d ( left ) In the three - chamber sociability test Grin2aS / S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a + / S and Grin2a + / + littermates . d ( right ) Occupancy heat maps in the three - chamber sociability test show a representative example of a Grin2aS / S mouse with a reduced preference for another mouse versus an inanimate object . The social preference is visible for Grin2a + / + and Grin2a + / S mice . The occupancy is color - coded separately for each group and translates to a % given in white numbers on the key for each genotype .

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d (left) In the three-chamber sociability test Grin2aS/S mutants had a significantly reduced preference to explore the stimulus mouse over the object compared to Grin2a+/S and Grin2a+/+ littermates. d (right) Occupancy heat maps in the three-chamber sociability test show a representative example of a Grin2aS/S mouse with a reduced preference for another mouse versus an inanimate object. The social preference is visible for Grin2a+/+ and Grin2a+/S mice. The occupancy is color-coded separately for each group and translates to a % given in white numbers on the key for each genotype.


f During the sample run in the Y - maze Grin2aS / S mice made significantly more total arm entries . In the test run , Grin2a + / + and Grins2a + / S showed a preference for the unexplored novel Y - maze arm , whereas Grin2aS / S mice showed a lack of novelty preference . The numbers of mice are shown in brackets or below the bars of bar graphs . Chance levels ( Ch . ) are indicated by dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 7 ) .

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f During the sample run in the Y-maze Grin2aS/S mice made significantly more total arm entries. In the test run, Grin2a+/+ and Grins2a+/S showed a preference for the unexplored novel Y-maze arm, whereas Grin2aS/S mice showed a lack of novelty preference. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 7).


c Morris watermaze . In the standard Morris watermaze task ( left graph ) the path length to reach the hidden platform decreased across training blocks for Grin2a + / + and Grin2a + / S mice but not for Grin2aS / S mice . Two probe trials were conducted after 12 and 24 training trials ( P1 and P2 , respectively ) during which the platform was removed from the pool . At both P1 and P2 ( right bar graphs ) the Grin2aS / S mice failed to search for the platform in the target quadrant . AdjL adjacent left , Target fixed location of the hidden escape platform during acquisition , AdjR adjacent right , Opp opposite of target quadrant . Dashed lines indicate chance levels .

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c Morris watermaze. In the standard Morris watermaze task (left graph) the path length to reach the hidden platform decreased across training blocks for Grin2a+/+ and Grin2a+/S mice but not for Grin2aS/S mice. Two probe trials were conducted after 12 and 24 training trials (P1 and P2, respectively) during which the platform was removed from the pool. At both P1 and P2 (right bar graphs) the Grin2aS/S mice failed to search for the platform in the target quadrant. AdjL adjacent left, Target fixed location of the hidden escape platform during acquisition, AdjR adjacent right, Opp opposite of target quadrant. Dashed lines indicate chance levels.


d Rewarded alternation . ( left ) In the T - maze rewarded alternation task ( right ) spatial working memory performance was substantially impaired in Grin2aS / S mice .

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d Rewarded alternation. (left) In the T-maze rewarded alternation task (right) spatial working memory performance was substantially impaired in Grin2aS/S mice.


e Contiguous task . In the contiguous version of the conditional T - maze task ( with floor inserts covering the entire T - maze ; white Perspex versus gray wire mesh ) , Grin2a + / + and Grin2aS / S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm .

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e Contiguous task. In the contiguous version of the conditional T-maze task (with floor inserts covering the entire T-maze; white Perspex versus gray wire mesh), Grin2a+/+ and Grin2aS/S mice were able to associate a particular floor insert with the location of the reward in either the left or the right goal arm.


f Discontiguous task . Separate groups of mice were trained in the discontiguous version of this conditional task , in which the floor insert cues were now limited to the start arm only . Grin2a + / + mice readily acquired the task , but Grin2aS / S mice failed to learn . The numbers of mice are shown in brackets or below the bars of bar graphs . Chance levels ( Ch . ) are indicated by dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 8 ) .

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f Discontiguous task. Separate groups of mice were trained in the discontiguous version of this conditional task, in which the floor insert cues were now limited to the start arm only. Grin2a+/+ mice readily acquired the task, but Grin2aS/S mice failed to learn. The numbers of mice are shown in brackets or below the bars of bar graphs. Chance levels (Ch.) are indicated by dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 8).


A Heart rate ( HR ) of WT and Adcy9 - / - mice averaged over 5 min of non - active versus sustained activity on home cage running wheels . Student ' s t - test P values for indicated comparisons are shown . Circles represent data from individual animals . The boxplot central band is the median while whiskers represent the 10th and 90th percentiles .

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A Heart rate (HR) of WT and Adcy9-/- mice averaged over 5 min of non-active versus sustained activity on home cage running wheels. Student's t-test P values for indicated comparisons are shown. Circles represent data from individual animals. The boxplot central band is the median while whiskers represent the 10th and 90th percentiles.


