D ) HeLa C1 cells transfected with CFP or CFP - TBR were treated with D / D solubiliser ( Clontech ) or D / D solubiliser plus 2 . 5 μg / ml Brefeldin A ( BFA ) for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . Results are expressed as a percentage of GFP levels at t = 0 , and the mean of 3 independent experiments ± s . e . m .

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

D) HeLa C1 cells transfected with CFP or CFP-TBR were treated with D/D solubiliser (Clontech) or D/D solubiliser plus 2.5 μg/ml Brefeldin A (BFA) for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. Results are expressed as a percentage of GFP levels at t=0, and the mean of 3 independent experiments ± s.e.m.


A ) HEK293A cells expressing GFP , GFP - TBC1D14 224 - 669 or GFP - TBR were treated in duplicate with EBSS , EBSS plus 100 nM BafA1 or not for two hours , lysed and subjected to immunoblotting for LC3B , tubulin and GFP . The amount of LC3B / tubulin for each condition from three independent experiments is shown on the bar graph , ± s . e . m . * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test .

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

A) HEK293A cells expressing GFP, GFP-TBC1D14 224-669 or GFP-TBR were treated in duplicate with EBSS, EBSS plus 100 nM BafA1 or not for two hours, lysed and subjected to immunoblotting for LC3B, tubulin and GFP. The amount of LC3B/tubulin for each condition from three independent experiments is shown on the bar graph, ±s.e.m. * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


B ) HEK293A GFP - LC3B ( 2GL9 ) cells were transfected with mCherry or mCherry - TBR , treated with EBSS for two hours , fixed and analysed by confocal microscopy .

O O O O O O O O O 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) HEK293A GFP-LC3B (2GL9) cells were transfected with mCherry or mCherry-TBR, treated with EBSS for two hours, fixed and analysed by confocal microscopy.


C ) HEK293A cells were treated as in ( B ) , fixed , stained for WIPI2 and analysed by confocal microscopy .

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

C) HEK293A cells were treated as in (B), fixed, stained for WIPI2 and analysed by confocal microscopy.


D ) HEK293A cells stably expressing GFP - DFCP1 were treated as in ( B ) , fixed and analysed by confocal microscopy . Scale bars = 20 μm . For quantification of ( B ) , ( C ) and ( D ) , 10 fields of view containing transfected cells were imaged for each of three experiments , and the number of GFP - LC3B , WIPI2 or GFP - DFCP1 puncta per cell enumerated using Imaris software ( Bitplane ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells stably expressing GFP-DFCP1 were treated as in (B), fixed and analysed by confocal microscopy. Scale bars = 20 μm. For quantification of (B), (C) and (D), 10 fields of view containing transfected cells were imaged for each of three experiments, and the number of GFP-LC3B, WIPI2 or GFP-DFCP1 puncta per cell enumerated using Imaris software (Bitplane).


B ) Samples prepared according to ( A ) ( 8 × 107 HEK293A cells per transfection ) were lysed in TNTE , split equally and denatured with 1 % SDS ( D ) or not ( ND ) and subjected to 1 hour streptavidin resin pulldown at room temperature . The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Samples prepared according to (A) (8 × 107 HEK293A cells per transfection) were lysed in TNTE, split equally and denatured with 1% SDS (D) or not (ND) and subjected to 1 hour streptavidin resin pulldown at room temperature. The bound proteins were eluted in 2x Laemmli sample buffer plus 3 mM Biotin and immunoblotted for the indicated proteins.


C ) siRNA transfected control cells ( RISC Free ) or TRAPPC8 knockdown ( TRAPPC8 ) ( 1x10cm plate per condition ) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST - TBR . Bound proteins were subjected to immunoblot analysis using TRAPPC8 , TRAPPC4 and tubulin antibodies . Ponceau staining was used to verify loading of the GST fusion proteins ( GST - TBR indicated with arrowhead ) . The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments , error bars are ± s . e . m . * * p < 0 . 01 , unpaired T -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) siRNA transfected control cells (RISC Free) or TRAPPC8 knockdown (TRAPPC8) (1x10cm plate per condition) were lysed in TNTE and used in pulldown assays with immobilised recombinant GST or GST-TBR. Bound proteins were subjected to immunoblot analysis using TRAPPC8, TRAPPC4 and tubulin antibodies. Ponceau staining was used to verify loading of the GST fusion proteins (GST-TBR indicated with arrowhead). The bar chart expresses the relative amount of TRAPPC4 isolated from siTRAPPC8 compared to siRISC Free control cells from three independent experiments, error bars are ± s.e.m. ** p<0.01, unpaired T-test


D ) HEK293T cells stably depleted of TRAPPC8 ( shC8 ) or not ( shCtrl ) were transfected with constructs encoding GFP or GFP - TRAPPC3 and subjected to GFP Trap IP . Precipitated proteins were immunoblotted for GFP , TRAPPC4 and TRAPPC8 . Bar chart : the amount of TRAPPC4 isolated was normalised to the amount of GFP - TRAPPC3 precipitated from three independent experiments and plotted ± s . e . m . , ns = non - significant , unpaired T - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293T cells stably depleted of TRAPPC8 (shC8) or not (shCtrl) were transfected with constructs encoding GFP or GFP-TRAPPC3 and subjected to GFP Trap IP. Precipitated proteins were immunoblotted for GFP, TRAPPC4 and TRAPPC8. Bar chart: the amount of TRAPPC4 isolated was normalised to the amount of GFP-TRAPPC3 precipitated from three independent experiments and plotted ± s.e.m., ns = non-significant, unpaired T-test.


A ) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B ( knockdown confirmed by immunoblot analysis ) were fed with Alexa - 647 Transferrin for 15 minutes in full medium , and the fluorescent transferrin chased out for the indicated time periods . At the end of the time course , cells were trypsinised , fixed and the Alexa - 647 fluorescence analysed by flow cytometry . Results are plotted as percentage of transferrin fluorescence at t = 0 for the RISC free control cells , expressed as the mean of three independent experiments ± s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells treated with transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 and RAB11A and B (knockdown confirmed by immunoblot analysis) were fed with Alexa-647 Transferrin for 15 minutes in full medium, and the fluorescent transferrin chased out for the indicated time periods. At the end of the time course, cells were trypsinised, fixed and the Alexa-647 fluorescence analysed by flow cytometry. Results are plotted as percentage of transferrin fluorescence at t=0 for the RISC free control cells, expressed as the mean of three independent experiments ± s.e.m.


