Relative gene expression of Id1 , specifically induced by activated SMAD1 / 5 , Pai1 , specifically induced by activated SMAD2 / 3 , in total muscle tissues 1 week after implantation with V Low - , V Med - V High - expressing myoblast clones or control ( Ctrl ) , expressed as fold - change versus Ctrl group . Data represent values , with mean ± ( n = 3 or 4 , as indicated ) ; * * P < 0 . 01 * * * P < 0 . 001 by one - way ANOVA with Bonferroni multiple comparisons , after data normalization by logarithmic transformation ; V Low versus Ctrl P = 0 . 0002 ; V Low versus V Med P = 0 . 005 ; V Low versus V High P = 0 . 0016 .

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Relative gene expression of Id1, specifically induced by activated SMAD1/5, and Pai1, specifically induced by activated SMAD2/3, in total muscle tissues 1 week after implantation with V Low-, V Med- and V High-expressing myoblast clones or control cells (Ctrl), expressed as fold-change versus the Ctrl group. Data represent individual values, with mean ± SEM (n = 3 or 4, as indicated); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; V Low versus Ctrl P = 0.0002; V Low versus V Med P = 0.005; V Low versus V High P = 0.0016.


Immunofluorescence of endothelium ( CD31 , in red ) , nuclei ( DAPI , in blue ) , phosphorylated SMAD2 / 3 ( in cyan ) on cryosections of similarly injected confirmed that endothelial SMAD2 / 3 activation was inhibited by increasing VEGF doses . Scale bar = 10 μm ; n = 3 .

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Immunofluorescence staining of endothelium (CD31, in red), nuclei (DAPI, in blue), and phosphorylated SMAD2/3 (in cyan) on cryosections of similarly injected muscles confirmed that endothelial SMAD2/3 activation was inhibited by increasing VEGF doses. Scale bar = 10 μm; n = 3.


A - C Endothelial Sema3A expression is directly inhibited by VEGF stimulated by TGF - β1 in vitro . Mouse aortic endothelial were stimulated with increasing doses of recombinant VEGF164 or TGF - β1 for 24 h . expression of Sema3a Tgfb1 was quantified by qRT - PCR expressed as fold - change versus non - stimulated . VEGF dose dependently inhibited Sema3a expression ( A ) , but did not affect that of Tgfb1 ( B ) , while TGF - β1 upregulated Sema3a expression in a dose - dependent fashion ( C ) . Data represent values , with mean ± ( n = 4 ) ; * * P < 0 . 01 * * * P < 0 . 001 by one - way ANOVA with Bonferroni multiple comparisons , after data normalization by logarithmic transformation ; Sema3A expression upon VEGF stimulation : 40 versus 0 P = 0 . 0041 ; TGF - β1 upon VEGF stimulation : no significant differences ; Sema3A expression upon TGF - β1 stimulation : 1 versus 0 P < 0 . 0001 ; 2 . 5 versus 0 P < 0 . 0001 ; 40 versus 0 P < 0 . 0001 .

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A-C Endothelial Sema3A expression is directly inhibited by VEGF and stimulated by TGF-β1 in vitro. Mouse aortic endothelial cells were stimulated with increasing doses of recombinant VEGF164 or TGF-β1 for 24 h. The expression of Sema3a and Tgfb1 was quantified by qRT-PCR and expressed as fold-change versus the non-stimulated cells. VEGF dose dependently inhibited Sema3a expression (A), but did not affect that of Tgfb1 (B), while TGF-β1 upregulated Sema3a expression in a dose-dependent fashion (C). Data represent individual values, with mean ± SEM (n = 4); **P < 0.01 and ***P < 0.001 by one-way ANOVA with Bonferroni multiple comparisons test, after data normalization by logarithmic transformation; Sema3A expression upon VEGF stimulation: 40 versus 0 P = 0.0041; TGF-β1 upon VEGF stimulation: no significant differences; Sema3A expression upon TGF-β1 stimulation: 1 versus 0 P < 0.0001; 2.5 versus 0 P < 0.0001; 40 versus 0 P < 0.0001.


D , E Inhibition of TGF - β1 abolishes Sema3A production NEM recruitment in vivo . Mice , implanted with control ( Ctrl ) or myoblasts expressing low ( V Low ) VEGF , were treated with a TGF - β1 blocking antibody . Analyses were preformed after 1 week . ( D ) Immunohistochemistry for Sema3A on frozen muscle sections with H & amp ; amp ; E counterstaining . of Sema3A stain was quantified as reciprocal on a scale out of 250 . Scale bar = 50 μm . Data represent mean ± of ( n ) acquired from three / group : No stain , n = 10 ; Ctrl IgG1 , n = 17 ; Ctrl AbαTGF - β1 , n = 16 ; V Low IgG1 , n = 23 ; V Low AbαTGF - β1 , n = 28 . * * P < 0 . 01 * * * P < 0 . 001 by Kruskal - Wallis with Dunn ' s multiple comparisons : V Low IgG1 versus No stain P = 0 . 0069 ; V Low IgG1 versus Ctrl IgG1 P < 0 . 0001 ; V Low AbαTGF - β1 versus V Low IgG1 P = 0 . 0002 .

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D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (D) Immunohistochemistry for Sema3A protein on frozen muscle sections with H&amp;E counterstaining. The intensity of Sema3A stain was quantified as reciprocal intensity on a scale out of 250. Scale bar = 50 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: No stain, n = 10; Ctrl IgG1, n = 17; Ctrl AbαTGF-β1, n = 16; V Low IgG1, n = 23; V Low AbαTGF-β1, n = 28. **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus No stain P = 0.0069; V Low IgG1 versus Ctrl IgG1 P < 0.0001; V Low AbαTGF-β1 versus V Low IgG1 P = 0.0002.


D , E Inhibition of TGF - β1 abolishes Sema3A production NEM recruitment in vivo . Mice , implanted with control ( Ctrl ) or myoblasts expressing low ( V Low ) VEGF , were treated with a TGF - β1 blocking antibody . Analyses were preformed after 1 week . ( E ) of recruited NEM ( number of CD11b + / mm2 of ) . Scale bar = 25 μm . Data represent mean ± of ( n ) acquired from three / group : Ctrl IgG1 , n = 20 ; Ctrl AbαTGF - β1 , n = 19 ; V Low IgG1 , n = 20 ; V Low AbαTGF - β1 , n = 27 ; * * * P < 0 . 001 by Kruskal - Wallis with Dunn ' s multiple comparisons : V Low IgG1 versus Ctrl IgG1 P = 0 . 0005 ; V Low AbαTGF - β1 versus V Low IgG1 P < 0 . 0001 .

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D, E Inhibition of TGF-β1 abolishes Sema3A production and NEM recruitment in vivo. Mice, implanted with control cells (Ctrl) or myoblasts expressing low (V Low) VEGF levels, were treated with a TGF-β1 blocking antibody. Analyses were preformed after 1 week. (E) Quantification of recruited NEM (number of CD11b+ cells/mm2 of area). Scale bar = 25 μm. Data represent the mean ± SEM of individual images (n) acquired from three muscles/group: Ctrl IgG1, n = 20; Ctrl AbαTGF-β1, n = 19; V Low IgG1, n = 20; V Low AbαTGF-β1, n = 27; ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low IgG1 versus Ctrl IgG1 P = 0.0005; V Low AbαTGF-β1 versus V Low IgG1 P < 0.0001.


A - C Mice , implanted with myoblasts expressing low ( V Low ) or medium ( V Med ) VEGF , were treated on days 4 6 after implantation with intramuscular injections of Sema3A - Fc ( 0 . 1 or 10 mg / kg of average muscle tissue ) , or with control Fc . ( A ) of number of NEM / cm of vessel in sites of new angiogenesis after 1 week . Data represent mean ± of ( n ) acquired from 3 to 5 / group : V Low Fc , n = 21 ; V Low 0 . 1 mg / kg , n = 22 ; V Low 10 mg / kg , n = 28 ; V Med Fc , n = 20 ; V Med 0 . 1 mg / kg , n = 30 ; V Med 10 mg / kg , n = 25 ; * * P < 0 . 01 * * * P < 0 . 001 by Kruskal - Wallis with Dunn ' s multiple comparisons : V Low Fc versus V Low 10 mg / kg P = 0 . 0027 ; V Med Fc versus V Med 0 . 1 mg / kg P = 0 . 001 ; V Med Fc versus V Med 10 mg / kg P < 0 . 0001 . ( B ) of vessel density ( VLD ) in treated after 1 week . Data represent mean ± of ( n ) acquired from 3 to 4 / group : V Low Fc , n = 8 ; V Low 0 . 1 mg / kg , n = 20 ; V Low 10 mg / kg , n = 18 ; V Med Fc , n = 8 ; V Med 0 . 1 mg / kg , n = 23 ; V Med 10 mg / kg , n = 27 . Data were subjected to Kruskal - Wallis with Dunn ' s multiple comparisons , no significant differences were detected . ( C ) Vascular stabilization rate was determined 2 after implantation by measuring vessel density after abrogation of VEGF signaling by VEGF - Trap . Treatment with 10 mg / kg Sema3A - Fc did not affect stabilization of angiogenesis induced by low VEGF , but significantly increased resistant fraction of vessels induced by medium VEGF . Data represent mean ± of ( n ) acquired from 3 to 4 / group : V Low Fc , n = 32 ; V Low Sema3A , n = 14 ; V Med Fc , n = 28 ; V Med Sema3A , n = 33 ; * P < 0 . 05 by one - way ANOVA with Bonferroni multiple comparisons : V Med Fc versus V Med Sema3A P = 0 . 0458 .

