monocytes-macrophages treated for 6 h with various combinations (bottom) of poly(dA:dT) (b) or nigericin (c), plus 3-MA and amino-acid starvation. *P 0.01 (unpaired t test).


( d ) Confocal microscopy of LPS - primed human monocytes - macrophages transfected for 3 h with poly ( dA : dT ) ( top ) , treated for 3 h with nigericin ( middle ) or left unstimulated ( bottom ) , immunostained for p62 ( green ) and ASC ( red ) ; arrows indicate colocalized proteins . Second row , three - dimensional volume rendering reconstructed from confocal z - stack images . Scale bars , 10 μm . Data are representative of at least two experiments .

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(d) Confocal microscopy of LPS-primed human monocytes-macrophages transfected for 3 h with poly(dA:dT) (top), treated for 3 h with nigericin (middle) or left unstimulated (bottom), immunostained for p62 (green) and ASC (red); arrows indicate colocalized proteins. Second row, three-dimensional volume rendering reconstructed from confocal z-stack images. Scale bars, 10 μm. Data are representative of at least two experiments.


( a ) Immunoblot analysis of lysates of LPS ( 20 ng / ml ) - primed mouse BMDMs exposed to LPS ( 500 ng / ml ) and ATP ( 3 μM ) , poly ( dA : dT ) ( 1 . 5 μg / ml ) or uric acid crystals ( 50 μg / ml ) , detecting GTP - bound RalB ( collected by RaBP1 - agarose affinity purification ) and RalB in total lysates .

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(a) Immunoblot analysis of lysates of LPS (20 ng/ml)-primed mouse BMDMs exposed to LPS (500 ng/ml) and ATP (3 μM), poly(dA:dT) (1.5 μg/ml) or uric acid crystals (50 μg/ml), detecting GTP-bound RalB (collected by RaBP1-agarose affinity purification) and RalB in total lysates.


( b ) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB - specific siRNA on 2 sequential days , then primed with LPS and exposed for 6 h to poly ( dA : dT ) ( 1 . 5 μg / ml ) , probed for RalB , LC3 and IL - 1β ; supernatants were collected after overnight culture in serum - free medium ( numbers below lanes as in Fig . 1d ) . Nonsp , nonspecific band .

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(b) Immunoblot analysis of lysates and supernatants of mouse BMDMs transfected with control or RalB-specific siRNA on 2 sequential days, then primed with LPS and exposed for 6 h to poly(dA:dT) (1.5 μg/ml), probed for RalB, LC3 and IL-1β; supernatants were collected after overnight culture in serum-free medium (numbers below lanes as in Fig. 1d). Nonsp, nonspecific band.


( c ) Immunoblot analysis of lysates of wild - type and Aim2 − / − mouse BMDMs transfected with control or AIM2 - specific siRNA on 2 sequential days , primed for 2 h with LPS and then exposed for 1 h to poly ( dA : dT ) ( numbers below lanes as in Fig . 1d ) .

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

(c) Immunoblot analysis of lysates of wild-type and Aim2−/− mouse BMDMs transfected with control or AIM2-specific siRNA on 2 sequential days, primed for 2 h with LPS and then exposed for 1 h to poly(dA:dT) (numbers below lanes as in Fig. 1d).


( d ) Immunoblot analysis of ASC immunoprecipitates from LPS - primed mouse BMDMs left untreated or transfected for 1 h with poly ( dA : dT ) and treated for 30 min with LPS and ATP . Mock , immunoprecipitation with control antibody .

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(d) Immunoblot analysis of ASC immunoprecipitates from LPS-primed mouse BMDMs left untreated or transfected for 1 h with poly(dA:dT) and treated for 30 min with LPS and ATP. Mock, immunoprecipitation with control antibody.


( e ) Confocal microscopy of LPS - primed mouse BMDMs left untransfected or transfected for 30 min with poly ( dA : dT ) , immunostained for RalB and AIM2 ( left three columns ) ; RalB and ASC ( middle columns ) ; and beclin - 1 and AIM2 ( right two columns ) . Bottom row , 3 × electronic enlargement of areas outlined above . Scale bars , 10 μm . Data are representative of at least two experiments .

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(e) Confocal microscopy of LPS-primed mouse BMDMs left untransfected or transfected for 30 min with poly(dA:dT), immunostained for RalB and AIM2 (left three columns); RalB and ASC (middle columns); and beclin-1 and AIM2 (right two columns). Bottom row, 3× electronic enlargement of areas outlined above. Scale bars, 10 μm. Data are representative of at least two experiments.