B ) Cells transiently transfected with RISC Free control siRNA , or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis - Golgi markers GM130 or RAB1B and analysed by confocal microscopy . Scale bars = 20 μm in main panels , 10 μm in inset panels . Bar graph : > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments . Error bars ± s . e . m . , * p < 0 . 05 , * * p < 0 . 01 , one way ANOVA with Sidak ' s multiple comparison test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Cells transiently transfected with RISC Free control siRNA, or siRNA duplexes directed against TRAPPC8 or RAB1A and B were stained for the cis-Golgi markers GM130 or RAB1B and analysed by confocal microscopy. Scale bars = 20 μm in main panels, 10 μm in inset panels. Bar graph: > 100 cells per siRNA were scored for fragmented or normal juxtanuclear Golgi stacks from three independent experiments. Error bars ± s.e.m., * p<0.05, ** p<0.01, one way ANOVA with Sidak's multiple comparison test.


C ) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B ( knockdown confirmed by immunoblot analysis ) , were treated with D / D solubiliser for the indicated time periods , trypsinised , fixed and their GFP content analysed by flow cytometry . The line graph shows the GFP fluorescence as a percentage of t = 0 , ± s . e . m . from three independent experiments .

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

C) HeLa C1 cells transiently transfected with RISC free control siRNA or siRNA duplexes directed against TRAPPC8 or RAB1A and B (knockdown confirmed by immunoblot analysis), were treated with D/D solubiliser for the indicated time periods, trypsinised, fixed and their GFP content analysed by flow cytometry. The line graph shows the GFP fluorescence as a percentage of t=0, ± s.e.m. from three independent experiments.


A ) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed ( F ) or starved ( S ) HEK293A cells using a monoclonal hamster anti - ATG9 ( ATG9 ) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody , TRAPPC8 , TRAPPC4 and actin .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ATG9 positive membranes were immunoisolated from approximately 2 × 107 fed (F) or starved (S) HEK293A cells using a monoclonal hamster anti-ATG9 (ATG9) or control hamster IgM and immunoblotted for ATG9 using a rabbit polyclonal antibody, TRAPPC8, TRAPPC4 and actin.


B ) HEK293A cells transfected with GFP or GFP - TBR ( green ) were stained for ATG9 using hamster anti - ATG9 ( red ) and analysed by confocal microscopy . Scale bar = 20

O O O O O O O O O O O O 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) HEK293A cells transfected with GFP or GFP-TBR (green) were stained for ATG9 using hamster anti-ATG9 (red) and analysed by confocal microscopy. Scale bar = 20 μm


C ) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B ( green ) and ATG9 using hamster anti - ATG9 ( red ) and imaged using confocal microscopy .

O O O O O O O O O O O O O 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) HEK293A cells transiently transfected with RISC Free siRNA or siRNA against TRAPPC8 were stained for RAB1B (green) and ATG9 using hamster anti-ATG9 (red) and imaged using confocal microscopy.


D ) Stills at t = 30 minutes from time lapse imaging of 9B9 cells ( stably expressing GFP - LC3 and mRFP - ATG9 ) expressing either CFP or CFP - TBR ( blue ) . Scale bars = 20 μm . Inset panels show mRFP - ATG9 and GFP - LC3 contact events at the indicated time points . Bar chart shows number of times a GFP - LC3 spot was within 1 μm of an mRFP - ATG9 spot ( a contact event ) , expressed as total mRFP - ATG9 contacts per GFP - LC3 spot , n = 18 cells per condition pooled from 4 independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stills at t = 30 minutes from time lapse imaging of 9B9 cells (stably expressing GFP-LC3 and mRFP-ATG9) expressing either CFP or CFP-TBR (blue). Scale bars = 20 μm. Inset panels show mRFP-ATG9 and GFP-LC3 contact events at the indicated time points. Bar chart shows number of times a GFP-LC3 spot was within 1 μm of an mRFP-ATG9 spot (a contact event), expressed as total mRFP-ATG9 contacts per GFP-LC3 spot, n = 18 cells per condition pooled from 4 independent experiments.


A ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with GFP or GFP - TBR ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) . Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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

A) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with GFP or GFP-TBR (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red). Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


B ) Cells depleted of ULK1 ( siULK1 ) or not ( RISC Free ) were transfected with myc - RAB1B or myc - RAB1B ( S22N ) ( green ) , starved ( EBSS ) or not ( - EBSS ) and stained for ATG9 using hamster anti - ATG9 ( red ) Arrows indicate transfected cells . Western blot indicates ULK1 knockdown . Scale bars = 20 μm .

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

B) Cells depleted of ULK1 (siULK1) or not (RISC Free) were transfected with myc-RAB1B or myc-RAB1B (S22N) (green), starved (EBSS) or not (-EBSS) and stained for ATG9 using hamster anti-ATG9 (red) Arrows indicate transfected cells. Western blot indicates ULK1 knockdown. Scale bars = 20 μm.


C Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP . Conserved histidines in octarepeats 2 - 5 are coloured blue and missense mutations are shown in red . Overproduction of C2 fragment is indicated by + + signs to the right of the panel .

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sequences of synthetic PrP alleles differing in the octarepeat region of mousePrP. Conserved histidines in octarepeats 2-5 are coloured blue and missense mutations are shown in red. Overproduction of C2 fragment is indicated by ++ signs to the right of the panel.


A Lysates from RK13 cells transfected with WT , S1 , S3 and S3 . F88W PrP were probed with antibody Sha31 , 9A2 and 12B2 after digestion with PNGaseF .

O O O O O O O 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 Lysates from RK13 cells transfected with WT, S1, S3 and S3.F88W PrP were probed with antibody Sha31, 9A2 and 12B2 after digestion with PNGaseF.


B , C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates . A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of ( C ) .

O O O O O O O O O O O O O O O O O O O O O O O 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, C Additional PrP mutants were examined by Western blot with Sha31 after PNGaseF digestion of RK13 cell lysates. A schematic of the different lengths of C2 caused by single mutations in the OR is shown in the lower portion of (C).


D Lysates of N2a , HEK , SMB - PS and SH - SY5Y cells not transfected ( − ) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31 .

O O O O O O O O O O O O O O O O O O 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 Lysates of N2a, HEK, SMB-PS and SH-SY5Y cells not transfected (−) and transiently transfected with WT PrP or S3 PrP plasmids were digested with PNGaseF and analysed by Western blot using Sha31.


E Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP , S1 PrP and S3 PrP . Lysates were PNGaseF - digested before Western blot using the Sha31 antibody . F1 : A114F , F2 : G118F , F3 : G122F , F4 :

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Analysis of lysates from RK13 cells transfected with plasmids incorporating Phe substitutions into the hydrophobic domain of WT PrP, S1 PrP and S3 PrP. Lysates were PNGaseF-digested before Western blot using the Sha31 antibody. F1: A114F, F2: G118F, F3: G122F, F4: G126F


A Brain homogenates of TgPrP ( WT ) , TgPrP ( S1 ) - 17 and TgPrP ( S3 . F88W ) - 35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31 , 12B2 and 9A2 . Prnp0 / 0 , and WT brain homogenates were used as controls .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Brain homogenates of TgPrP(WT), TgPrP(S1)-17 and TgPrP(S3.F88W)-35 mice were analysed by Western blot before and after PNGaseF digestion using the PrP antibodies Sha31, 12B2 and 9A2. Prnp0/0, and WT brain homogenates were used as controls.