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A-C Mice, implanted with myoblasts expressing low (V Low) or medium (V Med) VEGF levels, were treated on days 4 and 6 after cell implantation with intramuscular injections of Sema3A-Fc (0.1 or 10 mg/kg of average muscle tissue weight), or with control Fc protein. (A) Quantification of the number of NEM/cm of vessel length in sites of new angiogenesis after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 5 muscles/group: V Low Fc, n = 21; V Low 0.1 mg/kg, n = 22; V Low 10 mg/kg, n = 28; V Med Fc, n = 20; V Med 0.1 mg/kg, n = 30; V Med 10 mg/kg, n = 25; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: V Low Fc versus V Low 10 mg/kg P = 0.0027; V Med Fc versus V Med 0.1 mg/kg P = 0.001; V Med Fc versus V Med 10 mg/kg P < 0.0001. (B) Quantification of vessel length density (VLD) in treated muscles after 1 week. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 8; V Low 0.1 mg/kg, n = 20; V Low 10 mg/kg, n = 18; V Med Fc, n = 8; V Med 0.1 mg/kg, n = 23; and V Med 10 mg/kg, n = 27. Data were subjected to Kruskal-Wallis analysis with Dunn's multiple comparisons test, and no significant differences were detected. (C) Vascular stabilization rate was determined 2 weeks after cell implantation by measuring vessel length density after abrogation of VEGF signaling by VEGF-Trap. Treatment with 10 mg/kg Sema3A-Fc did not affect the stabilization of angiogenesis induced by low VEGF, but significantly increased the resistant fraction of vessels induced by medium VEGF. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: V Low Fc, n = 32; V Low Sema3A, n = 14; V Med Fc, n = 28; V Med Sema3A, n = 33; *P < 0.05 by one-way ANOVA with Bonferroni multiple comparisons test: V Med Fc versus V Med Sema3A P = 0.0458.


D - F Mice received intramuscular injections of adenovirus expressing VEGF ( AdVIC ) or CD8 control ( AdCD8 ) were treated on days 4 6 after vector delivery with intramuscular injections of Sema3A - Fc ( 10 mg / kg of average muscle tissue ) or control Fc . ( D ) Immunofluorescence of endothelium ( CD31 , in red ) , pericytes ( NG2 , in green ) , smooth muscle ( α - SMA , in cyan ) , nuclei ( DAPI , in blue ) , showing vessel density 1 week after injection of adenoviral vectors alone ( no treatment ) or after 3 with Fc or Sema3A - Fc treatment . Scale bar = 50 μm . ( E ) of vessel density ( VLD ) on same samples shows that treatment with Sema3A - Fc accelerated stabilization of vessels induced by transient uncontrolled VEGF expression . Data represent mean ± of ( n ) acquired from 3 to 4 / group : Uninjected , n = 72 ; AdCD8 1 week , n = 68 ; AdVIC 1 week , n = 53 ; AdVIC + Fc 3 , n = 78 ; AdVIC + Sema3AFc 3 , n = 91 ; * * P < 0 . 01 * * * P < 0 . 001 by Kruskal - Wallis with Dunn ' s multiple comparisons : AdCD8 1 week versus AdVIC + Fc 3 P = 0 . 0037 ; other comparisons indicated P < 0 . 0001 .

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D-F Mice received intramuscular injections of adenovirus expressing VEGF (AdVIC) or CD8 control (AdCD8) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A-Fc (10 mg/kg of average muscle tissue weight) or control Fc protein. (D) Immunofluorescence staining of endothelium (CD31, in red), pericytes (NG2, in green), smooth muscle cells (α-SMA, in cyan), and nuclei (DAPI, in blue), showing vessel density and morphology 1 week after injection of adenoviral vectors alone (no treatment) or after 3 weeks and with Fc or Sema3A-Fc treatment. Scale bar = 50 μm. (E) Quantification of vessel length density (VLD) on the same samples shows that treatment with Sema3A-Fc accelerated stabilization of vessels induced by transient and uncontrolled VEGF expression. Data represent the mean ± SEM of individual images (n) acquired from 3 to 4 muscles/group: Uninjected muscles, n = 72; AdCD8 1 week, n = 68; AdVIC 1 week, n = 53; AdVIC+Fc 3 weeks, n = 78; AdVIC+Sema3AFc 3 weeks, n = 91; **P < 0.01 and ***P < 0.001 by Kruskal-Wallis analysis with Dunn's multiple comparisons test: AdCD8 1 week versus AdVIC+Fc 3 weeks P = 0.0037; all other comparisons indicated P < 0.0001.


D - F Mice received intramuscular injections of adenovirus expressing VEGF ( AdVIC ) or CD8 control ( AdCD8 ) were treated on days 4 6 after vector delivery with intramuscular injections of Sema3A - Fc ( 10 mg / kg of average muscle tissue ) or control Fc . ( F ) Immunofluorescence of endothelial ( CD31 , in red ) NEM ( CD11b , in green ) on frozen sections of 3 after injection of adenoviral vectors with Fc or Sema3A - Fc treatment . arrows indicate NEM . Scale bar = 50 μm .

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D-F Mice received intramuscular injections of adenovirus expressing VEGF (AdVIC) or CD8 control (AdCD8) and were treated on days 4 and 6 after vector delivery with intramuscular injections of Sema3A-Fc (10 mg/kg of average muscle tissue weight) or control Fc protein. (F) Immunofluorescence staining of endothelial cells (CD31, in red) and NEM (CD11b, in green) on frozen sections of muscles 3 weeks after injection of adenoviral vectors and with Fc or Sema3A-Fc treatment. White arrows indicate NEM. Scale bar = 50 μm.


A , Representative of controls from siRNA screen for HU - induced RPA accumulation . GFP - RPA1 RFP - PCNA reporter were transfected with siRNAs 48 hrs later treated with HU ( 3 mM ) for 2 hrs prior to pre - extraction , fixation imaging .

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A, Representative images of controls from the siRNA screen for HU-induced RPA accumulation. GFP-RPA1 and RFP-PCNA reporter cells were transfected with siRNAs and 48 hrs later treated with HU (3 mM) for 2 hrs prior to pre-extraction, fixation and imaging.


B , Gene ranking on siRNA scores from two independent screens . 219 genes were targeted by three siRNAs ranked on P - values derived from RSA Fisher ' s . Selected hits are highlighted .

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B, Gene ranking based on siRNA scores from two independent screens. 219 genes were targeted by three individual siRNAs and ranked based on P-values derived from RSA and Fisher's analysis. Selected hits are highlighted.


C , Validation of selected genes by single of GFP - RPA1 in pre - extracted . Only S - phase positive for RFP - PCNA were analysed . a , b , c denote independent siRNAs ; Ctrl , Control . Median with interquartile range of relative per is shown , n > 4000 . Mann - Whitney : * * * p < 10 - 4 , n . s . non - significant . One representative experiment out of two biological replicas is shown .

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C, Validation of selected genes by single cell analysis of GFP-RPA1 intensity in pre-extracted cells. Only S-phase cells positive for RFP-PCNA were analysed. a, b, c denote independent siRNAs; Ctrl, Control. Median with interquartile range of relative intensity per cell is shown, n > 4000. Mann-Whitney: *** p < 10-4, n.s. non-significant. One representative experiment out of two biological replicas is shown.


D , of synthesis rate . were pulsed 15 min with EdU EdU was analysed in S - phase positive for RFP - PCNA . EdU incorporation is shown relative to siCtrl .

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D, Analysis of DNA synthesis rate. Cells were pulsed 15 min with EdU and EdU intensity was analysed in S-phase cells positive for RFP-PCNA. EdU incorporation is shown relative to siCtrl.


A , replication measured by EdU incorporation . RPF - PCNA reporter were transfected with indicated siRNAs pulsed with EdU for 20 min . S - phase positive for RFP - PCNA were analysed . n > 150 . Error bars , SD ; n = 3 biological replicas .

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A, DNA replication measured by EdU incorporation. RPF-PCNA reporter cells were transfected with indicated siRNAs and pulsed with EdU for 20 min. S-phase cells positive for RFP-PCNA were analysed. n > 150. Error bars, SD; n= 3 biological replicas.


B , Complementation of synthesis . Stable lines expressing BRPF3 resistant to BRPF3 / a siRNA control ( lacZ - V5 ) were siRNA transfected analysed for EdU incorporation as in A . Error - bars , SD ; n = 6 biological replicas . Two - t , * * p < 10 - 2 .

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B, Complementation analysis of DNA synthesis. Stable cell lines expressing BRPF3 resistant to the BRPF3/a siRNA and control (lacZ-V5) were siRNA transfected and analysed for EdU incorporation as in A. Error-bars, SD; n = 6 biological replicas. Two-tailed t test, ** p<10-2.


C , Comparison of native BRPF1 BRPF3 complexes purified from K562 . ( left ) mass - spec ( spectral / total peptides identified ) ( right ) verification of associated by western blot .

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C, Comparison of native BRPF1 and BRPF3 complexes purified from K562 cells. (left) mass-spec analysis (spectral counts/total peptides identified) and (right) verification of associated protein by western blot.


D , Immunoprecipitation of BRPF3 deletion mutants . One representative experiment out of two is shown .