D : KD - values of wild - type , mutant and phosphorylated peptides binding to clathrin NTD ( HURP832 - 846 ) , RANBP2 RanBD4 ( DHX38260 - 275 ) and SNX27 PDZ ( CDC25C30 - 44 ) . Significant differences indicated by the Mann - Whitney Test ( * * : p ≤ 0 . 01 ) . Measurements are from two independent protein preparations with two or three technical replicates each .

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D: KD-values of wild-type, mutant and phosphorylated peptides binding to clathrin NTD (HURP832-846), RANBP2 RanBD4 (DHX38260-275) and SNX27 PDZ (CDC25C30-44). Significant differences indicated by the Mann-Whitney Test (**: p≤0.01). Measurements are from two independent protein preparations with two or three technical replicates each.


E : Displacement curves of the KPNA4 ARM domain with the RanBP3114 - 129 peptides ( wild - type , phosphomimetic and phosphorylated ) sampling the effects of two distinct phosphosites ( T124 and S125 ) . IC50 - values for the different peptides are indicated in µM and with SEM . Measurements were obtained in technical triplicates .

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E: Displacement curves of the KPNA4 ARM domain with the RanBP3114-129 peptides (wild-type, phosphomimetic and phosphorylated) sampling the effects of two distinct phosphosites (T124 and S125). IC50-values for the different peptides are indicated in µM and with SEM. Measurements were obtained in technical triplicates.


B : Displacement curves of the three different bait domains and the indicated peptides using wild - type , phosphomimetic and phosphorylated peptide . IC50 values of the different peptides are indicated in µM and with SEM . Measurements were in technical triplicates .

O O O O O O O O O O O O O O O O O O O O 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: Displacement curves of the three different bait domains and the indicated peptides using wild-type, phosphomimetic and phosphorylated peptide. IC50 values of the different peptides are indicated in µM and with SEM. Measurements were in technical triplicates.


A : Displacement curves of the FP experiment using clathrin NTD and wild - type , phosphomimetic and phosphorylated HURP peptides . IC50 values are indicated in in µM and with SEM . Measurements were in four to five technical replicates .

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

A: Displacement curves of the FP experiment using clathrin NTD and wild-type, phosphomimetic and phosphorylated HURP peptides. IC50 values are indicated in in µM and with SEM. Measurements were in four to five technical replicates.


F : Co - IP of clathrin with Venus - tagged HURPWT and HURP ∆ MUT constructs in asynchronous and mitotic HeLa cells .

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F: Co-IP of clathrin with Venus-tagged HURPWT and HURP∆MUT constructs in asynchronous and mitotic HeLa cells.


G Co - IP of clathrin with Venus - tagged HURPWT , HURP ∆ MUT and HURPS839A constructs in mitotic HeLa cells .

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G Co-IP of clathrin with Venus-tagged HURPWT, HURP∆MUT and HURPS839A constructs in mitotic HeLa cells.


H : Representative images of HURPWT , HURP ∆ MUT and HURPS839A in the different mitotic stages .

H: Representative images of HURPWT, HURP∆MUT and HURPS839A in the different mitotic stages.


I - J : RNAi rescue experiments ( n = 2 ) , which included the knockdown of endogenous HURP and re - expressing either RNAi resistant HURPWT , HURP ∆ MUT and HURPS839A . In order to assess mitotic behaviour of the cells , the time required of control , HURP knockdown , HURPWT , HURP ∆ MUT and HURPS839A HeLa cells to progress through mitosis ( NEBD - anaphase ) was established using time - lapse microscopy . Every circle represents one analysed cell . The number of cells analysed is indicated as n in I . Mann - Whitney test was performed to assess significant differences in the median time required for mitotic progression ( * : p - value ≤ 0 . 05 , * * * : p - value : ≤ 0 . 001 , * * * * : p - value ≤ 0 . 0001 ; ns : non - significant )

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I-J: RNAi rescue experiments (n=2), which included the knockdown of endogenous HURP and re-expressing either RNAi resistant HURPWT, HURP∆MUT and HURPS839A. In order to assess mitotic behaviour of the cells, the time required of control, HURP knockdown, HURPWT, HURP∆MUT and HURPS839A HeLa cells to progress through mitosis (NEBD-anaphase) was established using time-lapse microscopy. Every circle represents one analysed cell. The number of cells analysed is indicated as n in I. Mann-Whitney test was performed to assess significant differences in the median time required for mitotic progression (*: p-value≤0.05, ***: p-value: ≤0.001, ****: p-value ≤0.0001; ns: non-significant)


( A ) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10 - wk - old WT and RDEB mice . ( B ) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn , 4 - wk - old and 10 - wk - old WT and RDEB mice . Plotted as fold increase compared to mean of age - matched WT paws ; replicates per age = 5 - 6 . ( Data information : Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

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(A) Plot from flow cytometry analysis of neutrophils in forepaw skin from 10-wk-old WT and RDEB mice. (B) Bar graph of flow cytometry analyses as in A of neutrophils in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age = 5-6. ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.