B The ratio of C2 to full - length PrP in TgPrP ( S3 . F88W ) - 35 and WT mice ( left panel , n = 7 , P = 1 . 46E - 06 ) and in RK13 cells expressing WT and S3 PrP ( right panel , n = 5 , P = 4 . 01E - 05 ) is shown . Values are presented as mean ± SEM . Unpaired , two - tailed t - test , * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The ratio of C2 to full-length PrP in TgPrP(S3.F88W)-35 and WT mice (left panel, n = 7, P = 1.46E-06) and in RK13 cells expressing WT and S3 PrP (right panel, n = 5, P = 4.01E-05) is shown. Values are presented as mean ± SEM. Unpaired, two-tailed t-test, ****P < 0.0001.


C Different tissue homogenates from a TgPrP ( S3 . F88W ) - 35 mouse were PNGaseF - digested and analysed for PrP using the antibody 1A6 . FL , full - length PrP ; ns , non - specific signal detected in the immunoblotting procedure .

C Different tissue homogenates from a TgPrP(S3.F88W)-35 mouse were PNGaseF-digested and analysed for PrP using the antibody 1A6. FL, full-length PrP; ns, non-specific signal detected in the immunoblotting procedure.


A Sections of sciatic nerve from TgPrP ( S3 . F88W ) - 14 female mice ( all 610 days old ) and TgPrP ( S3 . F88W ) - 35 female mice ( 614 , 570 and 614 days old , respectively ) were analysed using toluidine blue staining ( upper row ) , with the third panels for each line showing a hypermyelinated phenotype . Scale bar in left panel represents 50 μm . The lower rows show EM analyses of mice of the indicated genotypes . Scale bar is 10 μm for panels 1 - 4 , with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3 . F88W PrP ( scale bar , 5 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sections of sciatic nerve from TgPrP(S3.F88W)-14 female mice (all 610 days old) and TgPrP(S3.F88W)-35 female mice (614, 570 and 614 days old, respectively) were analysed using toluidine blue staining (upper row), with the third panels for each line showing a hypermyelinated phenotype. Scale bar in left panel represents 50 μm. The lower rows show EM analyses of mice of the indicated genotypes. Scale bar is 10 μm for panels 1-4, with the fifth panel showing a high power view to illustrate hypermyelinated fibres in a mouse expressing S3.F88W PrP (scale bar, 5 μm).


B Western blot analysis of sciatic nerve protein extracts ( 10 μg ) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain ( see Fig3 ) . The asterisk indicates a greater than full - length fragment present in TgPrP ( S3 . F88W ) mice , possibly corresponding to incomplete removal of the N - terminal signal peptide .

B Western blot analysis of sciatic nerve protein extracts (10 μg) from female mice after PNGaseF treatment shows a similar hierarchy of PrP expression in Tg lines to that of brain (see Fig3). The asterisk indicates a greater than full-length fragment present in TgPrP(S3.F88W) mice, possibly corresponding to incomplete removal of the N-terminal signal peptide.


C Quantification of fibre morphology in aged animals ( 492 - 608 days old ) , representing the percentage of the nerve occupied by fibres versus the total cross - sectional area of the nerve . The S1 and S3 . F88W alleles rescue Prnp0 / 0 nerves to a similar percentage as the WT allele , but the variance and P - value is greater for the two Tg lines expressing the TgPrP ( S3 . F88W ) allele . Unpaired , two - tailed t - test , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . Sample sizes and exact P - values compared with Prnp0 / 0 ( n = 9 ) were as follows : TgPrP ( WT ) n = 6 , P = 3 . 63E - 05 ; Prnp0 / 0 , n = 9 ; TgPrP ( S1 ) - 17 n = 5 , P = 3 . 54E - 05 ; TgPrP ( S1 ) - 19 n = 3 , P = 6 . 35E - 06 ; TgPrP ( S3 . F88W ) - 14 n = 3 , P = 3 . 70E - 03 ; and TgPrP ( S3 . F88W ) - 35 n = 7 , P = 7 . 18E - 03 .

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

C Quantification of fibre morphology in aged animals (492-608 days old), representing the percentage of the nerve occupied by fibres versus the total cross-sectional area of the nerve. The S1 and S3.F88W alleles rescue Prnp0/0 nerves to a similar percentage as the WT allele, but the variance and P-value is greater for the two Tg lines expressing the TgPrP(S3.F88W) allele. Unpaired, two-tailed t-test, **P < 0.01, ***P < 0.001, ****P < 0.0001. Sample sizes and exact P-values compared with Prnp0/0 (n = 9) were as follows: TgPrP(WT) n = 6, P = 3.63E-05; Prnp0/0, n = 9; TgPrP(S1)-17 n = 5, P = 3.54E-05; TgPrP(S1)-19 n = 3, P = 6.35E-06; TgPrP(S3.F88W)-14 n = 3, P = 3.70E-03; and TgPrP(S3.F88W)-35 n = 7, P = 7.18E-03.


A , B Infected brain homogenates were PK - digested and analysed by Western blot using ( A ) Sha31 antibody and ( B ) after PK and PNGaseF digestion using Sha31 .

O O O O O O O O O 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, B Infected brain homogenates were PK-digested and analysed by Western blot using (A) Sha31 antibody and (B) after PK and PNGaseF digestion using Sha31.


C Conformational stability assay on RML - infected brain homogenates incubated with increasing concentrations of GdnHCl . The best - fit alignments of the data are presented . Genotypes are noted on the individual panels . Source data are available online for this figure .

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

C Conformational stability assay on RML-infected brain homogenates incubated with increasing concentrations of GdnHCl. The best-fit alignments of the data are presented. Genotypes are noted on the individual panels.Source data are available online for this figure.


Pathology of RML - infected miceA - H The pattern of PrPSc deposition ( A - D ) and GFAP immunostaining ( E - H ) is presented . I - P A higher power view of H & amp ; amp ; E stained hippocampus ( I - L ) and thalamus ( M - P ) to reveal vacuolation is presented . Data information : Animals of the same genotype are shown within a column , and incubation times of the presented animals with the RML prion isolate are shown ( days ) . Mice expressing PrP S1 and S3 alleles ( central columns ) exhibit attenuated PrPSc deposition , gliosis and spongy change . Pathological features of the RML isolate are not altered upon sequential passage through TgPrP ( S3 . 88W ) - 35 mice ( see schematic at bottom of the diagram indicating sequential passages and finally , passage back into WT mice , compare the data in first and fourth columns ) .