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D, Immunoprecipitation analysis of BRPF3 deletion mutants. One representative experiment out of two is shown.


E , Histone acetyltransferase ( HAT ) with purified BRPF1 / 3 complexes on free histones mononucleosomes .

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E, Histone acetyltransferase assay (HAT) with purified BRPF1/3 complexes on free histones and mononucleosomes.


F , HAT with purified BRPF1 / 3 complexes on H3 peptides unmodified or acetylated ( ac ) / methylated ( me ) at K14 K27 , respectively .

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F, HAT assay with purified BRPF1/3 complexes on H3 peptides unmodified or acetylated (ac)/methylated (me) at K14 and K27, respectively.


G , of histone acetylation in BRPF3 depleted . ( left ) Western blot of siRNA treated U - 2 - OS . TSA treatment ( 1 hr ) was included as a positive control . 2x , double amount of extract loaded in 1x . ( right ) H3K14 acetylation quantified relative to total H3 . Error bars , SD ; n = 3 biological replicates .

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G, Analysis of histone acetylation in BRPF3 depleted cells. (left) Western blot of siRNA treated U-2-OS cells. TSA treatment (1 hr) was included as a positive control. 2x, double amount of extract loaded in 1x. (right) H3K14 acetylation levels quantified relative to total H3. Error bars, SD; n = 3 biological replicates.


H , Complementation of H3K14ac . Total extracts of siRNA treated lacZ - V5 BRPF3 - V5 ( siBRPF3 / a resistant ) expressing were analysed by western blotting . One representative experiment out of four biological replicas is shown .

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H, Complementation analysis of H3K14ac. Total cell extracts of siRNA treated lacZ-V5 and BRPF3-V5 (siBRPF3/a resistant) expressing cells were analysed by western blotting. One representative experiment out of four biological replicas is shown.


A - B , Single - molecule of replication by combing of siRNA transfected - labelled with IdU ( 10 min ) CIdU ( 20 min ) . ( A ) Distribution of inter - track distances . Bars represent median . n > 100 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . ( B ) distribution of CldU track . Bars represent median . n > 300 . Mann - Whitney : * * * P < 10 - 3 . One representative experiment out of two biological replicas is shown . C , Distribution of intertrack distances measured by IdU / CIdU labelling fiber . Bars represent median . n > 110 . Mann - Whitney : * * * * P < 10 - 4 . One representative experiment out of two biological replicas is shown .

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A-B, Single-molecule analysis of DNA replication by DNA combing of siRNA transfected cells pulse-labelled with IdU (10 min) and CIdU (20 min). (A) Distribution of inter-track distances. Bars represent the median. n > 100. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. (B) Size distribution of CldU track length. Bars represent the median. n > 300. Mann-Whitney: *** P < 10-3. One representative experiment out of two biological replicas is shown. C, Distribution of intertrack distances measured by IdU/CIdU pulse labelling and DNA fiber analysis. Bars represent the median. n > 110. Mann-Whitney: **** P < 10-4. One representative experiment out of two biological replicas is shown.


D , Scatter plot of - bound GFP - RPA1 per . GFP - RPA1 RFP - PCNA reporter were transfected with indicated siRNAs treated 2 hrs with UCN - 01 ( 300 nM ) prior to pre - extraction fixation . GFP - RPA1 were measured in PCNA positive . Lines represent medians . n > 150 . t - : * * * * P < 10 - 4 , * P < 0 . 05 ; a . u . , arbitrary unit .

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D, Scatter plot of chromatin-bound GFP-RPA1 intensity per cell. GFP-RPA1 and RFP-PCNA reporter cells were transfected with indicated siRNAs and treated 2 hrs with UCN-01 (300 nM) prior to pre-extraction and fixation. GFP-RPA1 levels were measured in PCNA positive cells. Lines represent medians. n > 150. t-tests: **** P < 10-4, * P < 0.05; a.u., arbitrary unit.


E , of replication factor loading . Soluble from siRNA transfected treated as in D where removed by CSK - T extraction pellets analysed by western blotting . One representative experiment out of three independent biological replicas is shown . F , of - bound MCM2 ( left ) CDC45 ( right ) from three independent experiments described in ( E ) . MCM2 CDC45 were normalized to histone H4 shown relative to untreated control . t - : * * * P < 10 - 3 , * * P < 0 . 01 , * P < 0 . 05 , n . s . non - significant .

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E, Analysis of replication factor loading. Soluble proteins from siRNA transfected cells treated as in D where removed by CSK-T extraction and chromatin pellets analysed by western blotting. One representative experiment out of three independent biological replicas is shown. F, Quantification of chromatin-bound MCM2 (left) and CDC45 (right) from the three independent experiments described in (E). MCM2 and CDC45 levels were normalized to histone H4 levels and shown relative to untreated control. t-tests: *** P < 10-3, ** P < 0.01, * P < 0.05, n.s. non-significant.


G , of - bound in HBO1 depleted as in E . One representative experiment out of two biological replicas is shown .

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G, Analysis of chromatin-bound proteins in HBO1 depleted cells as in E. One representative experiment out of two biological replicas is shown.


A , ChIP - seq of BRPF3 binding on human genome . BRPF3 enrichment signal obtained by ChIP - seq with asynchronous ( AS ) , synchronized ( S ) released into HU ( S + HU ) RKO , on MCM4 ( left ) Lamin B2 ( right ) replication origins . Signals were compared to published enrichment signals for ORC1 in HeLa ( ( Dellino et al , 2013 ) , GSE37583 ) , HBO1 in RKO ( Avvakumov et al . , 2012 , GSE33221 ) , H3K14ac in IMR90 ( GSM521881 ) . negative control IgG signal shown is from S + HUchromatin .

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A, ChIP-seq analysis of BRPF3 binding on the human genome. BRPF3 enrichment signal obtained by ChIP-seq with asynchronous (AS), synchronized (S) and released into HU (S+HU) RKO cells, on the MCM4 (left) and Lamin B2 (right) replication origins. Signals were compared to the published enrichment signals for ORC1 in HeLa cells ((Dellino et al, 2013), GSE37583), HBO1 in RKO cells (Avvakumov et al., 2012, GSE33221), and H3K14ac in IMR90 cells (GSM521881). The negative control IgG signal shown is from the S+HUchromatin.


B , Venn diagrams illustrating overlap of genome binding sites between ( top ) BRPF3 , HBO1 ORC1 , ( bottom ) BRPF3 , BRPF1 / 2 ORC1 in human .

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B, Venn diagrams illustrating the overlap of genome binding sites between (top) BRPF3, HBO1 and ORC1, and (bottom) BRPF3, BRPF1/2 and ORC1 in human cells.


C , Heatmaps of BRPF3 , HBO1 H3K14ac ChIP - seq signal on ± 5kb surrounding TSS of genes . Genes were sorted by BRPF3 abundance from high to low . A . U . arbitrary unit .

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C, Heatmaps of BRPF3, HBO1 and H3K14ac ChIP-seq signal on ± 5kb surrounding the TSS of genes. Genes were sorted by BRPF3 abundance from high to low. A.U. arbitrary unit.


D , Average profiles of BRPF3 H3K14ac ChIP - seq signal on ± 2 . 5kb around centre of ( top ) BRPF3 ( bottom ) ORC1 genome binding sites .

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D, Average profiles of BRPF3 and H3K14ac ChIP-seq signal on ± 2.5kb around the centre of (top) BRPF3 and (bottom) ORC1 genome binding sites.


E , ChIP - qPCR of H3K14ac on well - characterized replication origins . H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated . Error bars , range ; n = 2 biological replicas .

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E, ChIP-qPCR analysis of H3K14ac on well-characterized replication origins. H3K14ac enrichments measured relative to IgG control are shown relative to control siRNA treated cells. Error bars, range; n= 2 biological replicas.


F , Expression of replication factors were not altered by lack of BRPF3 . Each point represents expression of a gene in control BRPF3 depleted ( GSE65065 ) . Replication factors were classified as described previously ( Alabert et al , 2014 ) . Total genes ( grey ) , replication factors ( colors ) BRPF3 ( black ) expression are shown . Continuous black lines mark 1 . 5 fold change in expression .

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F, Expression levels of DNA replication factors were not altered by the lack of BRPF3. Each point represents the expression levels of a gene in control and BRPF3 depleted cells (GSE65065). Replication factors were classified as described previously (Alabert et al, 2014). Total genes (grey), DNA replication factors (colors) and BRPF3 (black) expression levels are shown. Continuous black lines mark 1.5 fold change in expression levels.


A , Western blot of damage signalling in BRPF3 depleted . siRNA transfected U - 2 - OS were treated with HU for 2 h or 24 h . One representative experiment out of two biological experiments is shown .

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A, Western blot of DNA damage signalling in BRPF3 depleted cells. siRNA transfected U-2-OS cells were treated with HU for 2 h or 24 h. One representative experiment out of two biological experiments is shown.


B - C , Recovery from replication stress . siRNA treated were treated for 24 hrs with HU ( 0 h ) , released into normal medium for indicated times ( see also Fig . S6A , B ) . ( B ) replication rate measured by EdU incorporation . were pulsed with EdU for 15 min gated as shown in Figure S6A to analyse EdU positive . n > 1000 . Mann - Whitney : * * * * P < 10 - 4 . ( C ) cycle progression measured by FACS of content . One representative experiment out of two independent biological replicas is shown .