B , C HR and R - R interval of WT ( black bars ) and Adcy9 - / - mice ( red bars ) before and after ISO ( 1 μg / g )

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B,C HR and R-R interval of WT (black bars) and Adcy9-/- mice (red bars) before and after ISO (1 μg/g) injection


D Representative ECG recordings of 7 mo WT and Adcy9 - / - mice , ~ 55 min post ISO - injection .

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D Representative ECG recordings of 7 mo WT and Adcy9-/- mice, ~55 min post ISO-injection.


E , F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences ( RMSSD ; panel ( E ) ) and percentage of sequential R - R intervals differing by > 6 ms ( pNN6 ; panel ( F ) ) methods .

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E,F HR variability was calculated before and after ISO injection using the root mean square of successive RR interval differences (RMSSD; panel (E)) and percentage of sequential R-R intervals differing by >6 ms (pNN6; panel (F)) methods.


A , B PLA assay performed in neonatal cardiomyocytes expressing GFP - Flag or AC9 - Flag using antibodies against Flag and endogenous POPDC1 , POPDC2 or Gβγ . Images ( A ) ; scale bar is 20 um ; nuclear staining by DAPI shown in blue ) and quantitation ( B ) of PLA signal are shown .

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A,B PLA assay performed in neonatal cardiomyocytes expressing GFP-Flag or AC9-Flag using antibodies against Flag and endogenous POPDC1, POPDC2 or Gβγ. Images (A); scale bar is 20 um; nuclear staining by DAPI shown in blue) and quantitation (B) of PLA signal are shown.


( D ) Quantification of COS7 cells expressing BiFC constructs for AC9 , POPDC 1 - 3 . The expressed proteins tagged with VN ( top line ) and VC ( bottom line ) are shown .

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(D) Quantification of COS7 cells expressing BiFC constructs for AC9, POPDC 1-3. The expressed proteins tagged with VN (top line) and VC (bottom line) are shown.


( E ) Representative live - cell images of indicated BiFC combinations in HEK293 cells ( n > 20 cells ; scale bar : 10 μm ) . Quantification of POPDC1 - VN : AC9 - VC is shown in Fig 3 and 7 .

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(E) Representative live-cell images of indicated BiFC combinations in HEK293 cells (n>20 cells; scale bar: 10 μm). Quantification of POPDC1-VN:AC9-VC is shown in Fig 3 and 7.


A HEK293 cells expressing Flag - AC9 in the presence or absence of Myc - tagged POPDC1 or - 2 were subjected to co - immunoprecipitation ( Co - IP ) with anti - MYC and assayed for AC activity with 300 nM Gαs - GTPγS . Kruskal - Wallis One Way ANOVA analysis on Ranks was performed ( n = 6 experiments , P = 0 . 003 between all groups ) with multiple comparisons to AC9 control by Dunn ' s method and Mann - Whitney Rank Sum Test ( * * P = 0 . 002 ) .

A HEK293 cells expressing Flag-AC9 in the presence or absence of Myc-tagged POPDC1 or -2 were subjected to co-immunoprecipitation (Co-IP) with anti-MYC and assayed for AC activity with 300 nM Gαs-GTPγS. Kruskal-Wallis One Way ANOVA analysis on Ranks was performed (n=6 experiments, P=0.003 between all groups) with multiple comparisons to AC9 control by Dunn's method and Mann-Whitney Rank Sum Test (**P=0.002).


B A portion of the lysates and Co - IP from ( A ) were subjected to western blot ( WB ) analysis . Sf9 cells expressing Flag - AC9 served as a positive WB control . Molecular weight markers are denoted as M . Quantitation of Flag - AC9 WB by One Way ANOVA , n = 3 - 4 experiments , with comparisons by Tukey test , * * P = 0 . 003 , * * * P = 0 . 001 .

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B A portion of the lysates and Co-IP from (A) were subjected to western blot (WB) analysis. Sf9 cells expressing Flag-AC9 served as a positive WB control. Molecular weight markers are denoted as M. Quantitation of Flag-AC9 WB by One Way ANOVA, n=3-4 experiments, with comparisons by Tukey test, **P=0.003, ***P=0.001.


( C ) HEK293 cells expressing Flag - AC9 + / - POPDC1 - Myc or POPDC2 - Myc were subjected to Co - IP with anti - FLAG and subjected to WB analysis with anti - MYC and anti - FLAG . Quantitation of anti - MYC WB by One Way ANOVA , n = 4 experiments , with comparisons by Tukey test , * * P = 0 . 003 , * * * P < 0 . 001 ) .

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(C) HEK293 cells expressing Flag-AC9 +/- POPDC1-Myc or POPDC2-Myc were subjected to Co-IP with anti-FLAG and subjected to WB analysis with anti-MYC and anti-FLAG. Quantitation of anti-MYC WB by One Way ANOVA, n=4 experiments, with comparisons by Tukey test, **P=0.003, ***P<0.001).


