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 .

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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 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 .

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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 , 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 .

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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 , 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 .

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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 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 .

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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 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 .

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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 analysis 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 assay ( HAT ) with purified BRPF1 / 3 complexes on free histones and mononucleosomes .

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


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

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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 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 .

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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 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 .

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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 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 .

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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 , 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 .

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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 , Analysis of chromatin - bound proteins in HBO1 depleted cells 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 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 .

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

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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 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 .

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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 and H3K14ac ChIP - seq signal on ± 2 . 5kb around the centre of ( top ) BRPF3 and ( 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 cells were infected with WT P . aeruginosa for various time points and cell 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 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 .

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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 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 .

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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 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 .

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

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


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

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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 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 .

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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 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 .

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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 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 .

O O O B-CELL_LINE O O O O B-ORGANISM O O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_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 B-GENEPROD O O O O O O O O O O O O O O B-EXP_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

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

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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 + / + 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 .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-TISSUE O O O O B-TISSUE O B-TISSUE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C,D) 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 + / + 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 ) .

O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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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 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 .

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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 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 .

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

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


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

B-CELL_LINE O O B-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O B-EXP_ASSAY O

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 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 .

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 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 .

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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 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 .

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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 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 .

O O O B-CELL_LINE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O 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-GENEPROD B-SUBCELLULAR B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O B-EXP_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

(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 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 .

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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 , 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 .

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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 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 .

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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 ) 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 .

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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 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 .

O O O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O

(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 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 ) .

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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 cells were treated as in ( C ) , and 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 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 ) .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

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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 ) 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 ) .

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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 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O

(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 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 .

O O O O O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O

(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 , and its interactions with a series of IL - 2Ra proteins 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 cell 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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O

(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 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 .

O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O

(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 ( 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 .

O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O

(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 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 ) .

O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O

(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 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 .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_LINE O O B-EXP_ASSAY O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O

(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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O 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-SUBCELLULAR O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(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 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 .

O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O

(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 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 .

O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O O O O O O O O O O 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-SUBCELLULAR O

(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 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 .

O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_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

(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 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 .

O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O

(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 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 .

O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O

(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 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 .

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-ORGANISM O O O O O O O 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-SUBCELLULAR 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(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.


( C ) ER - mitochondria associations and the VAPB - PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice . Representative images of proximity ligation signals in 11 week old FUS and non - transgenic ( NTg ) littermate mice . Data were analysed by unpaired t - test ; N = 40 cells from 3 FUS and 3 non - transgenic littermates ( age 10 weeks ) .

O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-TISSUE I-TISSUE O B-GENEPROD O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O

(C) ER-mitochondria associations and the VAPB-PTPIP51 interaction are disrupted in lumbar motor neurons in spinal cords of FUS transgenic mice. Representative images of proximity ligation signals in 11 week old FUS and non-transgenic (NTg) littermate mice. Data were analysed by unpaired t-test; N=40 cells from 3 FUS and 3 non-transgenic littermates (age 10 weeks).


( A and B ) FUS disrupts Ca2 + homeostasis . HEK293 cells were transfected with M3R and either control vector ( CTRL ) , FUS , FUSR521C or FUSR518K as indicated . Release of ERCa2 + was induced by treatment of cells with OxoM . ( A ) shows cytosolicCa2 + levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right . Fluo4 fluorescence shows a transient increase in cytosolicCa2 + levels upon OxoM treatment but compared to control , wild - type and mutant FUS all increase peak cytosolicCa2 + levels . ( B ) shows mitochondrialCa2 + levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right . Data were analysed by one - way ANOVA and Tukey ' s post hoc test . ( A ) N = 49 - 52 cells from 3 experiments ; ( B ) N = 50 - 52 cells from 5 experiments , error bars are s . e . m . ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and B) FUS disrupts Ca2+ homeostasis. HEK293 cells were transfected with M3R and either control vector (CTRL), FUS, FUSR521C or FUSR518K as indicated. Release of ERCa2+ was induced by treatment of cells with OxoM. (A) shows cytosolicCa2+ levels with representative Fluo4 fluorescence traces on the left and normalized peak values on the right. Fluo4 fluorescence shows a transient increase in cytosolicCa2+ levels upon OxoM treatment but compared to control, wild-type and mutant FUS all increase peak cytosolicCa2+ levels. (B) shows mitochondrialCa2+ levels with representative Rhod2 fluorescence traces on the left and normalized peak values on the right. Data were analysed by one-way ANOVA and Tukey's post hoc test. (A) N=49-52 cells from 3 experiments; (B) N=50-52 cells from 5 experiments, error bars are s.e.m.; *p<0.05, **p<0.01, ***p<0.001.