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B-C, Recovery from replication stress. siRNA treated cells were treated for 24 hrs with HU (0 h), and released into normal medium for the indicated times (see also Fig. S6A, B). (B) DNA replication rate measured by EdU incorporation. Cells were pulsed with EdU for 15 min and gated as shown in Figure S6A to analyse EdU positive cells. n > 1000. Mann-Whitney: **** P < 10-4. (C) Cell cycle progression measured by FACS analysis of DNA content. One representative experiment out of two independent biological replicas is shown.


A549 stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( A ) were incubated for 2 hours in presence or absence of 50 nM of lysosomal inhibitor chloroquine ( CQ ) . lysates were evaluated by immunoblotting .

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A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (A) Cells were incubated for 2 hours in the presence or absence of 50 nM of lysosomal inhibitor chloroquine (CQ). Cell lysates were evaluated by immunoblotting.


A549 stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( B ) were infected , for 1 4 hours , with K . pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment . lysates were plated in LB agar for 18 hours CFU were determined . Data information : triangles dots represent ( three technical replicates per ) on control Atg7 knock down . bars represent mean CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . Statistical significance between assays were calculated using two - student ' s t with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (B) Cells were infected, for 1 and 4 hours, with K. pneumonia at MOI of 10 followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


A549 stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated . ( D - E ) were infected , for 1 - 4 hours , with WT ( D ) or ΔpscD P . aeruginosa ( E ) at MOI of 10 . infections were followed by 1 hour of gentamycin treatment . lysates were plated in LB agar for 18 hours CFU were determined . Data information : , triangles dots represent ( three technical replicates per ) on control Atg7 knock down . bars represent mean CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . Statistical significance between assays were calculated using two - student ' s t with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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A549 cells stably expressing lentivirus vector containing random shRNA or Atg7 specific shRNA were generated. (D-E) Cells were infected, for 1-4 hours, with WT (D) or ΔpscD P. aeruginosa (E) at MOI of 10. All infections were followed by 1 hour of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: , the triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells. The bars represent the mean CFU of each group (three biological replicates each group), error bars represent standard deviation. Statistical significance between assays were calculated using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


( A ) A549 were infected with WT P . aeruginosa for various time points lysates were evaluated by immunoblotting .

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(A) A549 cells were infected with WT P. aeruginosa for various time points and cell lysates were evaluated by immunoblotting.


( B ) A549 infected with WT or ΔpscD P . aeruginosa for 4 hours or left uninfected ( NI ) in presence of lysosomal inhibitor CQ ( 50 nM ) . lysates were evaluated by immunoblotting .

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(B) A549 cells infected with WT or ΔpscD P. aeruginosa for 4 hours or left uninfected (NI) in the presence of lysosomal inhibitor CQ (50 nM). The cell lysates were evaluated by immunoblotting.


( C ) A549 stably expressing GFP - LC3 were left non - infected or infected , for 4 hours , with WT or ΔpscD P . aeruginosa , evaluated by microscopy . nuclei were counterstained with DAPI ( blue ) autophagosome were visualized as green . Graph represents quantitative of GFP - LC3 positive autophagosome per , scale bars : 10 µm . Each bar represents mean value of three independent experiments , error bars represent standard deviation . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison . * * p ≤ 0 . 01 .

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(C) A549 cells stably expressing GFP-LC3 were left non-infected or infected, for 4 hours, with WT or ΔpscD P. aeruginosa, and evaluated by fluorescence microscopy. Cell nuclei were counterstained with DAPI (blue) and autophagosome were visualized as green puncta. Graph represents quantitative analysis of GFP-LC3 positive autophagosome per cell, scale bars: 10 µm. Each bar represents mean value of three independent experiments, error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. **p ≤ 0.01.


( D - E ) A549 were treated for 16 hours with rapamycin 0 . 8 μg / ml or DMSO , then infected with either WT ( D ) or ΔpscD P . aeruginosa followed by 1 hour of gentamycin treatment ( E ) . circles squares represent ( three technical replicates per ) with DMSO rapamycin treatment . bars represent means of CFU from three independent experiments , error bars represent standard deviation . significance of differences between different drug treatment was determined using two - student ' s t with Welch ' s correction . NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(D-E) A549 cells were treated for 16 hours with rapamycin 0.8 μg/ml or DMSO, then infected with either WT (D) or ΔpscD P. aeruginosa and followed by 1 hour of gentamycin treatment (E). The circles and squares represent the individual test (three technical replicates per individual test) with DMSO and rapamycin treatment. The bars represent the means of CFU from three independent experiments, error bars represent standard deviation. The significance of differences between different drug treatment was determined using two-tailed student's t test with Welch's correction. NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


A549 were infected with one of several P . aeruginosa mutants . lysates were evaluated by immunoblotting .

A549 cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.


( B ) human bronchial epithelial were infected with one of several P . aeruginosa mutants . lysates were evaluated by immunoblotting .

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(B) primary human bronchial epithelial cells were infected with one of several P. aeruginosa mutants. Cell lysates were evaluated by immunoblotting.


( C ) A549 , stably expressing GFP - LC3 , were infected with WT or P . aeruginosa mutants evaluated by microscopy , scale bars : 10 µm . GFP - LC3 representing autophagosomes was quantified presented in graph . Each bar represents mean value from three independent experiments error bars represent standard deviation . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 . P . aeruginosa mutant strains legend : ΔSTY ( mutant deficient for three cytotoxins ) , WT ( contains cytotoxin ) , ΔS ( deficient for ExoS but still express ExoT ExoY ) , ΔTY ( express ExoS but deficient for ExoT ExoY ) .

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(C) A549 cells, stably expressing GFP-LC3, were infected with WT or P. aeruginosa mutants and evaluated by fluorescence microscopy, scale bars: 10 µm. GFP-LC3 puncta representing the autophagosomes was quantified and presented in the graph. Each bar represents mean value from three independent experiments and error bars represent standard deviation. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01. P. aeruginosa mutant strains legend: ΔSTY (mutant deficient for the three cytotoxins), WT (contains all cytotoxin), ΔS (deficient for ExoS but still express ExoT and ExoY), ΔTY (express ExoS but deficient for ExoT and ExoY).


( D , E ) A549 , stably expressing GFP - LC3 , were infected for 4 hours with WT or with one of P . aeruginosa mutants ( ΔSTY , ΔS ΔTY ) evaluated by microscopy ( D ) . P . aeruginosa were visualized using specific antibody ( red ) colocalized signals with GFP - LC3 ( green ) were merged presented as yellow . Scale bars : 10 µm . ( E ) Quantitative of co - localization of internalized P . aeruginosa with GFP - LC3 positive . colocalization of these two signals was quantified with 10 independent visual fields of more than 100 . Data represent mean ± SD of three independent experiments . Statistical significance between assays was determined using One - way ANOVA followed by Dunn ' s Multiple Comparison . * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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(D,E) A549 cells, stably expressing GFP-LC3, were infected for 4 hours with WT or with one of P. aeruginosa mutants (ΔSTY, ΔS and ΔTY) and evaluated by fluorescence microscopy (D). P. aeruginosa were visualized using specific antibody (red) and the colocalized bacteria signals with GFP-LC3 puncta (green) were merged and presented as yellow. Scale bars: 10 µm. (E) Quantitative analysis of co-localization of internalized P. aeruginosa with GFP-LC3 positive puncta. The colocalization of these two signals was quantified with 10 independent visual fields of more than 100 cells. Data represent mean ± SD of three independent experiments. Statistical significance between assays was determined using One-way ANOVA followed by Dunn's Multiple Comparison Test. *p ≤ 0.05; **p ≤ 0.01.


( F ) Representative transmission electron microscopy of A549 infected with P . aeruginosa containing ExoS ( ΔTY ) or deficient for three T3SS effector ( ΔSTY ) for 4 hours . ΔSTY P . aeruginosa ( star ) was engulfed by multiple autophagosomes ( black arrow ) . ΔTY P . aeruginosa ( star ) were found in cytoplasm with single surrounded , scale bars : 0 . 5 µm .

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(F) Representative transmission electron microscopy images of A549 cells infected with P. aeruginosa containing ExoS (ΔTY) or deficient for the three T3SS effector proteins (ΔSTY) for 4 hours. ΔSTY P. aeruginosa (white star) was engulfed by multiple membrane autophagosomes (black arrow). ΔTY P. aeruginosa (white star) were found in cytoplasm with single membrane surrounded, scale bars: 0.5 µm.


( G ) A549 , stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA , were infected with WT or P . aeruginosa mutants ( ΔSTY , ΔTY ΔS ) at MOI of 10 . infections were followed by 1 hours of gentamycin treatment . lysates were plated in LB agar for 18 hours CFU were determined . Data information : triangles dots represent ( three technical replicates per ) on control Atg7 knock down from three biological replicates . bars represent means of CFU of each group ( three biological replicates each group ) , error bars represent standard deviation . significance of differences between different was determined using two - student ' s t with Welch ' s correction , NS : not significant ; * * p ≤ 0 . 01 .

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(G) A549 cells, stably expressing lentiviral vectors containing random shRNA or Atg7 shRNA, were infected with WT or P. aeruginosa mutants (ΔSTY, ΔTY and ΔS) at MOI of 10. All infections were followed by 1 hours of gentamycin treatment. Cell lysates were plated in LB agar for 18 hours and CFU were determined. Data information: The triangles and dots represent the individual test (three technical replicates per individual test) on control and Atg7 knock down cells from three biological replicates. The bars represent the means of CFU of each group (three biological replicates each group), error bars represent standard deviation. The significance of differences between different assay was determined using two-tailed student's t test with Welch's correction, NS: not significant; **p ≤ 0.01.