( D ) HEK293 cells expressing POPDC1 - Myc + / - GFP - tagged AC9 and control TM proteins EGFR , or LAMP1 were subjected to Co - IP with anti - MYC . Western blotting of lysates and Co - IPs for GFP ( top ) and MYC ( bottom ) are shown ( n = 3 experiments ) .

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(D) HEK293 cells expressing POPDC1-Myc +/- GFP-tagged AC9 and control TM proteins EGFR, or LAMP1 were subjected to Co-IP with anti-MYC. Western blotting of lysates and Co-IPs for GFP (top) and MYC (bottom) are shown (n=3 experiments).


( F ) BiFC of AC9 and indicated POPDC1 truncations in COS - 7 cells . Kruskal - Wallis One Way ANOVA analysis was performed ( n = 4 experiments ) with multiple comparisons by Tukey test , * * * P < 0 . 001 compared to VN control , * * P = 0 . 008 .

(F) BiFC of AC9 and indicated POPDC1 truncations in COS-7 cells. Kruskal-Wallis One Way ANOVA analysis was performed (n=4 experiments) with multiple comparisons by Tukey test, ***P<0.001 compared to VN control, **P=0.008.


( G ) Co - IP with anti - MYC in COS - 7 cells expressing Flag - AC9 and indicated Myc - tagged POPDC1 truncations . Western blotting with anti - AC9 and anti - MYC ( POPDC1 ) of Co - IP and lysates is shown . Kruskal - Wallis One Way ANOVA analysis was performed ( n = 3 - 5 experiments , P = 0 . 006 ) , with multiple comparisons by Dunn ' s method ( * P = 0 . 021 , * * P = 0 . 01 ) .

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(G) Co-IP with anti-MYC in COS-7 cells expressing Flag-AC9 and indicated Myc-tagged POPDC1 truncations. Western blotting with anti-AC9 and anti-MYC (POPDC1) of Co-IP and lysates is shown. Kruskal-Wallis One Way ANOVA analysis was performed (n=3-5 experiments, P=0.006), with multiple comparisons by Dunn's method (*P=0.021, **P=0.01).


A Co - localization of CFP - TREK - 1 with the BiFC signal from AC9 - VN : AC9 - VC homodimer ( top ) and POPDC1 - VN : AC9 - VC complex ( bottom ) in HEK293 cells .

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A Co-localization of CFP-TREK-1 with the BiFC signal from AC9-VN:AC9-VC homodimer (top) and POPDC1-VN:AC9-VC complex (bottom) in HEK293 cells.


B The lifetime distribution of Cerulean - tagged proteins + / - the indicated YFP - tagged proteins expressed in HEK293 cells . Mann - Whitney Rank Sum Test was performed ( * * * P < 0 . 001 ; n = cell number indicated on each bar ) .

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B The lifetime distribution of Cerulean-tagged proteins +/- the indicated YFP-tagged proteins expressed in HEK293 cells. Mann-Whitney Rank Sum Test was performed (***P<0.001; n=cell number indicated on each bar).


C Co - IP of Myc - tagged POPDC1 ( anti - MYC ) pulled down both Flag - AC9 and YFP - tagged TREK - 1 ( n = 3 experiments ) . One - way ANOVA was performed with multiple comparisons by Holm - Sidak method ( # P = 0 . 007 compared to control ) .

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C Co-IP of Myc-tagged POPDC1 (anti-MYC) pulled down both Flag-AC9 and YFP-tagged TREK-1 (n=3 experiments). One-way ANOVA was performed with multiple comparisons by Holm-Sidak method (#P=0.007 compared to control).


D Co - IP of TREK - 1 ( anti - GFP ) pulls down Flag - AC9 and endogenous POPDC1 . AKAP79 is not pulled down in complex ( n = 3 experiments ) .

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D Co-IP of TREK-1 (anti-GFP) pulls down Flag-AC9 and endogenous POPDC1. AKAP79 is not pulled down in complex (n=3 experiments).


A IP - AC assay with IgG versus anti - TREK - 1 in heart homogenates from WT and Adcy9 - / - mice ; AC activity stimulated with 300 nM Gαs - GTPγS . IP - AC with anti - AKAP150 is shown as a positive control . Two - way ANOVA ( overall P < 0 . 001 , n = 3 mice per genotype ) with multiple comparisons by Holm - Sidak test ; # P < 0 . 001 for indicated comparison or to respective IgG controls . IgG versus anti - TREK - 1 in Adcy9 - / - was analyzed by Student ' s t - test ( * * P = 0 . 009 , n = 3 ) . A portion of the co - IP was subjected to western blotting with anti - TREK - 1 and anti - AKAP150 .