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Pathology of RML-infected miceA-H The pattern of PrPSc deposition (A-D) and GFAP immunostaining (E-H) is presented.I-P A higher power view of H&amp;E stained hippocampus (I-L) and thalamus (M-P) to reveal vacuolation is presented.Data information: Animals of the same genotype are shown within a column, and incubation times of the presented animals with the RML prion isolate are shown (days). Mice expressing PrP S1 and S3 alleles (central columns) exhibit attenuated PrPSc deposition, gliosis and spongy change. Pathological features of the RML isolate are not altered upon sequential passage through TgPrP(S3.88W)-35 mice (see schematic at bottom of the diagram indicating sequential passages and finally, passage back into WT mice, compare the data in first and fourth columns).


A Spot counts in the scrapie cell assay using WT , TgPrP ( S1 ) - 17 and TgPrP ( S3 . F88W ) - 14 brain homogenates ( 0 . 1 % or 0 . 01 % ) from mice infected with RML are shown in the upper panel ( WT vs S1 P = 2 . 09E - 05 and vs S3 P = 4 . 62E - 08 ) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel ( WT vs S1 P = 1 . 04E - 13 and vs S3 P = 3 . 39E - 20 ; two - tailed t - test , n = 3 ) .

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A Spot counts in the scrapie cell assay using WT, TgPrP(S1)-17 and TgPrP(S3.F88W)-14 brain homogenates (0.1% or 0.01%) from mice infected with RML are shown in the upper panel (WT vs S1 P = 2.09E-05 and vs S3 P = 4.62E-08) with numbers corrected for PrPC expression levels as per Table1 presented in the lower panel (WT vs S1 P = 1.04E-13 and vs S3 P = 3.39E-20; two-tailed t-test, n = 3).


B A comparison of incubation time vs PrPC : PrPSc ratio for mice expressing different levels of WT PrP ( WT , TgPrP ( WT ) , tga20 ) , S1 PrP ( TgPrP ( S1 ) - 17 , TgPrP ( S1 ) - 19 , TgPrP ( S1 ) - 39 ) and S3 PrP ( TgPrP ( S3 . F88W ) - 14 , TgPrP ( S3 . F88W ) - 35 ) .

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B A comparison of incubation time vs PrPC:PrPSc ratio for mice expressing different levels of WT PrP (WT, TgPrP(WT), tga20), S1 PrP (TgPrP(S1)-17, TgPrP(S1)-19, TgPrP(S1)-39) and S3 PrP (TgPrP(S3.F88W)-14, TgPrP(S3.F88W)-35).


BATs were isolated from cold - exposed mice ( 4 ℃ , 72 h ) or control mice ( 30 ℃ , 72 h ) , then lysed and subjected to western blotting for analysis of the indicated proteins ( A ) . Relative protein expression levels , normalized to Actin , are shown in ( B ) .

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BATs were isolated from cold-exposed mice (4℃, 72 h) or control mice (30℃, 72 h), then lysed and subjected to western blotting for analysis of the indicated proteins (A). Relative protein expression levels, normalized to Actin, are shown in (B).


mRNA expression levels of the indicated genes were assayed by real - time PCR and normalized to Actb ( C ) .

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mRNA expression levels of the indicated genes were assayed by real-time PCR and normalized to Actb (C).


Mitochondrial DNA copy numbers were determined by real - time PCR and normalized to nuclear DNA standards ( D ) .

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Mitochondrial DNA copy numbers were determined by real-time PCR and normalized to nuclear DNA standards (D).


Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted ( E ) .

Relative increase of mitochondrial proteins was normalized to mtDNA copy numbers and plotted (E).


( F - G ) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30 ℃ or 4 ℃ for 72 h . Scale bar , 50 μm . The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining .

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(F-G) Representative images showing immunohistochemical detection of FUNDC1 in sections of BATs from mice kept at 30℃ or 4℃ for 72 h. Scale bar, 50 μm. The expression of FUNDC1 was quantified by analyzing and normalizing the average optical density of FUNDC1 staining.


Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30 ℃ ( Normal ) or following 72 h at 4 ℃ ( Cold ) ( A ) . Relative protein expression levels were normalized to Actin ( B ) .

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Western blotting analysis of proteins related to mitochondrial biogenesis in BATs from mice at 30℃ (Normal) or following 72 h at 4℃ (Cold) (A). Relative protein expression levels were normalized to Actin (B).


Flag - PGC - 1α was introduced into PGC - 1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins ( D ) . Expression levels of the indicated proteins were quantified ( E ) ( mean ± SEM ; n = 3 ) . Scramble , scramble shRNA .

Flag-PGC-1α was introduced into PGC-1α knockdown brown preadipocytes for 24 h and samples were collected for western blotting analyses of the indicated proteins (D). Expression levels of the indicated proteins were quantified (E) (mean ± SEM; n=3). Scramble, scramble shRNA.


Samples were collected for real - time PCR analysis . The expression of Fundc1 was normalized ( F ) .

Samples were collected for real-time PCR analysis. The expression of Fundc1 was normalized (F).


( G - H ) Scramble and PGC - 1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses ( G ) and quantification of the indicated proteins ( H )

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(G-H) Scramble and PGC-1α knockdown preadipocytes were induced to differentiate into mature adipocytes and samples were collected for western blotting analyses (G) and quantification of the indicated proteins (H)


( B ) Luciferase activity stimulated by the human Fundc1 promoter . The activity of Fundc1 promoter fragments , either wild type ( WT ) or with a mutation in the potential NRF1 binding site , was assayed using a dual luciferase reporter system .

(B) Luciferase activity stimulated by the human Fundc1 promoter. The activity of Fundc1 promoter fragments, either wild type (WT) or with a mutation in the potential NRF1 binding site, was assayed using a dual luciferase reporter system.


( C , D ) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 ( F1 probe or mut F1 probe ) , the confirmed NRF1 binding site in the promoter of VSNL1 ( positive probe ) . The probes were incubated with HeLa nuclear extracts , then run on a gel ( C ) . A super - shifted band ( asterisk ) is detected in the presence of an anti - NRF1 antibody ( D ) . Labeled probe : biotin labeled wild type Fundc1 probe ; Cold probe : wild type Fundc1 probe ; Mut probe : NRF1 binding site mutant Fundc1 probe ; asterisk indicates the supershifted band generated with antibody to NRF1 .