( A ) airway epithelial were isolated from tracheas of Atg7 + / + Atg7 - / - mouse were evaluated by immunoblotting .

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(A) Primary airway epithelial cells were isolated from tracheas of Atg7+/+ and Atg7-/- mouse and were evaluated by immunoblotting.


( B ) Sections of tracheas from Atg7 + / + Atg7 - / - mouse were evaluated by immunofluorescence microscopy following of with DAPI ( Blue ) , ciliated epithelial marker β - tubulin IV ( green ) Atg7 ( red ) . Scale bars : 20 µm .

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(B) Sections of tracheas from Atg7+/+ and Atg7-/- mouse were evaluated by immunofluorescence microscopy following staining of nuclear DNA with DAPI (Blue), ciliated epithelial cell marker β-tubulin IV (green) and Atg7 (red). Scale bars: 20 µm.


( C , D ) Atg7 + / + Atg7 - / - mice were intranasally injected with 3 x107 CFU of WT , ΔS , ΔTY or ΔSTY P . aeruginosa . After 24 hours , lungs ( C ) , spleens livers ( D ) were harvested , homogenized diluted for CFU counting . Each data point represents ( Log10 CFU / g ) from mouse . horizontal lines represent mean value from each group ( 6 mice each group ) , error bars represent standard deviation . significance of differences between different drug treatment was determined using One - way ANOVA Dunn ' s multiple comparisons . , NS : not significant ; * p ≤ 0 .

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(C,D) Atg7+/+ and Atg7-/- mice were intranasally injected with 3 x107 CFU of WT, ΔS, ΔTY or ΔSTY P. aeruginosa. After 24 hours, lungs (C), spleens and livers (D) were harvested, homogenized and diluted for CFU counting. Each data point represents bacterial count (Log10 CFU/g) from individual mouse. The horizontal lines represent the mean value from each group (6 mice each group), error bars represent standard deviation. The significance of differences between different drug treatment was determined using One-way ANOVA and Dunn's multiple comparisons test., NS: not significant; *p ≤ 0.05


( E , F ) Atg7 + / + Atg7 - / - mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY ( E ) ΔSTY ( F ) . Mice survival was monitored for 10 days post infection . Kaplan - Meyer survival curves were made on observation of 12 - 18 mice / group from two independent experiments . P - values were calculated using log - rank ( ΔTYAtg7 + / + vs ΔTYAtg7 - / - , P = 0 . 2186 ; ΔSTYAtg7 + / + vs ΔSTYAtg7 - / - , P = 0 . 0145 ) .

O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,F) Atg7+/+ and Atg7-/- mice were intranasally injected with PBS with or without 1 x109 CFU of ΔTY (E) ΔSTY (F). Mice survival was monitored for 10 days post infection. Kaplan-Meyer survival curves were made based on the observation of 12-18 mice/group from two independent experiments. The P-values were calculated using the log-rank test (ΔTYAtg7+/+ vs ΔTYAtg7-/-, P = 0.2186; ΔSTYAtg7+/+ vs ΔSTYAtg7-/-, P=0.0145).


( A A549 were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in GTPase activating domain ( SG - A + ) , in ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . lysates were evaluated by immunoblotting .

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(A A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( B ) A549 were transfected , for 24hours , with a plasmid expressing empty vector ( EV ) , WT GFP - ExoS , a GFP - ExoS mutant for ADPRT domain ( SG + A - ) or a GFP - ExoS mutant deficient in both GAP ADPRT domains ( SG - A - ) . lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24hours, with a plasmid expressing empty vector (EV), WT GFP-ExoS, a GFP-ExoS mutant for ADPRT domain (SG+A-) or a GFP-ExoS mutant deficient in both GAP and ADPRT domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( C ) A549 were treated for 24 hours with 100 µM of ADPRT activity inhibitor MIBG then left uninfected ( NI ) or infected with P . aeruginosa containing ExoS with ADPRT activity only ( SG - A + ) . lysates were evaluated by immunoblotting .

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(C) A549 cells were treated for 24 hours with 100 µM of the ADPRT activity inhibitor MIBG and then left uninfected (NI) or infected with P. aeruginosa containing ExoS with ADPRT activity only (SG-A+). Cell lysates were evaluated by immunoblotting.


A549 were infected for 4 hours with WT or P . aeruginosa mutants ( ΔpscD lysates were evaluated by immunoblotting .

A549 cells were infected for 4 hours with WT or P. aeruginosa mutants (ΔpscD Cell lysates were evaluated by immunoblotting.


A549 were infected for 4 hours with WT or P . aeruginosa mutants STY , ΔS ΔTY ) . lysates were evaluated by immunoblotting .

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A549 cells were infected for 4 hours with WT or P. aeruginosa mutants STY, ΔS and ΔTY). Cell lysates were evaluated by immunoblotting.


F ) A549 were infected for 4 hours with ExoS - deficient P . aeruginosa ( ΔS ) , ExoS containing P . aeruginosa ( SWT ) , P . aeruginosa containing ExoS with loss - of - function mutations in GTPase activating domain ( SG - A + ) , in ADP - ribosylation domain ( SG + A - ) , or in both domains ( SG - A - ) . lysates were evaluated by immunoblotting .

F) A549 cells were infected for 4 hours with ExoS-deficient P. aeruginosa (ΔS), ExoS containing P. aeruginosa (SWT), P. aeruginosa containing ExoS with loss-of-function mutations in the GTPase activating domain (SG-A+), in the ADP-ribosylation domain (SG+A-), or in both domains (SG-A-). Cell lysates were evaluated by immunoblotting.


( A ) A549 were transfected with empty vector ( VE ) vector contains Ras - G12V for 24 hours , followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . lysates were evaluated by immunoblotting .

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(A) A549 cells were transfected with empty vector (VE) and vector contains Ras-G12V for 24 hours, followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( B ) A549 were transfected , for 24 hours , with empty vector ( VE ) vectors contain Ras - G12V or Ras - G12V & R41K followed by a 4 - hours infection with P . aeruginosa ΔSTY or ΔTY . lysates were evaluated by immunoblotting .

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(B) A549 cells were transfected, for 24 hours, with empty vector (VE) and vectors contain Ras-G12V or Ras-G12V&R41K followed by a 4-hours infection with P. aeruginosa ΔSTY or ΔTY. Cell lysates were evaluated by immunoblotting.


( A ) A549 were transfected , for 48 hours , with a mCherry - DFCP1 plasmid then infected , for 4 hours , with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY , SG - A - , SG - A + or SG + A - ) mCherry number per was analyzed by microscopy . Representative immunofluorescence were showed , scale bars represent 10 µm . ( B ) Quantitative of mCherry - DFCP1 by visually more than 100 for each sample . Each bar represents mean value of from three independent experiments error bars represent standard deviation . significance of differences between assays was determined using ANOVA with Dunnett multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 ; * * p ≤ 0 . 01 .

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

(A) A549 cells were transfected, for 48 hours, with a mCherry-DFCP1 plasmid and then infected, for 4 hours, with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY, SG-A-, SG-A+ or SG+A-) and the mCherry puncta number per cell was analyzed by fluorescence microscopy. Representative immunofluorescence images were showed, and scale bars represent 10 µm. (B) Quantitative analysis of mCherry-DFCP1 puncta by visually count more than 100 cells for each sample. Each bar represents mean value of from three independent experiments and error bars represent standard deviation. The significance of differences between assays was determined using ANOVA with Dunnett multiple-comparison posttest, NS: not significant; *p ≤ 0.05; **p ≤ 0.01.


( A A549 , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa ( WT , ΔSTY , ΔS , ΔTY Flag - Atg14L - Vps34 was immunoprecipitated ( IP ) by Flag - antibody magnetic beads evaluated by immunoblotting .

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(A A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa (WT, ΔSTY, ΔS, ΔTY Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.


( B , IP complexes were subjected to in vitro kinase using phosphatidylinositol as substrate . products radiolabeled PI3P were separated by TLC detected by autoradiography . Data are mean ± SD of three independent experiments . significance of differences between different assays was determined using One - way ANOVA followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

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(B, The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


C ) A549 , transfected for 48 hours with vector encoding Flag - Atg14L , were infected with P . aeruginosa , ΔSTY SG - A - , SG - A + or SG + A - ) . Flag - Atg14L - Vps34 was immunoprecipitated ( IP ) by Flag - antibody magnetic beads evaluated by immunoblotting .

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C) A549 cells, transfected for 48 hours with vector encoding Flag-Atg14L, were infected with P. aeruginosa , ΔSTY SG-A-, SG-A+ or SG+A-). Flag-Atg14L-Vps34 complex was immunoprecipitated (IP) by Flag-antibody magnetic beads and evaluated by immunoblotting.


D ) IP complexes were subjected to in vitro kinase using phosphatidylinositol as substrate . products radiolabeled PI3P were separated by TLC detected by autoradiography . Data are mean ± SD of three independent experiments . significance of differences between different assays was determined using One - way ANOVA followed Dunn ' s multiple - comparison posttest , NS : not significant ; * p ≤ 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The IP complexes were subjected to in vitro kinase assay using phosphatidylinositol as substrate. The products radiolabeled PI3P were separated by TLC and detected by autoradiography. Data are mean ± SD of three independent experiments. The significance of differences between different assays was determined using One-way ANOVA analysis followed Dunn's multiple-comparison posttest, NS: not significant; *p ≤ 0.05.