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A IP-AC assay with IgG versus anti-TREK-1 in heart homogenates from WT and Adcy9-/- mice; AC activity stimulated with 300 nM Gαs-GTPγS. IP-AC with anti-AKAP150 is shown as a positive control. Two-way ANOVA (overall P<0.001, n=3 mice per genotype) with multiple comparisons by Holm-Sidak test; #P<0.001 for indicated comparison or to respective IgG controls. IgG versus anti-TREK-1 in Adcy9-/- was analyzed by Student's t-test (**P=0.009, n=3). A portion of the co-IP was subjected to western blotting with anti-TREK-1 and anti-AKAP150.


B IP - AC assay with IgG versus anti - TREK in WT and Popdc1 - / - mouse heart homogenates ; AC activity stimulated as in ( A ) . Statistics as in ( A ) , n = 4 mice per genotype , # P < 0 . 001 . Paired t - test within Popdc1 - / - , P = 0 . 123 .

B IP-AC assay with IgG versus anti-TREK in WT and Popdc1-/- mouse heart homogenates; AC activity stimulated as in (A). Statistics as in (A), n=4 mice per genotype, #P<0.001. Paired t-test within Popdc1-/-, P=0.123.


C Total AC activity in heart homogenates from WT or Popdc1 - / - ( P1 - / - ) mice stimulated with 300 nM Gαs - GTPγS or 100 μM calcium and 300 nM calmodulin .

C Total AC activity in heart homogenates from WT or Popdc1-/- (P1-/-) mice stimulated with 300 nM Gαs-GTPγS or 100 μM calcium and 300 nM calmodulin.


D IP - AC assay with anti - MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc - tagged POPDC1 . AC activity stimulated with 100 μM calcium and 300 nM calmodulin . Student ' s t - test ( n = 3 experiments , * * P = 0 . 0011 and * P = 0 . 012 ) .

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D IP-AC assay with anti-MYC from cell lysates expressing AC1 or AC8 in the presence or absence of Myc-tagged POPDC1. AC activity stimulated with 100 μM calcium and 300 nM calmodulin. Student's t-test (n=3 experiments, **P=0.0011 and *P=0.012).


E IP - AC assay with IgG versus anti - TREK - 1 in WT and Popdc1 - / - mouse heart homogenates ; AC activity stimulated as in ( D ) . Student ' s t - test ( * P = 0 . 03 ; * * P = 0 . 005 , n = 4 mice per genotype ) .

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E IP-AC assay with IgG versus anti-TREK-1 in WT and Popdc1-/- mouse heart homogenates; AC activity stimulated as in (D). Student's t-test (*P=0.03; **P=0.005, n=4 mice per genotype).


A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag - AC9 and POPDC1 - Myc pulled down by TREK - 1 co - IP .

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A Treatment of HEK293 cells for 10 min in the presence of 10 µM ISO reduced the amount of Flag-AC9 and POPDC1-Myc pulled down by TREK-1 co-IP.


B , C Quantification ( IP / input ) of Flag - AC9 ( B ) and POPDC1 - Myc ( C ) between ISO treatment groups by two - tailed paired Student ' s t - test ( * P = 0 . 028 , * * P = 0 . 007 ; n = 3 biological replicates ) .

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B, C Quantification (IP/input) of Flag-AC9 (B) and POPDC1-Myc (C) between ISO treatment groups by two-tailed paired Student's t-test (*P=0.028, **P=0.007; n=3 biological replicates).


, B BiFC signal between the indicated VN - and VC - tagged proteins ( POPDC1 , P1 ) . B Percent decrease of BiFC signal in ( A ) with ISO treatment ( 10 μM , 10 min at 37 ° C ) . Paired t - test for each condition ( vehicle versus ISO ) using raw data prior to calculation of percent of vehicle control ( n = 6 ; * P < 0 . 05 ; * * P < 0 .

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, B BiFC signal between the indicated VN- and VC-tagged proteins (POPDC1, P1). B Percent decrease of BiFC signal in (A) with ISO treatment (10 μM, 10 min at 37°C). Paired t-test for each condition (vehicle versus ISO) using raw data prior to calculation of percent of vehicle control (n=6; *P<0.05; **P<0.01


C ISO dose - response curves for BiFC interactions between POPDC1 : AC9 , TREK - 1 : AC9 , and TREK - 1 with a catalytically inactive AC9 ( TREK - 1 : AC9D ) .

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C ISO dose-response curves for BiFC interactions between POPDC1:AC9, TREK-1:AC9, and TREK-1 with a catalytically inactive AC9 (TREK-1:AC9D).


D TREK - 1 : AC9 BiFC signal is absent in COS - 7 cells lacking detectable POPDC1 . TREK - 1 : TREK - 1 BiFC is a positive control . One - way ANOVA with multiple comparisons by Holm Sidak method ( n = 3 experiments ; * * * P < 0 . 001 ) .