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(C,D) EMSA assay using biotinylated oligonucleotide probes containing the predicted NRF1 binding site or mutant NRF1 binding site in the promoter of Fundc1 (F1 probe or mut F1 probe) ,the confirmed NRF1 binding site in the promoter of VSNL1 (positive probe). The probes were incubated with HeLa nuclear extracts, then run on a gel (C). A super-shifted band (asterisk) is detected in the presence of an anti-NRF1 antibody (D). Labeled probe: biotin labeled wild type Fundc1 probe; Cold probe: wild type Fundc1 probe; Mut probe: NRF1 binding site mutant Fundc1 probe; asterisk indicates the supershifted band generated with antibody to NRF1.


( E , F ) ChIP assay to analyze the interaction of NRF1 ( E ) or PGC - 1α ( F ) proteins with the Fundc1 promoter .

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(E,F) ChIP assay to analyze the interaction of NRF1 (E) or PGC-1α (F) proteins with the Fundc1 promoter.


( G ) NRF1 - myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies . ( H ) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC - 1α - Flag for 24 h and samples were collected for western blotting with the indicated antibodies .

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(G) NRF1-myc was introduced into NRF1 shRNA knockdown cells for 24 h and scramble cells were used as control and samples were collected for western blotting with the indicated antibodies. (H) Scramble and NRF1 shRNA knockdown cells were transfected with vector and PGC-1α-Flag for 24 h and samples were collected for western blotting with the indicated antibodies.


( I ) ChIP analysis of the interaction of PGC - 1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells .

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(I) ChIP analysis of the interaction of PGC-1α protein with the Fundc1 promoter in scramble or NRF1 shRNA knockdown cells.


( A , B ) Western blotting analyses of the levels of autophagy - related proteins in BAT from mice at the normal temperature ( 30 ℃ ) or at 4 ℃ for 72 h ( A ) . Expression of the indicated proteins was analyzed before and after 72 h cold exposure ( 4 ℃ ) ( B ) .

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(A,B) Western blotting analyses of the levels of autophagy-related proteins in BAT from mice at the normal temperature (30℃) or at 4℃ for 72 h (A). Expression of the indicated proteins was analyzed before and after 72 h cold exposure (4℃) (B).


( C ) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h .

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(C) Representative images from immunofluorescence staining of LC3B and CYCS in BAT from mice at the normal temperature (30 ℃) or 4℃ for 72 h.


( D Representative transmission electron microscope ( TEM ) images of BAT cells isolated from mice at the normal temperature ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 1

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(D Representative transmission electron microscope (TEM) images of BAT cells isolated from mice at the normal temperature (30℃) or 4℃ for 72 h. Scale bar, 1 μm


( D ) . The number of mitophagosomes per 100 mitochondria was counted and normalized ( E ) .

(D).The number of mitophagosomes per 100 mitochondria was counted and normalized (E).


( F , G ) BATs from mice exposed to normal conditions ( 30 ℃ ) or cold ( 4 ℃ ) were lysed and immunoprecipitated with antibodies against FUNDC1 , LC3B or IgG , then immunoblotted with the indicated antibodies . The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted ( G ) .

(F,G) BATs from mice exposed to normal conditions (30℃) or cold (4℃) were lysed and immunoprecipitated with antibodies against FUNDC1, LC3B or IgG, then immunoblotted with the indicated antibodies. The ratio between immunoprecipitated LC3B to FUNDC1 or FUNDC1 to LC3B were quantified and plotted (G).


( A , B ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) mice at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . Representative images are shown , scale bar , 1μm ( A ) . The number of mitophagosomes per 100 mitochondria were counted and normalized ( B ) .

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(A,B) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) mice at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. Representative images are shown, scale bar, 1μm (A). The number of mitophagosomes per 100 mitochondria were counted and normalized (B).


( C , D ) . Representative fluorescence images of BAT sections from Fundc1fl / fl / Adiponectincre - ( Cre - ) and Fundc1fl / fl / Adiponectincre + ( Cre + ) mito - Keima mice at normal conditions ( 30 ℃ ) or 4 ℃ for 72 h . Scale bar , 50 μm ( C ) . Quantification of the ratio between red and green signals ( D ) .

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(C,D).Representative fluorescence images of BAT sections from Fundc1fl/fl/Adiponectincre- (Cre-) and Fundc1fl/fl/Adiponectincre+ (Cre+) mito-Keima mice at normal conditions (30℃) or 4℃ for 72 h. Scale bar, 50 μm (C). Quantification of the ratio between red and green signals (D).


( E ) Fundcfl / fl / Ucp1cre - ( Cre - ) and Fundcfl / fl / Ucpcre + ( Cre + ) mice were maintained at 30 ℃ ( Normal ) or exposed to 4 ℃ for 72 h ( Cold ) . Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards .

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(E) Fundcfl/fl/Ucp1cre- (Cre-) and Fundcfl/fl/Ucpcre+ (Cre+) mice were maintained at 30℃ (Normal) or exposed to 4℃ for 72 h (Cold). Mitochondrial DNA copy numbers were analyzed by null and normalized to nuclear DNA standards.


( F ) The relative total mito - keima signal ( green signal ) in Fundc1 deficiency and control mito - keima mice under normal condition or cold exposure were quantified and plotted .

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(F) The relative total mito-keima signal (green signal ) in Fundc1 deficiency and control mito-keima mice under normal condition or cold exposure were quantified and plotted.


( G ) BATs isolated from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) at 30 ℃ or following 72 h at 4 ℃ were analyzed by transmission electron microscopy . The percent of mitochondria without cristae is shown in the histogram .

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(G) BATs isolated from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) at 30℃ or following 72 h at 4℃ were analyzed by transmission electron microscopy. The percent of mitochondria without cristae is shown in the histogram.


Oxygen consumption rate ( OCR ) ( H ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h .

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Oxygen consumption rate (OCR) (H) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.


mitochondrial superoxide ( I ) were measured in BAT from Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucpcre + ( Cre + ) mice kept at 30 ℃ or at 4 ℃ for 72 h .

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mitochondrial superoxide (I) were measured in BAT from Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucpcre+ (Cre+) mice kept at 30℃ or at 4℃ for 72 h.


( A ) Body temperature measurements in Fundc1fl / fl / Ucp1cre - ( Cre - ) and Fundc1fl / fl / Ucp1cre + ( Cre + ) fasting male mice before and during cold exposure ( 4 ° C ) for the indicated time . Body temperature ( B ) before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice .

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(A) Body temperature measurements in Fundc1fl/fl/Ucp1cre- (Cre-) and Fundc1fl/fl/Ucp1cre+ (Cre+) fasting male mice before and during cold exposure (4°C) for the indicated time. Body temperature (B) before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.


BAT tissue weight ( C ) measurements before and after 72 h cold exposure ( 4 ° C ) of Cre - and Cre + male mice .

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BAT tissue weight (C) measurements before and after 72 h cold exposure (4°C) of Cre- and Cre+ male mice.


Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Western blotting analysis of BAT protein levels ( D ) . Protein expression levels were normalized to Actin ( E ) .

Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Western blotting analysis of BAT protein levels (D). Protein expression levels were normalized to Actin (E).


Fundc1fl / fl / Ucpcre - and Fundc1fl / fl / Ucp1cre + mice were exposed to 4 ℃ for 72 h ( Cold ) , and control mice were kept at 30 ℃ ( Normal ) . Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real - time PCR and normalized to Actb ( F ) .

Fundc1fl/fl/Ucpcre- and Fundc1fl/fl/Ucp1cre+ mice were exposed to 4℃ for 72 h (Cold), and control mice were kept at 30℃ (Normal). Levels of mRNAs encoding Fundc1 and Ucp1 were measured by real-time PCR and normalized to Actb (F).


( G ) Representative images of UCP1 immunohistochemistry of BAT sections from Cre - or Cre + mice in normal conditions ( 30 ℃ ) or following 72 h at 4 ℃ . Scale bar , 50 μm .

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(G) Representative images of UCP1 immunohistochemistry of BAT sections from Cre- or Cre+ mice in normal conditions (30℃) or following 72 h at 4℃. Scale bar, 50 μm.


( A ) Amylose pulldown from benzonase - treated HEK293T cell lysates expressing 2xMBP - BRCA2NT in untreated or irradiated cells ( 6Gy ; + IR ) . DDX5 and BRCA2NT ( MBP ) detected by immunoblot . StainFree images of the gels before transfer were used as loading

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(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control


( B ) Immunoprecipitation ( IP ) of endogenous BRCA2 from benzonase - treated HEK293T cell lysates left untreated or treated with IR ( 6Gy ) and harvested 4h post - IR , as indicated . Mouse IgG was used as negative control . Immunoblot of DDX5 and BRCA2 . StainFree image of the gels before transfer were used as loading control ( cropped image is shown ) . Asterisk ( * ) indicate a non - specific band detected by anti - DDX5 antibody .

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(B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody.


( C ) Left : Representative images of in situ proximity ligation assay ( PLA ) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated ( - ) or irradiated ( 4h post - IR ; 6 Gy ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from untreated siC cells are shown . Scale bar indicates 10 µm . Right : Quantification of the number of PLA spots per nucleus . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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(C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( D ) Diagram showing the BRCA2 N - terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N - terminal truncations ( BRCA2T1 , BRCA2LT2 , BRCA2LT3 ) or the 2xMBP tag . DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP , respectively . StainFree images of the gels before transfer were used as loading

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(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control


( E ) Left : GST pulldown assay using purified BRCA2T1 and DDX5 ; MBP antibody was used for the detection of both proteins . UB : unbound ; E : eluate . Right : SDS - PAGE showing 300 ng of purified MBP - DDX5 - GST and of 2xMBP - tagged BRCA2T1 used in the pulldown assay .

(E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.


( A ) Left : Representative images of S9 . 6 immunofluorescence of U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . The merged images show the signal of S9 . 6 , nucleolin ( nucleoli ) antibodies and DAPI staining . Scale bar indicates 25 µm . Right : Quantification of S9 . 6 average nuclear intensity of U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . The red line in the plot indicates the median and each symbol represents the value of a single cell . The statistical significance of the difference was calculated with Mann - Whitney U - test and the p - values show the significant difference . The data represent at least 235 cells per condition from three independent experiments .

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(A) Left: Representative images of S9.6 immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments.


( B ) Top : Representative images of in situ PLA performed with anti - DDX5 and S9 . 6 antibodies in EdU - labelled U2OS cells . Where indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Bottom : Quantification of PLA spots per nucleus in each condition as indicated . At least 300 cells per condition were counted from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test , the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of PLA spots per nucleus in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Representative screenshot of a specific genomic region showing DRIPc - seq profiles at Watson ( W ) and Crick ( C ) strands in K562 cells depleted of DDX5 ( siDDX5 ) or control cells ( siC ) from two independent experiments .

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

(C) Representative screenshot of a specific genomic region showing DRIPc-seq profiles at Watson (W) and Crick (C) strands in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments.


( D ) DNA - RNA hybrid distribution along protein - coding genes containing DRIPc - seq peaks in both conditions ( siC and siDDX5 ) and replicates . Gene metaplots represent the mean of antisense or sense mean DRIPc - seq signal from two independent experiments in K562 cells depleted of DDX5 ( siDDX5 ) or control cells ( siC ) as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated.


( A ) Left : Representative images of in situ PLA between S9 . 6 and γH2AX antibodies in U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from non - irradiated siC cells are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Right : Quantification of PLA spots per nucleus in each condition as indicated . At least 500 cells per condition were counted from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( B ) DRIP - qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . The experiment was performed in both untreated cells ( - OHT ) and after tamoxifen addition ( + OHT ) . The data represent the mean ± SEM from at least four independent experiments . The statistical significance of the difference was calculated with unpaired one - tail t - test and the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference.


( A ) Top : Scheme showing the experimental set up for laser irradiation in DDX5 - GFP transfected U2OS cells depleted of BRCA2 ( siBRCA2 ) or control cells ( siC ) . Middle : Live cell imaging of the recruitment of GFP - 53BP1 or DDX5 - GFP to DNA damage tracks at different time points as indicated . Exposure and processing were adjusted to best demonstrate stripes and anti - stripes . Scale bar indicates 10 µm . Bottom left : Western blot showing the siRNA mediated knock - down of BRCA2 from U2OS cells transfected with DDX5 - GFP . Bottom right : Quantification of the percentage of transfected cells that exhibit DDX5 - GFP " anti - stripe " pattern ( reduced GFP signal at DNA damage tracks compared to the signal in the nucleus ) at the times indicated in cells depleted of BRCA2 ( siBRCA2 ) or treated with control siRNA ( siC ) . The data represent the mean ± SEM from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Scheme showing the experimental set up for laser irradiation in DDX5-GFP transfected U2OS cells depleted of BRCA2 (siBRCA2) or control cells (siC). Middle: Live cell imaging of the recruitment of GFP-53BP1 or DDX5-GFP to DNA damage tracks at different time points as indicated. Exposure and processing were adjusted to best demonstrate stripes and anti-stripes. Scale bar indicates 10 µm. Bottom left: Western blot showing the siRNA mediated knock-down of BRCA2 from U2OS cells transfected with DDX5-GFP. Bottom right: Quantification of the percentage of transfected cells that exhibit DDX5-GFP "anti-stripe" pattern (reduced GFP signal at DNA damage tracks compared to the signal in the nucleus) at the times indicated in cells depleted of BRCA2 (siBRCA2) or treated with control siRNA (siC). The data represent the mean ± SEM from three independent experiments.