( C ) FUS reduces mitochondrial ATP production . ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1 . 03 and either control vector ( CTRL ) , HA - FUS , HA - FUSR521C or HA - FUSR518K . Cells were imaged in time - lapse prior to and after KCN treatment to inhibit oxidative phosphorylation . Representative traces of YFP / CFP ratios are shown for the different samples ; initial YFP / CFP ratios prior to KCN treatment and those after KCN treatment are indicated . The fall in YFP / CFP ratios correlates with ATP produced by oxidative phosphorylation . Bar chart shows relative ATP levels produced by oxidative phosphorylation ( OXPHOS ) in the different samples .

O O O B-GENEPROD O B-SUBCELLULAR B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O B-CELL_LINE O O O O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O

(C) FUS reduces mitochondrial ATP production. ATP levels were measured in NSC34 cells transfected with the ATP indicator AT1.03 and either control vector (CTRL), HA-FUS, HA-FUSR521C or HA-FUSR518K. Cells were imaged in time-lapse prior to and after KCN treatment to inhibit oxidative phosphorylation. Representative traces of YFP/CFP ratios are shown for the different samples; initial YFP/CFP ratios prior to KCN treatment and those after KCN treatment are indicated. The fall in YFP/CFP ratios correlates with ATP produced by oxidative phosphorylation. Bar chart shows relative ATP levels produced by oxidative phosphorylation (OXPHOS) in the different samples.


( A ) cells were transfected with either control vector ( CTRL ) , HA - FUS , HA - FUSR521C or HA FUSR518K and the samples probed on immunoblots for GSK - 3 phosphorylated on serine - 9 ( GSK - 3 - S9 ) , total GSK - 3 , FUS ( using FUS antibody ) and tubulin as a loading control . Phosphorylation of GSK - 3 serine - 9 is the principal mechanism for regulating its activity ; serine - 9 phosphorylation inhibits GSK - 3 activity . Bar chart shows relative levels of GSK - 3β serine - 9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK - 3β signals . Data were analysed by one - way ANOVA and Tukey ' s post hoc test . N = 4 , error bars are s . e . m ; * p < 0 . 05 .

O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O 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-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O 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) cells were transfected with either control vector (CTRL), HA-FUS, HA-FUSR521C or HA FUSR518K and the samples probed on immunoblots for GSK-3 phosphorylated on serine-9 (GSK-3-S9), total GSK-3, FUS (using FUS antibody) and tubulin as a loading control. Phosphorylation of GSK-3 serine-9 is the principal mechanism for regulating its activity; serine-9 phosphorylation inhibits GSK-3 activity. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by one-way ANOVA and Tukey's post hoc test. N=4, error bars are s.e.m; *p<0.05.


( B ) immunoblots of total and serine - 9 phosphorylated GSK - 3 in spinal cords from three 10 week - old FUS transgenic mice and their non - transgenic littermates . Samples were probed for tubulin as a loading control . Bar chart shows relative levels of GSK - 3β serine - 9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK - 3β signals . Data were analysed by unpaired t - test , error bars are s . e . m . ; * p < 0 . 05 .

O O O B-EXP_ASSAY O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O 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) immunoblots of total and serine-9 phosphorylated GSK-3 in spinal cords from three 10 week-old FUS transgenic mice and their non-transgenic littermates. Samples were probed for tubulin as a loading control. Bar chart shows relative levels of GSK-3β serine-9 phosphorylation following quantification of signals from immunoblots and normalization to total GSK-3β signals. Data were analysed by unpaired t-test, error bars are s.e.m.; *p<0.05.