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D TREK-1:AC9 BiFC signal is absent in COS-7 cells lacking detectable POPDC1. TREK-1:TREK-1 BiFC is a positive control. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3 experiments; ***P<0.001).


E Overexpression of POPDC1 truncation of Popeye domain ( POPDC1 - Δ172 ) , but not WT POPDC1 , abolishes ISO reduction of TREK - 1 : AC9 BiFC signal . One - way ANOVA with multiple comparisons by Holm Sidak method ( n = 3 - 4 experiments ; * * * P < 0 . 001 compared to control ) .

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E Overexpression of POPDC1 truncation of Popeye domain (POPDC1-Δ172), but not WT POPDC1, abolishes ISO reduction of TREK-1:AC9 BiFC signal. One-way ANOVA with multiple comparisons by Holm Sidak method (n=3-4 experiments; ***P<0.001 compared to control).


A Representative two - electrode voltage clamp measurements in Xenopus oocytes injected with TREK - 1 alone ( orange ) or TREK - 1 co - injected with cRNAs encoding the indicated proteins . Voltage was ramped from - 120 to + 40 mV . B Relative TREK - 1 current amplitudes at + 40 mV normalized to TREK - 1 injected alone . The number of oocytes is given within the bar graphs ( n = 37 - 97 ) . 3 - 5 batches of oocytes were used for each condition .

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A Representative two-electrode voltage clamp measurements in Xenopus oocytes injected with TREK-1 alone (orange) or TREK-1 co-injected with cRNAs encoding the indicated proteins. Voltage was ramped from -120 to +40 mV. B Relative TREK-1 current amplitudes at +40 mV normalized to TREK-1 injected alone. The number of oocytes is given within the bar graphs (n=37-97). 3-5 batches of oocytes were used for each condition.


C Relative currents normalized to TREK - 1 plus POPDC1 in the absence and presence of theophylline . Data are plotted as mean ± SEM .

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C Relative currents normalized to TREK-1 plus POPDC1 in the absence and presence of theophylline. Data are plotted as mean ± SEM.


D , E Oocytes were treated with 0 . 5 mM theophylline prior to voltage - clamp measurements as in ( A , B ) .

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D, E Oocytes were treated with 0.5 mM theophylline prior to voltage-clamp measurements as in (A, B).


( B ) Gating strategy . pDC were identified as lineage ( TCRb , CD19 , Ly6G , NK1 . 1 ) - CD11b - CD11clow / int CD317high cells . 5 . 2 vs 5 . 1 cells were finally discriminated within the pDC gate

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(B) Gating strategy. pDC were identified as lineage (TCRb, CD19, Ly6G, NK1.1)- CD11b- CD11clow/int CD317high cells. 5.2 vs 5.1 cells were finally discriminated within the pDC gate


( F ) Enrichment of the gene modules identified in ( E ) for independent gene sets previously established from the analysis of other microarray datasets ( blue font ) , belonging to specific signaling pathways ( red font ) , associated to specific functions or diseases ( violet font ) or regulated by the indicated upstream regulators ( black font ) , as assessed by using Ingenuity Pathway Analysis ( IPA ) . Data are represented as a heatmap , with gene modules identified in panel E as columns and gene sets as rows . The colored cells indicate the significance of the enrichment of each gene module for each gene set , as computed by IPA analysis and according to the color scale shown below the heatmap

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

(F) Enrichment of the gene modules identified in (E) for independent gene sets previously established from the analysis of other microarray datasets (blue font), belonging to specific signaling pathways (red font), associated to specific functions or diseases (violet font) or regulated by the indicated upstream regulators (black font), as assessed by using Ingenuity Pathway Analysis (IPA). Data are represented as a heatmap, with gene modules identified in panel E as columns and gene sets as rows. The colored cells indicate the significance of the enrichment of each gene module for each gene set, as computed by IPA analysis and according to the color scale shown below the heatmap


Representative genes were selected for modules identified on Venn diagram in Fig 1E and their expression pattern across all samples shown as a heatmap . Red font , pDC ISG ( module A ) ; green font , pDC MSG ( module D ) ; mixed red and green font , pDC ISG and MSG ( module E ) ; black font , non - ISG , non - MSG ( module B ) . The relative mRNA expression levels for each gene ( lanes ) across biological samples ( rows ) are represented by colors according to the scale shown above the heatmap . For the legend of the symbols depicting the classes of biological samples above each column please refer to the symbol key in Fig 1D .

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

Representative genes were selected for modules identified on Venn diagram in Fig 1E and their expression pattern across all samples shown as a heatmap. Red font, pDC ISG (module A); green font, pDC MSG (module D); mixed red and green font, pDC ISG and MSG (module E); black font, non-ISG, non-MSG (module B). The relative mRNA expression levels for each gene (lanes) across biological samples (rows) are represented by colors according to the scale shown above the heatmap. For the legend of the symbols depicting the classes of biological samples above each column please refer to the symbol key in Fig 1D.