( C ) Plot from flow cytometry analysis of CD38 + and Egr2 + macrophages in forepaw skin from 10 - wk - old WT and RDEB mice . ( D ) Bar graph of flow cytometry analyses as in C of inflammatory macrophages ( CD38 + Egr2 - ) or tissue - repair macrophages ( CD38 - Egr2 + ) macrophages in forepaw skin of newborn , 4 - wk - old and 10 - wk - old WT and RDEB mice . Plotted as fold increase compared to mean of age - matched WT paws ; replicates per age ; n = 5 - 6 ( CD38 + Egr2 - ) ; n = 4 - 5 ( CD38 - Egr2 + ) . ( Data information : Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

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(C) Plot from flow cytometry analysis of CD38+ and Egr2+ macrophages in forepaw skin from 10-wk-old WT and RDEB mice. (D) Bar graph of flow cytometry analyses as in C of inflammatory macrophages (CD38+Egr2-) or tissue-repair macrophages (CD38-Egr2+) macrophages in forepaw skin of newborn, 4-wk-old and 10-wk-old WT and RDEB mice. Plotted as fold increase compared to mean of age-matched WT paws; replicates per age; n = 5-6 (CD38+Egr2-); n = 4-5 (CD38-Egr2+). ( Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.


( F - G ) The mean fluorescence intensity ( MFI ) of MHC II staining on ( F ) neutrophils ( n = 3 - 4 biological replicates ) and ( G ) macrophages ( n = 6 - 7 biological replicates ) . Data information : Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction P values < 0 . 05 are considered significant .

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(F-G) The mean fluorescence intensity (MFI) of MHC II staining on (F) neutrophils (n = 3-4 biological replicates) and (G) macrophages (n = 6-7 biological replicates). Data information: Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction P values < 0.05 are considered significant.


( A - D ) Skin from healthy young ( 3 - 8 years old ) and adult donors ( > 25 years old ) and from donors with RDEB with different stages of fibrosis - early ( 0 - 3 years old ) , mild ( 6 - 10 years old ) and advanced ( > 15 years old ) - stained for CD68 ( green ) , CD3ε ( green ) , periostin ( green ) and tenascin - C ( red ) . Nuclei were counterstained with DAPI ( blue ) . A and B , scale bar = 50 μm ; C and D , scale bar = 100 μm . The bar graphs at the right to the stained sections , represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D , the mean intensity of staining from 3 - 6 donors per group . Individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction . P values < 0 . 05 are considered significant .

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(A-D) Skin from healthy young (3-8 years old) and adult donors ( > 25 years old) and from donors with RDEB with different stages of fibrosis - early (0-3 years old), mild (6-10 years old) and advanced ( > 15 years old) - stained for CD68 (green), CD3ε (green), periostin (green) and tenascin-C (red). Nuclei were counterstained with DAPI (blue). A and B, scale bar = 50 μm; C and D, scale bar = 100 μm. The bar graphs at the right to the stained sections, represent for A and B quantification of the number positive cells per mm2 analyzed from 3 donors for each group and for C and D, the mean intensity of staining from 3-6 donors per group. Individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction. P values < 0.05 are considered significant.


( A ) Human dermal RDEBF treated daily with 10 μM LY2109761 ( LY ) ( TGF - β Receptor I / II inhibitor ) , 10 μM 1D11 ( neutralizing antibody to all TGF - βs ) 100 nM Ang - ( 1 - 7 ) , or 10 μM losartan ( AT1R antagonist ) . After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins , THBS - 1 , thrombospondin - 1 , β - tubulin was used as loading control . ( B ) Densitometric quantification of blots as in A . The abundance was normalized to β - tubulin and then expressed as % of untreated ( - ) . P values are shown ; the data were analyzed with one - way ANOVA with Dunnett ' s correction ( n = 5 biological replicates ) . ( Data information : P values < 0 . 05 are considered significant and shown , ns = not significant . individual data points , mean ± S . E . M are shown .