( B ) DDX5 ChIP - qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells ( - OHT ) or after tamoxifen addition ( + OHT ) . The data represent the mean ± SEM from three independent experiments . The green line represents the background levels of DDX5 signal . The statistical significance of the difference was calculated with unpaired one - tail t - test and the p - values show the significant differences between untreated cells ( - OHT ) and after tamoxifen addition ( + OHT ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) DDX5 ChIP-qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT).


( C ) Top : Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) and either untreated cells ( - OHT ) or after tamoxifen addition ( + OHT ) , as indicated . Scale bar indicates 10 µm . Bottom : Quantification of the number of γH2AX foci per nucleus ( left ) and DDX5 nuclear intensity ( right ) . The data represent at least 800 cells per condition from three independent experiments . The red line in the plot indicates the median and each symbol represents the value of a single cell . The statistical significance of the difference was calculated with Mann - Whitney U - test and the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.


( A ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 2 nM purified EGFP - MBP - BRCA2 ( top gel left ) or 10 nM purified BRCA2LT3 ( top gel right ) or 50 nM purified BRCA2T1 ( bottom gel ) . Bottom : Quantification of the unwinding experiments showing the percentage of free RNA relative to the R - loop substrate ( unwound product ) as a function of DDX5 concentration alone ( black ) or in presence of BRCA2 ( red ) or BRCA2LT3 ( blue ) or BRCA2T1 ( pink ) . The data represent the mean ± SD of at least three independent experiments . Right : SDS - PAGE showing 500 ng of purified 2XMBP - BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2 (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay.


( B ) Left : Thin layer chromatography ( TLC ) plate showing a representative ATP hydrolysis assay in which purified MBP - DDX5 - GST ( 50 - 200 nM ) was incubated with R - loop synthetic substrate and [ γ32P ] ATP in presence or absence of or 6 nM purified BRCA2LT3 . Right : Quantification of the ATP hydrolysed in each condition . No protein control was used as background . The data represent the mean ± SD from three independent experiments .

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

(B) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.


( A ) Left : GFP pull - down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 ( WT ) or the variant T207A ( T207A ) . DLD1 BRCA2 + / + ( + / + ) cell extracts were used as control for the GFP - trap . DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2 , respectively . StainFree images of the gels before transfer was used as loading control ( cropped image is shown ) . Right : Quantification of the GFP - trap pull - down experiments calculated as the co - immunoprecipitated DDX5 with either BRCA2 WT or BRCA2 - T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP - BRCA2 . Results are presented as the fold change compared to the BRCA2 WT clone . The data represents the mean ± SD of three independent experiments . Statistical significance of the difference was calculated with unpaired t test and the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: GFP pull-down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 (WT) or the variant T207A (T207A). DLD1 BRCA2+/+ (+/+) cell extracts were used as control for the GFP-trap. DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2, respectively. StainFree images of the gels before transfer was used as loading control (cropped image is shown). Right: Quantification of the GFP-trap pull-down experiments calculated as the co-immunoprecipitated DDX5 with either BRCA2 WT or BRCA2-T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP-BRCA2. Results are presented as the fold change compared to the BRCA2 WT clone. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


( B ) Left : Representative images of S9 . 6 immunofluorescence of DLD1 cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . The merged images show the signal of S9 . 6 , nucleolin ( nucleoli ) antibodies and DAPI staining . Scale bar indicates 25 µm . Right : Quantification of the average nuclear intensity of S9 . 6 antibody in DLD1 BRCA2 - WT ( WT ) or BRCA2 - T207A ( T207A ) cells transfected with either a plasmid expressing GFP alone ( - RH ) or GFP - RNase H1 ( + RH ) , as indicated . The red line in the plot indicates the median and each symbol represents the value of a single cell . Data correspond to at least 170 cells per condition from two independent experiments . The statistical significance of the difference was calculated with Mann - Whitney U - test ; p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative images of S9.6 immunofluorescence of DLD1 cells bearing BRCA2 (WT) or BRCA2-T207A (T207A). The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of the average nuclear intensity of S9.6 antibody in DLD1 BRCA2-WT (WT) or BRCA2-T207A (T207A) cells transfected with either a plasmid expressing GFP alone (-RH) or GFP-RNase H1 (+RH), as indicated. The red line in the plot indicates the median and each symbol represents the value of a single cell. Data correspond to at least 170 cells per condition from two independent experiments. The statistical significance of the difference was calculated with Mann-Whitney U-test; p-values show the significant difference.


( C ) Relative DRIP - qPCR signal values ( respect to the siC level at each locus ) at the MALAT1 , RRPH1 and HIST1H2BG loci in DLD1 BRCA2 - / - cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) and treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . The data represent the mean ± SEM from seven independent experiments . The statistical significance of the difference was calculated with paired t - Student test ; the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative DRIP-qPCR signal values (respect to the siC level at each locus) at the MALAT1, RRPH1 and HIST1H2BG loci in DLD1 BRCA2-/- cells bearing BRCA2 (WT) or BRCA2-T207A (T207A) and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The data represent the mean ± SEM from seven independent experiments. The statistical significance of the difference was calculated with paired t-Student test; the p-values show the significant difference.


( D ) Top : Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) , as indicated . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 750 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 (WT) or BRCA2-T207A (T207A), as indicated. Cells were fixed directly or 4h post-irradiation (6Gy). Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 750 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( A ) Top : Representative images of In situ PLA performed between DDX5 and S9 . 6 ( DNA - RNA hybrids ) antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) or DLD1 stable clones expressing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 500 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot . ( B ) Top : Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 600 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of In situ PLA performed between DDX5 and S9.6 (DNA-RNA hybrids) antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) or DLD1 stable clones expressing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 500 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (B) Top: Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Left : Representative images of the recruitment of transient transfected GFP - MBP - BRCA2 ( WT and T207A ) to DNA damage tracks at 5 min post - irradiation in U2OS cells . γH2AX is used as a marker of DNA damage . Scale bar indicates 10 µm . Right : Quantification of the intensity of GFP signal at the laser tracks . At least 130 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the two samples we applied an unpaired t - test . The red line in the plot indicates the median and each symbol represents a single cell intensity value .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative images of the recruitment of transient transfected GFP-MBP-BRCA2 (WT and T207A) to DNA damage tracks at 5 min post-irradiation in U2OS cells. γH2AX is used as a marker of DNA damage. Scale bar indicates 10 µm. Right: Quantification of the intensity of GFP signal at the laser tracks. At least 130 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the two samples we applied an unpaired t-test. The red line in the plot indicates the median and each symbol represents a single cell intensity value.