( A ) Histogram representing CD86 expression in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - infected MBMC ; grey , 5 . 2 + and 5 . 1 + pDC from CTR MBMC ; red , 5 . 2 + Ifnar1 - / - pDC isolated from Ifnar1 - TST MBMC ; purple , 5 . 1 + WT pDC isolated from Ifnar1 - TST MBMC . Dotted line shows isotype control staining . One histogram representative of 6 animals from 3 independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histogram representing CD86 expression in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-infected MBMC; grey, 5.2+ and 5.1+ pDC from CTR MBMC; red, 5.2+ Ifnar1-/- pDC isolated from Ifnar1-TST MBMC; purple, 5.1+ WT pDC isolated from Ifnar1-TST MBMC. Dotted line shows isotype control staining. One histogram representative of 6 animals from 3 independent experiments is shown.


( B ) Dot plots represent intracellular IFN - I staining in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - infected MBMC . Isotype control ( IC ) staining was performed on a mixture of cells isolated from both types of MCMV - infected MBMC .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dot plots represent intracellular IFN-I staining in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-infected MBMC. Isotype control (IC) staining was performed on a mixture of cells isolated from both types of MCMV-infected MBMC.


The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from MCMV - infected Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red )

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

The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from MCMV-infected Black, CTR MBMC; red, Ifnar1-TST MBMC (red)


The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from CpG - stimulated mice . Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red ) Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group .

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

The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from CpG-stimulated mice. Black, CTR MBMC; red, Ifnar1-TST MBMC (red) Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.


The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from MCMV - infected Black , CTR MBMC pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group .

The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from MCMV-infected Black, CTR MBMC pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.


The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Results were expressed as 5 . 2 / 5 . 1 ratio of Mean Fluorescence Intensity ( MFI ) values for CD86 or of the percentages of IFN - I - producing cells obtained from CpG - stimulated mice . Black , CTR MBMC pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group .

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

The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Results were expressed as 5.2/5.1 ratio of Mean Fluorescence Intensity (MFI) values for CD86 or of the percentages of IFN-I-producing cells obtained from CpG-stimulated mice. Black, CTR MBMC pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.


The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Ratio of IL - 12 - and TNF - producing cells in pDC isolated from indicated MCMV - infected MBMC . Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red ) Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group .

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

The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Ratio of IL-12- and TNF-producing cells in pDC isolated from indicated MCMV-infected MBMC. Black, CTR MBMC; red, Ifnar1-TST MBMC (red) Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.


The expression of indicated markers was analyzed in 5 . 2 + vs 5 . 1 + pDC of each MBMC type . Ratio of IL - 12 - and TNF - producing cells in pDC isolated from indicated MCMV - infected MBMC . Black , CTR MBMC ; pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with at least 3 mice per group .

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

The expression of indicated markers was analyzed in 5.2+ vs 5.1+ pDC of each MBMC type. Ratio of IL-12- and TNF-producing cells in pDC isolated from indicated MCMV-infected MBMC. Black, CTR MBMC ; pale blue, Ap3b1-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with at least 3 mice per group.


( A ) Dot plots represent IFN - I vs GFP staining in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - GFP - infected MBMC . One staining representative of 6 animals from 2 independent experiments is shown

O O O O O O O O O O O O O O O O O O O O O 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) Dot plots represent IFN-I vs GFP staining in 5.2+ vs 5.1+ pDC isolated from indicated MCMV-GFP-infected MBMC. One staining representative of 6 animals from 2 independent experiments is shown


( B , C ) Percentages of GFP + cells within 5 . 2 + pDC ( B ) or of GFP + ( green ) vs GFP - ( white ) cells within 5 . 2 + IFN - I - producing pDC ( C ) isolated from indicated MCMV - GFP - infected MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Percentages of GFP+ cells within 5.2+ pDC (B) or of GFP+ (green) vs GFP- (white) cells within 5.2+ IFN-I-producing pDC (C) isolated from indicated MCMV-GFP-infected MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group


( D ) qPCR for the expression of the indicated genes was performed on whole mRNA isolated from the spleen of indicated MCMV - infected MBMC . CTR MBMC ( black ) and Myd88 - / - TST MBMC ( dark green ) were treated with isotype control ( IC , square ) or blocking α - IFNAR1 mAbs ( triangle ) . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 2 - 3 mice per group

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qPCR for the expression of the indicated genes was performed on whole mRNA isolated from the spleen of indicated MCMV-infected MBMC. CTR MBMC (black) and Myd88-/- TST MBMC (dark green) were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group


( E - G ) CD86 MFI in 5 . 2 + pDC of each indicated MBMC type ( E ) and 5 . 2 / 5 . 1 ratio of cytokine - producing pDC ( F , G ) . Data ( mean ± SEM ) are representative of 2 pooled independent experiments each with 2 - 3 mice per group