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(A) Human dermal RDEBF treated daily with 10 μM LY2109761 (LY) (TGF-β Receptor I/II inhibitor), 10 μM 1D11 (neutralizing antibody to all TGF-βs) 100 nM Ang-(1-7), or 10 μM losartan (AT1R antagonist). After five days of treatment cell and matrix lysates were extracted and analyzed by western blotting for the indicated proteins, THBS-1, thrombospondin-1, β-tubulin was used as loading control. (B) Densitometric quantification of blots as in A. The abundance was normalized to β-tubulin and then expressed as % of untreated (-). P values are shown; the data were analyzed with one-way ANOVA with Dunnett's correction (n = 5 biological replicates). ( Data information: P values < 0.05 are considered significant and shown, ns = not significant. individual data points, mean ± S.E.M are shown.


( F ) Western blots of fibroblast lysates from co - cultures as described in E . The blots were probed with antibodies detecting fibronectin , thrombospondin - 1 ( THBS - 1 ) and pSMAD - 2 / 3 . β - tubulin was used as loading control . ( G ) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments , n = 4 - 5 using cells from 4 donors with RDEB . The abundance was normalized to β - tubulin then and then expressed as % of both untreated . The data were analyzed by one - way ANOVA with Tukey ' s correction . Data information : P values < 0 . 05 are considered significant and shown , ns = not significant . For B , D and G individual data points , mean ± S . E . M are shown .

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(F) Western blots of fibroblast lysates from co-cultures as described in E. The blots were probed with antibodies detecting fibronectin, thrombospondin-1 (THBS-1) and pSMAD-2/3. β-tubulin was used as loading control. (G) Densitometric quantification of western blots of fibroblast cell and matrix lysates as shown in F from multiple independent experiments, n = 4 -5 using cells from 4 donors with RDEB. The abundance was normalized to β-tubulin then and then expressed as % of both untreated. The data were analyzed by one-way ANOVA with Tukey's correction. Data information: P values < 0.05 are considered significant and shown, ns = not significant. For B, D and G individual data points, mean ± S.E.M are shown.


( B ) Kaplan Meier survival curve of mice treated as in A , P values as indicated , the data were tested with log - rank test - PBS treated vs . 0 . 1 mg / kg Ang - ( 1 - 7 ) treated and PBS treated vs . 1 . 0 mg / kg Ang - ( 1 - 7 ) treated ; n = 12 - 15 mice as indicated in the figure .

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(B) Kaplan Meier survival curve of mice treated as in A, P values as indicated, the data were tested with log-rank test - PBS treated vs. 0.1 mg/kg Ang-(1-7) treated and PBS treated vs. 1.0 mg/kg Ang-(1-7) treated; n = 12-15 mice as indicated in the figure.


( C ) Photos of left and right forepaws of RDEB mice before and after 7 weeks of daily injections with 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS . ( D ) Plot of numbers of toes lost during the 7 - week observation period , data tested with Kruskal - Wallis test , P values indicated , n = 20 - 24 paws as indicated in the figure . Median and range are shown . (

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(C) Photos of left and right forepaws of RDEB mice before and after 7 weeks of daily injections with 0.1, 1.0 mg/kg Ang-(1-7) or PBS. (D) Plot of numbers of toes lost during the 7-week observation period, data tested with Kruskal-Wallis test, P values indicated, n= 20-24 paws as indicated in the figure. Median and range are shown. (


( A ) Haemotoxylin and Eosin ( H & E ) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS for seven weeks . Inflammatory cells are elevated in the toes from the PBS and the two Ang - ( 1 - 7 ) treated RDEB mice compared to the WT counterpart . Scale bar = 100 μm . Data information scale bar = 100 μm .

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(A) Haemotoxylin and Eosin (H&E) staining of toes from forepaws from WT and RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Inflammatory cells are elevated in the toes from the PBS and the two Ang-(1-7) treated RDEB mice compared to the WT counterpart. Scale bar = 100 μm. Data information scale bar = 100 μm.


( B - D ) Flow cytometry analysis of the number of CD45 positive cells ( B ) , percentage of neutrophils ( C ) and of inflammatory macrophages ( CD38 + Egr2 - ) and tissue - repair macrophages ( CD38 - Egr2 + ) ( D ) in forepaw skin of RDEB mice treated as in A . Data information : individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant and shown ; n = 5 - 14 biological replicates .

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(B-D) Flow cytometry analysis of the number of CD45 positive cells (B), percentage of neutrophils (C) and of inflammatory macrophages (CD38+Egr2-) and tissue-repair macrophages (CD38-Egr2+) (D) in forepaw skin of RDEB mice treated as in A. Data information: individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.


( F ) Staining for IgG ( red ) in forepaws , nuclei counterstained with DAPI . ( G ) Quantification of fluorescence staining intensity of samples as in F . Data information : scale bar = 100 μm . , individual data points , mean ± S . E . M are shown . The data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant and shown ; n = 5 - 14 biological replicates .