( A ) UBQLN4 associates with polyubiquitinated proteasomal substrates . Flag - tagged UBQLN4 was affinity - purified from extracts of HeLa that were treated with ( + ) or without ( - ) 20 μM MG - 132 for 4 h before harvesting , Flag - precipitates ( IP : Flag ) were blotted with anti - polyubiquitin ( FK2 ) , anti - BAG6 , anti - Flag antibodies . Mock indicates empty vector transfection . Note that UBQLN4 - BAG6 co - precipitation can be observed exclusively in presence of polyubiquitinated substrates .

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(A) UBQLN4 associates with polyubiquitinated proteasomal substrates. Flag-tagged UBQLN4 protein was affinity-purified from extracts of HeLa cells that were treated with (+) or without (-) 20 μM MG-132 for 4 h before harvesting, and the Flag-precipitates (IP:Flag) were blotted with anti-polyubiquitin (FK2), anti-BAG6, and anti-Flag antibodies. Mock indicates empty vector transfection. Note that UBQLN4-BAG6 co-precipitation can be observed exclusively in the presence of polyubiquitinated substrates.


( B ) Polyubiquitinated associated with UBQLN4 are sensitive to translation inhibitor cycloheximide ( CHX ) . HeLa expressing Flag - tagged UBQLN4 were treated with 5 μM MG - 132 as well as CHX ( either 10 or 25 μg / mL ) for 4 h as indicated , then subjected to immunoprecipitation with an anti - Flag antibody as in ( A ) .

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(B) Polyubiquitinated proteins associated with UBQLN4 are sensitive to the translation inhibitor cycloheximide (CHX). HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 μM MG-132 as well as CHX (either 10 or 25 μg/mL) for 4 h as indicated, and then subjected to immunoprecipitation with an anti-Flag antibody as in (A).


( D ) HeLa were treated as in ( C ) , Flag - precipitates were blotted as in ( B ) .

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(D) HeLa cells were treated as in (C), and Flag-precipitates were blotted as in (B).


( A ) UBQLN4 co - localizes with after puromycin treatment . HeLa expressing Flag - tagged UBQLN4 were treated with 5 µg / mL puromycin for 2 h then followed by 1 % Triton X - 100 extraction 4 % paraformaldehyde fixation . Fixed were stained with anti - polyubiquitin FK2 ( shown in red to detect ubiquitin - positive , ALIS ) anti - Flag antibodies ( shown in green ) .

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(A) UBQLN4 co-localizes with cytoplasmic aggregates after puromycin treatment. HeLa cells expressing Flag-tagged UBQLN4 were treated with 5 µg/mL puromycin for 2 h and then followed by 1% Triton X-100 extraction and 4% paraformaldehyde fixation. Fixed cells were stained with anti-polyubiquitin FK2 (shown in red to detect ubiquitin-positive cytoplasmic aggregates, ALIS) and anti-Flag antibodies (shown in green).


( B , C ) Puromycin - treated with UBQLN4 siRNA contain increased number of ALIS . At 72 h after transfection of two distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 # 2 ) or control siRNA ( 5 nM each ) , were treated with 5 µg / mL puromycin for 2 h , then subjected to immunostaining with an anti - polyubiquitin FK2 antibody ( shown in green , B ) as in ( A ) . number of ALIS was determined . quantified data in ( C ) represent mean ± . n = 76 for control siRNA , n = 89 for UBQLN4 siRNA # 1 , n = 81 for UBQLN4 siRNA # 2 . P ‐ values were calculated by Welch ' s t - between siRNA ‐ transfected respective control condition ( * P < 0 . 01 ) .

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(B, C) Puromycin-treated cells with UBQLN4 siRNA contain increased number of ALIS. At 72 h after transfection of the two distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1 and #2) or control siRNA (5 nM each), the cells were treated with 5 µg/mL puromycin for 2 h, and then subjected to immunostaining with an anti-polyubiquitin FK2 antibody (shown in green, B) as in (A). The number of ALIS was determined. The quantified data in (C) represent mean ± SEM. n = 76 cells for control siRNA, n = 89 cells for UBQLN4 siRNA#1, and n = 81 cells for UBQLN4 siRNA #2. P‐values were calculated by Welch's t-test between the siRNA‐transfected and the respective control condition (*P < 0.01).


( D ) Depletion of UBQLN4 makes HeLa more sensitive to puromycin - induced . After 48 h of UBQLN4 siRNA treatment , were treated with 5 µg / mL puromycin for 15 . 5 h then viability was measured . data represent mean ± SD calculated from 3 independent experiments ( n = 3 ) . P ‐ values were calculated by Student ' s t - compared with puromycin - treated control siRNA ( * P < 0 . 01 ) .

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(D) Depletion of UBQLN4 makes HeLa cells more sensitive to puromycin-induced cell death. After 48 h of UBQLN4 siRNA treatment, the cells were treated with 5 µg/mL puromycin for 15.5 h and then viability was measured. The data represent mean ± SD calculated from 3 independent experiments (n=3). P‐values were calculated by Student's t-test compared with puromycin-treated control siRNA cells (*P < 0.01).


( B ) IL - 2Rα ΔSS mislocalized to fraction . HeLa , expressing Flag - tagged IL - 2Rα WT or ΔSS , were fractionated to ( Cyt . ) ( Mem . ) fractions . Tubulin was used as a marker , while calnexin was used for ER fraction .

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(B) IL-2Rα ΔSS mislocalized to the cytosolic fraction. HeLa cells, expressing Flag-tagged IL-2Rα WT or ΔSS, were fractionated to the cytosolic (Cyt.) and membrane (Mem.) fractions. Tubulin was used as a cytoplasmic marker, while calnexin was used for the ER membrane fraction.


( C ) IL - 2Rα ΔSS was not glycosylated . Flag - tagged IL - 2Rα WT or ΔSS was expressed in HeLa , immunoprecipitated with an anti - Flag antibody . precipitates were incubated with ( + ) or without ( - ) 10 unit of deglycosylation enzyme PNGase F for 2 h , subjected to western blot with an anti - Flag antibody . Glycosylated non - glycosylated signals are indicated .

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(C) IL-2Rα ΔSS was not glycosylated. Flag-tagged IL-2Rα WT or ΔSS proteins was expressed in HeLa cells, and immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F for 2 h, and subjected to western blot analysis with an anti-Flag antibody. Glycosylated and non-glycosylated signals are indicated.


( D ) glycosylated form of IL - 2Rα WT is a stable , while mislocalized IL - 2Rα ΔSS is degraded rapidly via its TMD . A series of IL - 2Ra deletion were expressed in HeLa then chased with 20 μg / mL CHX for indicated periods . Actin was used as a loading control . right panel is a quantified graph of left blot signals represent mean ± SD calculated from 4 independent experiments ( n = 4 ) .

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(D) The glycosylated form of IL-2Rα WT is a stable protein, while mislocalized IL-2Rα ΔSS is degraded rapidly via its TMD. A series of IL-2Ra deletion proteins were expressed in HeLa cells and then chased with 20 μg/mL CHX for the indicated periods. Actin was used as a loading control. The right panel is a quantified graph of the left blot signals represent mean ± SD calculated from 4 independent experiments (n=4).


( E ) IL - 2Rα ΔSS is degraded in a proteasome - dependent manner . HeLa expressing Flag - tagged IL - 2Rα ΔSS were treated with 20 μg / mL CHX as well as 0 . 1 % DMSO ( DM . ) , 10 μM MG - 132 ( MG . ) , 2 μM Bortezomib ( Bor . ) , 10 μM Leupeptin ( Leu . ) , 0 . 1 μM Bafilomycin A1 ( Baf . ) , or 10 μg / ml Pepstatin A ( pep . A ) / E64d for indicated periods . right panel indicates that quantified data of left blot signals represent mean ± SD calculated from 3 independent experiments ( n = 3 ) .

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(E) IL-2Rα ΔSS is degraded in a proteasome-dependent manner. HeLa cells expressing Flag-tagged IL-2Rα ΔSS were treated with 20 μg/mL CHX as well as 0.1% DMSO (DM.), 10 μM MG-132 (MG.), 2 μM Bortezomib (Bor.), 10 μM Leupeptin (Leu.), 0.1 μM Bafilomycin A1 (Baf.), or 10 μg/ml Pepstatin A (pep. A)/E64d for the indicated periods. The right panel indicates that the quantified data of the left blot signals represent mean ± SD calculated from 3 independent experiments (n=3).


( F ) Flag - tagged IL - 2Rα ΔSS mutant T7 - tagged ubiquitin were co - expressed in HeLa were treated with ( + ) or without ( - ) 10 μM MG - 132 . After 4 h , Flag - precipitates were blotted with anti - T7 anti - Flag antibodies .

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(F) The Flag-tagged IL-2Rα ΔSS mutant and T7-tagged ubiquitin were co-expressed in HeLa cells and the cells were treated with (+) or without (-) 10 μM MG-132. After 4 h, Flag-precipitates were blotted with anti-T7 and anti-Flag antibodies.


( G ) HeLa expressing Flag - tagged IL - 2RaWT , ΔSS , ΔSSΔTM were treated with ( + ) or without ( - ) 10 μM MG - 132 . At 4 h after MG - 132 treatment , were lysed analyzed by immunoblotting using indicated antibodies . Note that a higher migrating form of non - glycosylated ( thus defective ) IL - 2Ra WT ( indicated by arrowhead ) is detected strongly in presence of MG - 132 .