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G) CD86 MFI in 5.2+ pDC of each indicated MBMC type (E) and 5.2/ 5.1 ratio of cytokine-producing pDC (F, G). Data (mean±SEM) are representative of 2 pooled independent experiments each with 2-3 mice per group


CD86 MFI and percentages of cytokine - producing cells within pDC of each indicated mouse strain . Black , C57BL6 ; red , Ifnar1 - / - ; orange , Ifnar1 - / - x Myd88 - / -

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

CD86 MFI and percentages of cytokine-producing cells within pDC of each indicated mouse strain. Black, C57BL6; red, Ifnar1-/-; orange, Ifnar1-/-x Myd88-/-


CD86 MFI and percentages of cytokine - producing cells within pDC of each indicated mouse strain . Black , C57BL6 green , Tlr9 - / - and pale green , Tlr7 - / - . In ( K - L ) indicated mice were treated with IC ( square ) or blocking α - IFNAR1 mAbs ( triangle ) . Data depicted ( mean ± SEM ) are from 2 pooled independent experiments each with 2 - 3 mice per group .

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

CD86 MFI and percentages of cytokine-producing cells within pDC of each indicated mouse strain. Black, C57BL6 green, Tlr9-/- and pale green, Tlr7-/-. In (K-L) indicated mice were treated with IC (square) or blocking α-IFNAR1 mAbs (triangle). Data depicted (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group.


( A ) IRF7 protein expression in pDC isolated from Irf7 - / - ( pink , bottom ) , C57BL / 6 ( WT , black , top ) and Ifnar1 - / - ( red , middle ) uninfected ( empty histograms , Ø , left ) vs MCMV - infected mice ( filled histograms , MCMV , right ) . For each condition histograms are representative of one out of 4 - 6 mice from 2 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

(A) IRF7 protein expression in pDC isolated from Irf7-/- (pink, bottom), C57BL/6 (WT, black, top) and Ifnar1-/- (red, middle) uninfected (empty histograms, Ø, left) vs MCMV-infected mice (filled histograms, MCMV, right). For each condition histograms are representative of one out of 4-6 mice from 2 independent experiments


( B ) IRF7 protein expression in pDC ( left ) , cDC ( middle ) and conventional monocytes ( cMo , right ) isolated from uninfected ( Ø , empty ) or MCMV - infected ( MCMV , filled ) C57BL / 6 ( black ) or Ifnar1 - / - ( red ) mice . Data are shown as net IRF7 MFI , after subtraction of the MFI obtained using isotype control mAbs . Data shown ( mean ± SEM ) are pooled from 2 independent experiments with 5 - 6 mice for each group

(B) IRF7 protein expression in pDC (left), cDC (middle) and conventional monocytes (cMo, right) isolated from uninfected (Ø, empty) or MCMV-infected (MCMV, filled) C57BL/6 (black) or Ifnar1-/- (red) mice. Data are shown as net IRF7 MFI, after subtraction of the MFI obtained using isotype control mAbs. Data shown (mean±SEM) are pooled from 2 independent experiments with 5-6 mice for each group


( C - E ) The expression of CD86 ( C ) or of indicated cytokines ( D - E ) was analyzed , as described in Fig 2 . Black , CTR MBMC ; pink , Irf7 - TST MBMC . Mice were treated with isotype control ( IC , square ) or blocking α - IFNAR1 mAbs ( triangle ) . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 2 - 3 mice per

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) The expression of CD86 (C) or of indicated cytokines (D-E) was analyzed, as described in Fig 2. Black, CTR MBMC; pink, Irf7-TST MBMC. Mice were treated with isotype control (IC, square) or blocking α-IFNAR1 mAbs (triangle). Data shown (mean±SEM) are from 2 pooled independent experiments each with 2-3 mice per group


( A , B ) Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from MCMV - infected ( A MBMC of each 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

(A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected (A MBMC of each indicated type


( A , B ) Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated fro CpG - stimulated ( B ) MBMC of each 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

(A, B) Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated fro CpG-stimulated (B) MBMC of each indicated type


( C ) Ratio of IL - 12 - or TNF - producing cells in 5 . 2 + vs 5 . 1 + pDC of indicated MCMV - infected MBMC mice

O O O O O O O O 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) Ratio of IL-12- or TNF-producing cells in 5.2+ vs 5.1+ pDC of indicated MCMV-infected MBMC mice


Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from MCMV - infected MBMC of each indicated type . Black , CTR MBMC ; pale pink , Itgal - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group .

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

Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from MCMV-infected MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.


Ratio of CD86 expression or IFN - I production in 5 . 2 + vs 5 . 1 + pDC isolated from CpG - stimulated ( B ) MBMC of each indicated type . Black , CTR MBMC ; pale pink , Itgal - TST MBMC . Data shown ( mean ± SEM ) are from 2 pooled independent experiments each with 3 mice per group .