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(F) Staining for IgG (red) in forepaws, nuclei counterstained with DAPI. (G) Quantification of fluorescence staining intensity of samples as in F. Data information: scale bar = 100 μm. , individual data points, mean ± S.E.M are shown. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant and shown; n = 5-14 biological replicates.


( A ) Picrosirius red staining visualized under polarized light and Elastica van Gieson ( EvG ) staining ) of forepaw toes from 12 - wk - old WT or RDEB mice treated with daily injections of 0 . 1 , 1 . 0 mg / kg Ang - ( 1 - 7 ) or PBS for seven weeks . Note the reduced signal for picrosirus red in RDEB mice treated with 0 . 1 mg / kg Ang - ( 1 - 7 ) indicating less parallel alignment of and / or thinner collagen fibrils , and additionally the more organized and less disrupted appearance of elastic fibers ( black in the EvG staining ) . ( B ) Quantification of picrosirius red stained forepaws visualized as in A . A digital threshold was applied to the red channel before quantification . The data were analyzed by one - way ANOVA with Tukey ' s correction , P values < 0 . 05 are considered significant ; n = 10 - 15 . Individual data points , mean ± S . E . M are shown . ( Data information scale bar = 100

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(A) Picrosirius red staining visualized under polarized light and Elastica van Gieson (EvG) staining) of forepaw toes from 12-wk-old WT or RDEB mice treated with daily injections of 0.1, 1.0 mg/kg Ang-(1-7) or PBS for seven weeks. Note the reduced signal for picrosirus red in RDEB mice treated with 0.1 mg/kg Ang-(1-7) indicating less parallel alignment of and/or thinner collagen fibrils, and additionally the more organized and less disrupted appearance of elastic fibers (black in the EvG staining). (B) Quantification of picrosirius red stained forepaws visualized as in A. A digital threshold was applied to the red channel before quantification. The data were analyzed by one-way ANOVA with Tukey's correction, P values < 0.05 are considered significant; n = 10-15. Individual data points, mean ± S.E.M are shown. ( Data information scale bar = 100 μm


( C ) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A . α - SMA , α - smooth muscle actin ; Thbs - 1 , thrombospondin - 1 . Data information : β - actin or β - tubulin was used as loading

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(C) Western blotting for the indicated proteins on tissue lysates from forepaws from RDEB mice treated as in A. α-SMA, α-smooth muscle actin; Thbs-1, thrombospondin-1. Data information: β- actin or β-tubulin was used as loading control


( D ) Dermal β - arrestin - 1 / 2 ( red ) and NFκB ( red ) staining in forepaws from WT and RDEB mice treated as in A . Nuclei counterstained with DAPI ( blue ) . Data information scale bar = 20 μm .

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(D) Dermal β-arrestin-1/2 (red) and NFκB (red) staining in forepaws from WT and RDEB mice treated as in A. Nuclei counterstained with DAPI (blue). Data information scale bar = 20 μm.


( A ) Mouse embryos were immunostained with anti - neurofilament antibody ( blue ) , showing mesencephalic projection . Dispersed axon projections ( arrows point to the area of the superior colliculus ) and decreased axon fibers ( arrowheads point to the area of the pretectal commissure ) were observed in Plcg1f / f ; Nes - Cre mice at E11 . 5 ( n = 20 ) . The areas of the superior colliculus is shown magnified in each inset ( scale bar = 200 μm )

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(A) Mouse embryos were immunostained with anti-neurofilament antibody (blue), showing mesencephalic projection. Dispersed axon projections (arrows point to the area of the superior colliculus) and decreased axon fibers (arrowheads point to the area of the pretectal commissure) were observed in Plcg1f/f;Nes-Cre mice at E11.5 (n = 20). The areas of the superior colliculus is shown magnified in each inset (scale bar = 200 μm)


( B ) Representative images between the posterior and anterior borderlines of the superior colliculus ( a and b ) . The red boxes were drawn as rostrocaudal region of interest ( ROI ) based on the upper part of the pretectal commissure in the embryonic mesencephalon ( scale bar = 100 μm )

(B) Representative images between the posterior and anterior borderlines of the superior colliculus (a and b). The red boxes were drawn as rostrocaudal region of interest (ROI) based on the upper part of the pretectal commissure in the embryonic mesencephalon (scale bar = 100 μm)


( C and D ) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI , and the distribution of axons was statistically analyzed ( n = 5 ; t - test ; * * P < 0 . 005 )

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(C and D) The axons passing through the ROI was quantified by measuring the fluorescence intensity in the ROI, and the distribution of axons was statistically analyzed (n = 5; t-test; **P < 0.005)