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(G) HeLa cells expressing Flag-tagged IL-2RaWT, ΔSS, and ΔSSΔTM proteins were treated with (+) or without (-) 10 μM MG-132. At 4 h after MG-132 treatment, the cells were lysed and analyzed by immunoblotting using the indicated antibodies. Note that a higher migrating form of non-glycosylated (and thus defective) IL-2Ra WT (indicated by the arrowhead) is detected strongly in the presence of MG-132.


( A , B ) IL - 2Rα ΔSS is stabilized in UBQLN4 - knockdown . HeLa were transfected with three distinct siRNA duplexes for UBQLN4 ( UBQLN4 siRNA # 1 ~ # 3 ) or control siRNA . At 48 h after siRNA transfection , Flag tagged - IL - 2Rα ΔSS was expressed in . At 24 h after IL - 2RaSS transfection , were chased with 20 μg / mL CHX harvested at indicated time after CHX addition . Anti - Flag signals in control or UBQLN4 siRNA - treated ( siRNA # 1 ~ # 3 ) were quantified at indicated time points . data represent mean ± calculated from 4 independent experiments ( n = 4 ) . UBQLN4 signal is marked by an arrowhead , while band just below it ( indicated by an asterisk ) is a non - specific band , since this signal was never affected by any of UBQLN4 siRNAs . Actin was used as a loading control .

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(A, B) IL-2Rα ΔSS is stabilized in UBQLN4-knockdown cells. HeLa cells were transfected with three distinct siRNA duplexes for UBQLN4 (UBQLN4 siRNA#1~#3) or control siRNA. At 48 h after siRNA transfection, Flag tagged-IL-2Rα ΔSS was expressed in the cells. At 24 h after IL-2RaSS transfection, the cells were chased with 20 μg/mL CHX and harvested at the indicated time after CHX addition. Anti-Flag signals in the control or UBQLN4 siRNA-treated cells (siRNA#1~#3) were quantified at the indicated time points. The data represent mean ± SEM calculated from 4 independent experiments (n=4). The UBQLN4 signal is marked by an arrowhead, while the band just below it (indicated by an asterisk) is a non-specific band, since this signal was never affected by any of the UBQLN4 siRNAs. Actin was used as a loading control.


( C ) Polyubiquitin modification of IL - 2Rα ΔSS was not diminished , but rather strengthened , in UBQLN4 knockdown . At 48 h after transfection of HeLa with siRNA for UBQLN4 or BAG6 , Flag - tagged IL - 2Rα ΔSS T7 - tagged ubiquitin were expressed with ( + ) or without ( - ) 10 mM MG - 132 . From these , Flag - tagged IL - 2Rα ΔSS was affinity - purified using Hot lysis , Flag - precipitates were blotted with an anti - T7 antibody to detect polyubiquitination of IL - 2Rα ΔSS .

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(C) Polyubiquitin modification of IL-2Rα ΔSS was not diminished, but rather strengthened, in UBQLN4 knockdown cells. At 48 h after transfection of HeLa cells with siRNA for UBQLN4 or BAG6, Flag-tagged IL-2Rα ΔSS and T7-tagged ubiquitin were expressed with (+) or without (-) 10 mM MG-132. From these cells, Flag-tagged IL-2Rα ΔSS was affinity-purified using Hot lysis analysis, and Flag-precipitates were blotted with an anti-T7 antibody to detect polyubiquitination of IL-2Rα ΔSS.


( A ) A series of Flag - tagged truncated mutants of IL - 2Ra substrates were expressed in HeLa with T7 - tagged UBQLN4 treated with ( + ) or without ( - ) 10 µM MG - 132 for 4 h . Flag - IL - 2Ra substrates were immunoprecipitated their co - precipitation with UBQLN4 was analyzed .

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(A) A series of Flag-tagged truncated mutants of IL-2Ra substrates were expressed in HeLa cells with T7-tagged UBQLN4 and treated with (+) or without (-) 10 µM MG-132 for 4 h. Flag-IL-2Ra substrates were immunoprecipitated and their co-precipitation with UBQLN4 was analyzed.


( B ) T7 - UBQLN4 was immunoprecipitated , its interactions with a series of IL - 2Ra were detected .

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(B) T7-UBQLN4 was immunoprecipitated, and its interactions with a series of IL-2Ra proteins were detected.


( B ) UBQLN4 dominantly co - precipitates IL - 2Rα ΔSS over UBQLN1 . Precipitates of Flag - tagged UBQLN1 or UBQLN4 from HeLa lysates were probed with IL - 2Rα ΔSS .

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(B) UBQLN4 dominantly co-precipitates IL-2Rα ΔSS over UBQLN1. Precipitates of Flag-tagged UBQLN1 or UBQLN4 from HeLa cell lysates were probed with IL-2Rα ΔSS.


( C ) complete acid sequences of human UBQLN1 UBQLN4 were analyzed using disorder / order structure prediction program PONDR - FIT . STI - II region of UBQLN4 is indicated by red box , that of UBQLN1 is boxed blue . Note that disorder tendency of UBL , STI - I , UBA regions is nearly identical between UBQLN1 UBQLN4 ( indicated by black green boxes ) , while that of STI - II region is quite distinct , STI - II of UBQLN4 shows a high probability of an ordered structure . numbers denote corresponding acid positions in these .

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(C) The complete amino acid sequences of human UBQLN1 and UBQLN4 were analyzed using the disorder/order structure prediction program PONDR-FIT. The STI-II region of UBQLN4 is indicated by the red box, and that of UBQLN1 is boxed blue. Note that the disorder tendency of the UBL, STI-I, and UBA regions is nearly identical between UBQLN1 and UBQLN4 (indicated by black and green boxes), while that of the STI-II region is quite distinct, and the STI-II of UBQLN4 shows a high probability of an ordered structure. The numbers denote the corresponding amino acid positions in these proteins.


( E ) Substitution of STI - II of UBQLN4 with that of UBQLN1 ( designated as 4 - STI - II - 1 mutant ) abolished its high affinity to IL - 2Rα ΔSS client .

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(E) Substitution of the STI-II sequence of UBQLN4 with that of UBQLN1 (designated as the 4-STI-II-1 mutant protein) abolished its high affinity to the IL-2Rα ΔSS client protein.


( F ) Substitution of STI - II of UBQLN1 with that of UBQLN4 ( designated as 1 - STI - II - 4 ) did not enable UBQLN1 to recognize IL - 2Rα ΔSS .

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(F) Substitution of the STI-II sequence of UBQLN1 with that of UBQLN4 (designated as the 1-STI-II-4) did not enable UBQLN1 to recognize IL-2Rα ΔSS.


( B , C D ) wild - type ( WT ) form of Flag - tagged UBQLN4 its truncated derivatives were expressed in HeLa with T7 - tagged IL - 2RαΔSS ( B C ) . At 4 h after addition of 10 μM MG - 132 , UBQLN4 was immunoprecipitated with an anti - Flag antibody precipitates were blotted with an anti - T7 antibody .

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(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2RαΔSS (B and C). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.


( B , C D ) wild - type ( WT ) form of Flag - tagged UBQLN4 its truncated derivatives were expressed in HeLa with T7 - tagged IL - 2Rα ΔSS ( B C ) T7 - tagged ubiquitin ( D ) . At 4 h after addition of 10 μM MG - 132 , UBQLN4 was immunoprecipitated with an anti - Flag antibody precipitates were blotted with an anti - T7 antibody .

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(B, C and D) The wild-type (WT) form of Flag-tagged UBQLN4 and its truncated derivatives were expressed in HeLa cells with T7-tagged IL-2Rα ΔSS (B and C) and T7-tagged ubiquitin (D). At 4 h after the addition of 10 μM MG-132, UBQLN4 was immunoprecipitated with an anti-Flag antibody and the precipitates were blotted with an anti-T7 antibody.


( E ) GST - tagged purified UBQLN4 its truncated derivatives ( ΔUBA ΔSTI - II ) were incubated with K48 - linked polyubiquitin chains . After in vitro GST pull - down , precipitates were probed with an anti - polyubiquitin ( FK2 ) antibody .

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(E) GST-tagged and purified UBQLN4 and its truncated derivatives (ΔUBA and ΔSTI-II) were incubated with K48-linked polyubiquitin chains. After in vitro GST pull-down, the precipitates were probed with an anti-polyubiquitin (FK2) antibody.


( A ) UBQLN4 interacts with non - glycosylated ( thus defective ) IL - 2Ra WT in SRP54 - knockdown . After treatment of HeLa with SRP54 siRNA ( 10 nM ) for 48 h , Flag - tagged IL - 2RaWT T7 - tagged UBQLN4 were expressed , were treated with ( + ) or without ( - ) 10 µM MG - 132 at 4 h before harvesting . UBQLN4 was affinity - purified from extracts probed with an anti - Flag antibody . Note that of non - glycosylated IL - 2RaWT ( indicated as Defective ) increased , while glycosylated form of this ( indicated as Assembled ) decreased in SRP54 knockdown .