( D ) Top : Representative images of in situ PLA performed between S9 . 6 ( DNA - RNA hybrids ) and anti - γH2AX antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Bottom : Quantification of the number of PLA spots per nucleus . At least 600 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( E ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 50 nM purified BRCA2T1 - T207A ( bottom right ) . Bottom left : Quantification of the unwinding experiments showing the percentage of free RNA relative to the R - loop substrate ( unwound product ) as a function of DDX5 concentration alone ( black ) or in presence of BRCA2T1 - T207A ( green ) . The data represent the mean ± SD of at least three independent experiments . Bottom right : SDS - PAGE showing 650 ng of purified 2XMBP - BRCA2T1 - T207A used in the unwinding assay .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 50 nM purified BRCA2T1-T207A (bottom right). Bottom left: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2T1-T207A (green). The data represent the mean ± SD of at least three independent experiments. Bottom right: SDS-PAGE showing 650 ng of purified 2XMBP-BRCA2T1-T207A used in the unwinding assay.


( A ) Top : Representative immunofluorescence images of cells stained for RAD51 ( green ) in U2OS cells depleted of DDX5 ( siDDX5 ) and in control cells ( siC ) in non - treated ( - ) or different time points after exposure to IR ( 6Gy ) , as indicated . Scale bar indicates 10 µm . Bottom : Graph showing the average number of RAD51 repair foci in both cell lines . The data represent the mean ± SEM of three independent experiments .

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

(A) Top: Representative immunofluorescence images of cells stained for RAD51 (green) in U2OS cells depleted of DDX5 (siDDX5) and in control cells (siC) in non-treated (-) or different time points after exposure to IR (6Gy), as indicated. Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in both cell lines. The data represent the mean ± SEM of three independent experiments.


( B ) Top : Representative immunofluorescence images of DLD1 BRCA2 deficient cells ( BRCA2 - / - ) or BRCA2 - / - cells expressing BRCA2 WT ( WT ) or BRCA2 - T207A ( T207A ) in non - treated or at different time points post - IR ( 6 Gy ) , as indicated ; hybridized with anti - RAD51 antibody ( green ) . Scale bar indicates 10 µm . Bottom : Graph showing the average number of RAD51 repair foci in the three cell lines . The data represent the mean ± SEM of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Representative immunofluorescence images of DLD1 BRCA2 deficient cells (BRCA2-/-) or BRCA2-/- cells expressing BRCA2 WT (WT) or BRCA2-T207A (T207A) in non-treated or at different time points post-IR (6 Gy), as indicated; hybridized with anti-RAD51 antibody (green). Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in the three cell lines. The data represent the mean ± SEM of three independent experiments.


( C ) Left : Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2 - T207A cells 4h post - irradiation ( 6Gy ) hybridized with anti - γH2AX and anti - RPA antibodies . Right : Quantification of the number of γH2AX and RPA foci per nucleus , as indicated . The data represent at least 400 cells per condition from two independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single focus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2-T207A cells 4h post-irradiation (6Gy) hybridized with anti-γH2AX and anti-RPA antibodies. Right: Quantification of the number of γH2AX and RPA foci per nucleus, as indicated. The data represent at least 400 cells per condition from two independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.


( D ) Left : Representative immunofluorescence images of DLD1 BRCA2 - T207A cells 2h post - irradiation ( 6Gy ) , as indicated , hybridized with anti - γH2AX and anti - RAD51 antibodies . ( RH ) RNase H . Right : Quantification of the number of γH2AX foci ( left ) or RAD51 foci ( right ) per nucleus . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . The data shown is from at least 400 cells per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single focus .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative immunofluorescence images of DLD1 BRCA2-T207A cells 2h post-irradiation (6Gy), as indicated, hybridized with anti-γH2AX and anti-RAD51 antibodies. (RH) RNase H. Right: Quantification of the number of γH2AX foci (left) or RAD51 foci (right) per nucleus. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. The data shown is from at least 400 cells per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.


( E ) Top : Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2 - T207A ( T207A ) in chromatin fraction . When indicated , cells were irradiated ( 2h treatment ) and / or transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . α - tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively . Bottom : quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE . Results are presented as the fold change compared to the untreated sample . The data represents the mean ± SD of three independent experiments . Statistical significance of the difference was calculated with unpaired t test and the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2-T207A (T207A) in chromatin fraction. When indicated, cells were irradiated (2h treatment) and/or transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. α-tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively. Bottom: quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE. Results are presented as the fold change compared to the untreated sample. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


Cell viability analysis using CellTiter - Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs ( n = 4 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . SM represents SM - 164 . Error bars represent SD and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 .

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

Cell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


TNF - induced complex - II immuno - precipitation . BN175 cells were treated with the indicated agents for 8 hrs . FADD immuno - precipitation was performed followed by western blot analysis ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) .

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

TNF-induced complex-II immuno-precipitation. BN175 cells were treated with the indicated agents for 8 hrs. FADD immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).


Proximity ligation assay between Ripk1 and Casp - 8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs ( n = 3 biological replicates , shown are representative images ) . Scale bar represents 10 μm . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) .

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

Proximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs (n = 3 biological replicates, shown are representative images). Scale bar represents 10 μm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).


DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . Error bars represent SD and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 .

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

DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.


Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 knockouts ( KO ) cells treated with the indicated agents for 18 and 48 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

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

Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


TNF - induced complex - II immuno - precipitation . HT1080 cells were treated with the indicated agents for 8 hrs . CASP - 8 immuno - precipitation was performed followed by western blot analysis ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) .

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

TNF-induced complex-II immuno-precipitation. HT1080 cells were treated with the indicated agents for 8 hrs. CASP-8 immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml).


Cell death analysis measured by Celigo of PI - positive HT1080 cells , cells were treated with the indicated agents for 24 hrs . ( n = 3 biological replicates ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and an unpaired t - test was performed , * * * * P ≤ 0 . 0001 .

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

Cell death analysis measured by Celigo of PI-positive HT1080 cells, cells were treated with the indicated agents for 24 hrs. (n = 3 biological replicates). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and an unpaired t-test was performed, **** P ≤ 0.0001.


Cell death analysis by Celigo of PI - positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed , * * * * P ≤ 0 . 0001 .

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

Cell death analysis by Celigo of PI-positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed, **** P ≤ 0.0001.


TNF - induced complex - II immuno - precipitation . A375 cells were treated with the indicated agents for 6 hrs . Caspase - 8 immuno - precipitation was performed followed by western blot analysis . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) ( n = 3 biological replicates , shown is a representative experiment ) .

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

TNF-induced complex-II immuno-precipitation. A375 cells were treated with the indicated agents for 6 hrs. Caspase-8 immuno-precipitation was performed followed by western blot analysis. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) (n = 3 biological replicates, shown is a representative experiment).