( E ) Whole - mount immunostaining images show neurofilament ( red ) , netrin - 1 ( purple ) , and DCC ( green ) , respectively . An E11 . 5 Plcg1f / f ; Nes - Cre embryo showed shortened dorsal ramus and opthalmic nerves ( white and yellow boxes ) . Scale bar : 200

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(E) Whole-mount immunostaining images show neurofilament (red), netrin-1 (purple), and DCC (green), respectively. An E11.5 Plcg1f/f;Nes-Cre embryo showed shortened dorsal ramus and opthalmic nerves (white and yellow boxes). Scale bar: 200 μm


( F - G ) Enlarged views of the opthalmic nerve ( yellow arrow heads ) and dorsal ramus ( white arrow heads ) ( scale bar : 50 μm )

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(F - G) Enlarged views of the opthalmic nerve (yellow arrow heads) and dorsal ramus (white arrow heads) (scale bar: 50 μm)


( H ) Nerve length measurements ; Op : ophthalmic nerve ; DR : dorsal ramus ( n = 3 ; t - test ; * * P < 0 . 005 ) . Data are presented as the mean ± s . e .

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(H) Nerve length measurements; Op: ophthalmic nerve; DR: dorsal ramus (n = 3; t-test; **P < 0.005). Data are presented as the mean ± s.e.m


( I ) Double immunostaining images . The pattern of expression and colocalization of DCC ( green ) - and TH ( red ) - positive axon bundles extending toward the alar thalamus . Scale bar : 100

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(I) Double immunostaining images. The pattern of expression and colocalization of DCC (green)- and TH (red)-positive axon bundles extending toward the alar thalamus. Scale bar: 100 μm


( A ) In primary mesencephalic neurons , the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin - 1 concentration . The lower graph presents quantification of the band intensities in ( A ) ( n = 3 )

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(A) In primary mesencephalic neurons, the phosphorylation of PLCγ1 on Y783 was increased proportionally to the netrin-1 concentration. The lower graph presents quantification of the band intensities in (A) (n = 3)


( B ) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA ( siDcc ) ( N = 3 )

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(B) Phosphorylation of PLCγ1 on Y783 was decreased after knockdown of the DCC receptor by siRNA (siDcc) (N = 3)


( C ) Western blot for the NTN1 secretion test . Lane # 1 : transfection - free lysate , # 2 : transfection - free supernatant , # 3 : NTN1 - transfected cell lyasate , # 3 and # 4 : NTN - 1 - transfected

(C) Western blot for the NTN1 secretion test. Lane #1: transfection-free lysate, #2: transfection-free supernatant, #3: NTN1-transfected cell lyasate, #3 and #4: NTN-1- transfected supernatant


( D ) Ventral mesencephalic ( A9 ) cultures ( daggers ) derived from control or Plcg1f / f ; Nes - Cre mice , were co - cultured with netrin - 1 - releasing 293T cell aggregates ( asterisks ; scale bar = 100 μm ) . The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets ( n = 4 )

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(D) Ventral mesencephalic (A9) cultures (daggers) derived from control or Plcg1f/f;Nes-Cre mice, were co-cultured with netrin-1-releasing 293T cell aggregates (asterisks; scale bar = 100 μm). The areas delineated by the white boxes between the mesencephalon tissue and 293T cell aggregates are magnified in the respective insets (n = 4)


(F) Mean angle of the axon extension


( G ) Quantification of the ventral mesencephalic axons . The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin - 1 - releasing cell aggregates ( n = 200 ) . Each experimental set was performed in

(G) Quantification of the ventral mesencephalic axons. The bar graphs indicate the percentages of proper targeting of ventral mesencephalic axons correctly targeting the netrin-1-releasing cell aggregates (n = 200). Each experimental set was performed in quadruplicate


( H ) Anti - tyrosine hydroxylase ( TH ) immunostaining in ventral mesencephalic ( VM ) tissue . ( scale bar = 200 μm )

(H) Anti-tyrosine hydroxylase (TH) immunostaining in ventral mesencephalic (VM) tissue. (scale bar = 200 μm)


( I ) Primary mesencephalic neuron culture in the absence or presence of NTN1 - conditioned media ( n = 100 ; scale bar : 500μm )

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(I) Primary mesencephalic neuron culture in the absence or presence of NTN1-conditioned media (n = 100; scale bar: 500μm)


( K ) Mean length of neurites ( F - value : 1 . 82 , t - test , P * * < 0 . 005 )

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(K) Mean length of neurites (F-value:1.82, t-test, P**<0.005)


( A ) Co - immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor ( n = 2 , N = 3 ) .