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(A) UBQLN4 interacts with non-glycosylated (and thus defective) IL-2Ra WT in SRP54-knockdown cells. After treatment of HeLa cells with SRP54 siRNA (10 nM) for 48 h, Flag-tagged IL-2RaWT and T7-tagged UBQLN4 proteins were expressed, and the cells were treated with (+) or without (-) 10 µM MG-132 at 4 h before harvesting. UBQLN4 was affinity-purified from cell extracts and probed with an anti-Flag antibody. Note that the levels of non-glycosylated IL-2RaWT (indicated as Defective) increased, while the glycosylated form of this protein (indicated as Assembled) decreased in SRP54 knockdown cells.


( B , C ) SRP54 knockdown stimulates polyubiquitinated co - precipitation of UBQLN4 . Flag - tagged UBQLN4 T7 - tagged ubiquitin were expressed in SRP54 siRNA - treated , Flag precipitates were probed with an anti - T7 antibody to detect polyubiquitinated clients co - precipitation with UBQLN4 under no addition of MG - 132 ( B ) . Note that interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown treated with MG - 132 for 4 h before harvesting ( C ) .

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(B, C) SRP54 knockdown stimulates polyubiquitinated proteins co-precipitation of UBQLN4. Flag-tagged UBQLN4 and T7-tagged ubiquitin were expressed in SRP54 siRNA-treated cells, and Flag precipitates were probed with an anti-T7 antibody to detect polyubiquitinated clients co-precipitation with UBQLN4 under no addition of MG-132 (B). Note that the interaction of UBQLN4 with polyubiquitinated substrates was further increased in SRP54 knockdown cells treated with MG-132 for 4 h before harvesting (C).


( A ) expression of FUS does not alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in transfected NSC34 . Immunoblots of NSC34 transfected with EGFP as a control ( CTRL ) , or wild - type or mutant EGFP - FUS . Transfected were purified via EGFP using a sorter samples probed on immunoblots as indicated . On FUS immunoblot , samples were probed with FUS antibody to show endogenous transfected ; tubulin is shown as a loading control .

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(A) expression of FUS does not alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in transfected NSC34 cells. Immunoblots of NSC34 cells transfected with EGFP as a control (CTRL), or wild-type or mutant EGFP-FUS. Transfected cells were purified via EGFP using a cell sorter and the samples probed on immunoblots as indicated. On the FUS immunoblot, samples were probed with FUS antibody to show endogenous and transfected proteins; tubulin is shown as a loading control.


( B ) representative electron microscopy of ER - mitochondria associations in NSC34 transfected with control EGFP vector ( CTRL ) , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K as indicated ; arrowheads with loops show regions of association . Scale bar = 200 nm . Bar chart shows % of mitochondrial surface closely apposed to ER in different samples . Data were analysed by one - way of variance ( ANOVA ) followed by Tukey ' s multiple comparison . N = 27 - 30 247 - 424 mitochondria , error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) representative electron microscopy of ER-mitochondria associations in NSC34 cells transfected with control EGFP vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K as indicated; arrowheads with loops show regions of association. Scale bar=200 nm. Bar chart shows % of the mitochondrial surface closely apposed to ER in the different samples. Data were analysed by one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. N=27-30 cells and 247-424 mitochondria, error bars are s.e.m.; ***p<0.001.


( C D ) siRNA loss of FUS does not affect ER - mitochondria associations or alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in NSC34 . ( C ) immunoblots of either mock transfected or treated with control ( CTRL ) or FUS siRNAs ; GAPDH is shown as a loading control .

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(C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (C) immunoblots of cells either mock transfected or treated with control (CTRL) or FUS siRNAs; GAPDH is shown as a loading control.


( C D ) siRNA loss of FUS does not affect ER - mitochondria associations or alter expression of VAPB , PTPIP51 or mitofusin - 2 ( MFN2 ) in NSC34 . ( D ) representative electron microscopy of ER - mitochondria associations in control ( CTRL ) FUS siRNA treated . Arrowheads with loops show regions of association . Scale bar = 200 nm . Data analysed by unpaired t - . N = 27 - 28 193 - 202 mitochondria .

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(C and D) siRNA loss of FUS does not affect ER-mitochondria associations or alter expression of VAPB, PTPIP51 or mitofusin-2 (MFN2) in NSC34 cells. (D) representative electron microscopy of ER-mitochondria associations in control (CTRL) and FUS siRNA treated cells. Arrowheads with loops show regions of association. Scale bar=200 nm. Data analysed by unpaired t-test. N=27-28 cells and 193-202 mitochondria.


( A ) FU - induced reductions in ER - mitochondria associations can be detected using SIM . NSC34 were transfected with either EGFP control vector , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K immunostained for TOM20 PDI to label mitochondria ( Mito ) ER respectively ; FUS were detected via their EGFP tags . Merge ( ZOOM ) show zoomed of boxed regions co - localisation shows co - localised pixels . Scale bar = 2 m . Bar chart shows ER - mitochondria co - localisation ( Manders coefficient ) normalized to control in different samples . Data were analysed by one - way ANOVA with Tukey ' s post - hoc . 10 - 14 were analysed per condition from 3 independent experiments ; error bars are s . e . m . , * * p < 0 . 01 * * * p < 0 . 001 .

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(A) FU-induced reductions in ER-mitochondria associations can be detected using SIM. NSC34 cells were transfected with either EGFP control vector, EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and immunostained for TOM20 and PDI to label mitochondria (Mito) and ER respectively; FUS were detected via their EGFP tags. Merge (ZOOM) show zoomed images of boxed regions and co-localisation shows co-localised pixels. Scale bar=2 m. Bar chart shows ER-mitochondria co-localisation (Manders coefficient) normalized to control in the different samples. Data were analysed by one-way ANOVA with Tukey's post-hoc test. 10-14 cells were analysed per condition from 3 independent experiments; error bars are s.e.m., **p<0.01 and ***p<0.001.


( B ) ER - mitochondria associations VAPB - PTPIP51 interaction are disrupted by wild - type ALS / FTD mutant FUS . NSC34 were transfected with EGFP control vector ( CTRL ) , EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K proximity ligation assays performed using VAPB PTPIP51 antibodies . FUS were detected via their EGFP tags . Scale bar = 10 m . Bar chart shows relative number of proximity ligation signals / . Data were analysed by one - way ANOVA Tukey ' s post - hoc ; n = 47 - 53 from 5 experiments . Error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted by wild-type and ALS/FTD mutant FUS. NSC34 cells were transfected with EGFP control vector (CTRL), EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K and proximity ligation assays performed using VAPB and PTPIP51 antibodies. FUS were detected via their EGFP tags. Scale bar=10 m. Bar chart shows relative number of proximity ligation assay signals/cell. Data were analysed by one-way ANOVA and Tukey's post-hoc test; n=47-53 cells from 5 experiments. Error bars are s.e.m.; ***p<0.001.


( C ) overexpression of FUS reduces binding of VAPB to PTPIP51 in transfected . were transfected as indicated with either control empty vector ( CTRL ) , HA - PTPIP51 + CTRL , or HA - PTPIP51 + either EGFP - FUS , EGFP - FUSR521C or EGFP - FUSR518K . PTPIP51 was immunoprecipitated using HA tag amounts of endogenous bound VAPB detected by immunoblotting . Both inputs immunoprecipitations ( IP ) are shown no immunoprecipitating VAPB signals were obtained in absence of HA - PTPIP51 . Bar chart shows relative of VAPB bound to PTPIP51 in immunoprecipitations following of signals from immunoblots . VAPB signals were normalized to immunoprecipitated PTPIP51 - HA signals .

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(C) overexpression of FUS reduces the binding of VAPB to PTPIP51 in transfected cells. Cells were transfected as indicated with either control empty vector (CTRL), HA-PTPIP51+CTRL, or HA-PTPIP51+ either EGFP-FUS, EGFP-FUSR521C or EGFP-FUSR518K. PTPIP51 was immunoprecipitated using the HA tag and the amounts of endogenous bound VAPB detected by immunoblotting. Both inputs and immunoprecipitations (IP) are shown and no immunoprecipitating VAPB signals were obtained in the absence of HA-PTPIP51. Bar chart shows relative levels of VAPB bound to PTPIP51 in the immunoprecipitations following quantification of signals from immunoblots. VAPB signals were normalized to immunoprecipitated PTPIP51-HA signals.


( A ) overexpression of FUS does not affect expression of VAPB , PTPIP51 or mitofusin - 2 . Immunoblots of spinal cord from three 10 week old FUS transgenic mice three - matched are shown ; FUS was detected via its HA - tag . Tubulin was used as a loading control .

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(A) overexpression of FUS does not affect expression of VAPB, PTPIP51 or mitofusin-2. Immunoblots of spinal cord proteins from three 10 week old FUS transgenic mice and three age-matched littermates are shown; FUS was detected via its HA-tag. Tubulin was used as a loading control.


( B ) representative electron microscopy of ER - mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice their non - transgenic ; arrowheads with loops show regions of association . Scale bar is 200 nm . Bar chart shows % of mitochondrial surface closely apposed to ER in two samples . Data were analysed by unpaired t - . N = 67 - 88 438 - 749 mitochondria , error bars are s . e . m . ; * * * p < 0 . 001 .

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(B) representative electron microscopy of ER-mitochondria associations in lumbar spinal cord motor neurons of FUS transgenic mice and their non-transgenic littermates; arrowheads with loops show regions of association. Scale bar is 200 nm. Bar chart shows % of mitochondrial surface closely apposed to ER in the two samples. Data were analysed by unpaired t-test. N=67-88 cells and 438-749 mitochondria, error bars are s.e.m.; ***p<0.001.