(A) Co-immunoprecipitation data showed that PLCγ1 did not interact with the DCC receptor (n = 2, N = 3).


( B ) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1 . Rac1 is a positive interacting control for PLCγ1 ( N = 3 ) .

(B) Immunoprecipitation assay showed that Src kinase interacted with PLCγ1. Rac1 is a positive interacting control for PLCγ1 (N = 3).


( C ) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1 . ( D ) Quantification of the band intensities in ( C ) ( N = 3 ) .

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(C) In vitro kinase assay confirmed that Src kinase directly phosphorylated Y783 of PLCγ1. (D) Quantification of the band intensities in (C) (N = 3).


( E ) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 ( arrow ; Src I IC50 conc = 44 nM ; N = 3 ) .

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(E) Src kinase inhibitor reduced the specific phosphorylation of PLCγ1 on Y783 (arrow; Src I IC50 conc = 44 nM; N = 3).


( F ) Rac1 activity assay ( N = 3 ) . ( G ) Quantification of Rac1 activity levels in ( F ) .

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(F) Rac1 activity assay (N = 3). (G) Quantification of Rac1 activity levels in (F).


( H ) Representative immunoblot of cultured Plcg1f / f ( n = 2 , N = 3 ) and Plcg1f / f ; Nes - Cre ( n = 2 , N = 3 ) mesencephalic neurons stimulated with conditioned media containing Netrin - 1 .

(H) Representative immunoblot of cultured Plcg1f/f (n = 2, N = 3) and Plcg1f/f;Nes-Cre (n = 2, N = 3) mesencephalic neurons stimulated with conditioned media containing Netrin-1.


( B ) Immunohistochemical staining of control and Plcg1 conditional knockout ( cKO ) sagittal brain sections . The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f / f ; Nes - Cre ( d ) mice compared to the controls ( a ) ( n = 4 ; scale bar = 1 mm ) . The areas delineated by the white boxes ( insets ) are magnified images of the OT ( b and e ) , and the yellow boxes are magnified images of the SI ( c and f ) . ( C ) Quantification of the axon bundle distribution . Green boxes in ( b ) and ( e ) were selected as tegions of interest ( ROIs ) using the ZEN software ( n = 4 ) ( one - way ANOVA , F - value : 118 . 5745 , P * * < 0 . 005 ) .

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(B) Immunohistochemical staining of control and Plcg1 conditional knockout (cKO) sagittal brain sections. The deletion of Plcg1 caused structural changes in SI and OT projections in the Plcg1f/f;Nes-Cre (d) mice compared to the controls (a) (n = 4; scale bar = 1 mm). The areas delineated by the white boxes (insets) are magnified images of the OT (b and e), and the yellow boxes are magnified images of the SI (c and f). (C) Quantification of the axon bundle distribution. Green boxes in (b) and (e) were selected as tegions of interest (ROIs) using the ZEN software (n = 4) (one-way ANOVA, F-value: 118.5745, P**<0.005).


( D ) Representative images of tyrosine hydroxylase ( TH ) - positive neurons ( scale bar = 1 mm ) . The areas delineated by the white boxes are magnified in the insets . The yellow arrow indicated misoriented TH - positive axons ( magenta : TH ) .

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(D) Representative images of tyrosine hydroxylase (TH)- positive neurons (scale bar = 1 mm). The areas delineated by the white boxes are magnified in the insets. The yellow arrow indicated misoriented TH-positive axons (magenta: TH).


( E ) Fluorescence intensities in the ROIs were measured according to distance ( n = 4 ) ( one - way ANOVA , F - value : 271 . 8 , * * P < 0 . 005 ) . Cumulative values of fluorescence intensities per section are shown in the lower graph ( S : section ) .

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(E) Fluorescence intensities in the ROIs were measured according to distance (n = 4) (one-way ANOVA, F-value: 271.8, **P<0.005). Cumulative values of fluorescence intensities per section are shown in the lower graph (S: section).


( F ) In situ hybridization of PLCγ1 in mDA neurons ( scale bar = 50 μm ) ( green : TH , violet : DIG - labeled probe ) .

(F) In situ hybridization of PLCγ1 in mDA neurons (scale bar = 50 μm) (green: TH, violet: DIG-labeled probe).


( G ) Representative images of the CC axon bundles in control ( left ) and a Plcg1f / f ; Nes - Cre ( right ) mice ( n = 4 ) . The tip of the CC was triangular in the control mouse , but oval in the Plcg1f / f ; Nes - Cre mouse . The areas delineated by the white boxes are magnified in the insets ( red : neurofilament , green : glial fibrillary acidic protein , blue : Hoechst ; scale bar = 1 mm ) .

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(G) Representative images of the CC axon bundles in control (left) and a Plcg1f/f;Nes-Cre (right) mice (n = 4). The tip of the CC was triangular in the control mouse, but oval in the Plcg1f/f;Nes-Cre mouse. The areas delineated by the white boxes are magnified in the insets (red: neurofilament, green: glial fibrillary acidic protein, blue: Hoechst; scale bar = 1 mm).


( H ) The area of the CC in the Plcg1f / f ; Nes - Cre mouse was 36 % smaller than that in the control mouse ( scale bar = 500 μm ) ( I ) Quantitative comparison of the CC areas in the control and Plcg1f / f ; Nes - Cre mice ( n = 4 ; t - test ; * * p < 0 . 005 ) . Data are presented as the mean ± s . e . m .

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(H) The area of the CC in the Plcg1f/f;Nes-Cre mouse was 36% smaller than that in the control mouse (scale bar = 500 μm) (I) Quantitative comparison of the CC areas in the control and Plcg1f/f;Nes-Cre mice (n = 4; t-test; **p < 0.005). Data are presented as the mean ± s.e.m.


( A ) Cumulative histograms based on fractional anisotropy ( FA ) values . ( B ) , show the CC volume relative to FA values . As FA values increased , the structural ( CC volume differences increased between the control and Plcg1f / f ; Nes - Cre mice . ( n = 4 ; Shapiro - Wilk and t - test ; * p < 0 . 05 ) . Data are presented as the mean ± s . e . m .

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(A) Cumulative histograms based on fractional anisotropy (FA) values. (B), show the CC volume relative to FA values. As FA values increased, the structural (CC volume differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


( C ) , Cumulative histograms based on fractional anisotropy ( FA ) values . ( D ) , show the number of tensors relative to FA values . As FA values increased , the structural number of tensors ) differences increased between the control and Plcg1f / f ; Nes - Cre mice . ( n = 4 ; Shapiro - Wilk and t - test ; * p < 0 . 05 ) . Data are presented as the mean ± s . e . m .

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(C), Cumulative histograms based on fractional anisotropy (FA) values. (D), show the number of tensors relative to FA values. As FA values increased, the structural number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


( E ) Cumulative histograms based on fractional anisotropy ( FA ) values . ( F ) show the CC volume and the number of tensors relative to FA values . As FA values increased , the structural ( CC volume and the number of tensors ) differences increased between the control and Plcg1f / f ; Nes - Cre mice . Depending on the number of tensors , the length in the CC appeared to be shortened ( E , F ) . ( n = 4 ; Shapiro - Wilk and t - test ; * p < 0 . 05 ) . Data are presented as the mean ± s . e . m .

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(E) Cumulative histograms based on fractional anisotropy (FA) values. (F) show the CC volume and the number of tensors relative to FA values. As FA values increased, the structural (CC volume and the number of tensors) differences increased between the control and Plcg1f/f;Nes-Cre mice. Depending on the number of tensors, the length in the CC appeared to be shortened (E, F). (n = 4; Shapiro-Wilk and t-test; *p < 0.05). Data are presented as the mean ± s.e.m.


B Induction of NF - κB targets in Jurkat T cells in response to 20 ng / ml TNF treatment for 1 , 2 , and 4 hours as measured by RT - qPCR . Target values were normalized to GAPDH and are reported as fold change relative to basal expression . Data are presented as mean ± standard deviation ( SD ) of 3 biological replicates .

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B Induction of NF-κB targets in Jurkat T cells in response to 20 ng/ml TNF treatment for 1, 2, and 4 hours as measured by RT-qPCR. Target values were normalized to GAPDH and are reported as fold change relative to basal expression. Data are presented as mean ± standard deviation (SD) of 3 biological replicates.


D Maximum intensity projections of smFISH fluorescence microscopy z - stacks of basal Jurkat T cells stained for the indicated genes . Nfkbia , Tnfaip3 , Tnf , and Il6 were labeled with Quasar 670 , and Il8 and Csf2 were labeled with fluorescein . All images were filtered as described in Materials and Methods . Brightness and contrast were enhanced for visualization . Scale bars : 10 μm .

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D Maximum intensity projections of smFISH fluorescence microscopy z-stacks of basal Jurkat T cells stained for the indicated genes. Nfkbia, Tnfaip3, Tnf, and Il6 were labeled with Quasar 670, and Il8 and Csf2 were labeled with fluorescein. All images were filtered as described in Materials and Methods. Brightness and contrast were enhanced for visualization. Scale bars: 10 μm.