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

Ratio of CD86 expression or IFN-I production in 5.2+ vs 5.1+ pDC isolated from CpG-stimulated (B) MBMC of each indicated type. Black, CTR MBMC; pale pink, Itgal-TST MBMC. Data shown (mean±SEM) are from 2 pooled independent experiments each with 3 mice per group.


( C ) EYFP expression in splenocytes isolated from uninfected ( NI ) vs MCMV - infected ( MCMV ) IFNbtm1 ( EYFP ) reporter mice . The expression of indicated markers was analyzed on YFP + cells . Plots are representative of one out of 6 mice from 2 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

(C) EYFP expression in splenocytes isolated from uninfected (NI) vs MCMV-infected (MCMV) IFNbtm1(EYFP) reporter mice. The expression of indicated markers was analyzed on YFP+ cells. Plots are representative of one out of 6 mice from 2 independent experiments.


( D - F ) Immunohistological analysis of splenic sections stained with mAb against MOMA1 / CD169 ( blue ) , GFP / EYFP ( green ) , whose expression correlates with that of IFN - β , and MCMV IE - 1 ( red ) . Scale bars , 100µm . The image shown is one representative of 15 obtained from 5 different mice . Optical magnification was 20x , and images were scanned on the microscope at zoom x2 for ( E , F ) . White dotted lines delimit marginal zone . Yellow squares in ( D ) defines zoomed region in ( E , F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) Immunohistological analysis of splenic sections stained with mAb against MOMA1/CD169 (blue), GFP/EYFP (green), whose expression correlates with that of IFN-β, and MCMV IE-1 (red). Scale bars, 100µm. The image shown is one representative of 15 obtained from 5 different mice. Optical magnification was 20x, and images were scanned on the microscope at zoom x2 for (E, F). White dotted lines delimit marginal zone. Yellow squares in (D) defines zoomed region in (E, F).


A . Significantly recurrently mutated genes identified by MutSigCV analysis based on whole - exome sequencing data of 25 STAD patients ' samples .

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. Significantly recurrently mutated genes identified by MutSigCV analysis based on whole-exome sequencing data of 25 STAD patients' samples.


B . Distribution of PWWP2B mutations identified in 25 STAD patients . Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset ( Network , 2014 ) . Distribution of PWWP2B mutations identified in a large - scale colorectal adenocarcinoma genomic study .

O O O O O O O O O O O O O O O O O O O O O O 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. Distribution of PWWP2B mutations identified in 25 STAD patients. Distribution of PWWP2B mutations identified in the TCGA PanCancer Atlas stomach adenocarcinoma dataset (Network, 2014). Distribution of PWWP2B mutations identified in a large-scale colorectal adenocarcinoma genomic study.


E . Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations .

E. Overall survival curve of 25 gastric cancer patients with and without PWWP2B mutations.


B . The interaction between exogenous PWWP2B and UHRF1 . The indicated plasmids were transfected into 293T cells . After 24 hours , the transfected cell lysates were immunoprecipitated ( IP ) using an anti - Myc antibody and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

B. The interaction between exogenous PWWP2B and UHRF1. The indicated plasmids were transfected into 293T cells. After 24 hours, the transfected cell lysates were immunoprecipitated (IP) using an anti-Myc antibody and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


C . The interaction between endogenous PWWP2B and UHRF1 . Immunoprecipitation reactions were performed using rabbit IgG or anti - PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O 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. The interaction between endogenous PWWP2B and UHRF1. Immunoprecipitation reactions were performed using rabbit IgG or anti-PWWP2B antibodies and subjected to Western blotting analysis using the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


E . Myc - UHRF1 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with the anti - Myc antibody and then immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O 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. Myc-UHRF1 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with the anti-Myc antibody and then immunoblotted with the indicated antibodies.


G . SFB - PWWP2B WT and either Myc - UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti - FLAG and then immunoblotted with the indicated antibodies .

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

G. SFB-PWWP2B WT and either Myc-UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti-FLAG and then immunoblotted with the indicated antibodies.


I Cell lysates of 293T cells transfected with Myc - UHRF1 expression vector were incubated with 2 of purified GST , GST - PWWP2B - N , or GST - PWWP2B - C recombinant proteins for 1 h at 4 ° C . After pull - down with GST - beads , the bound complexes were separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( SDS - PAGE ) and subjected to Western blot analysis using the indicated antibodies ( upper panel ) . The Coomassie blue stain shows the purified GST fusion 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 O O O O O O O O O O O O O O O O O O O O O O O O

I Cell lysates of 293T cells transfected with Myc-UHRF1 expression vector were incubated with 2 of purified GST, GST-PWWP2B-N, or GST-PWWP2B-C recombinant proteins for 1 h at 4 °C. After pull-down with GST-beads, the bound complexes were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to Western blot analysis using the indicated antibodies (upper panel). The Coomassie blue stain shows the purified GST fusion proteins.


Cell lysates of 293T expressing Myc - UHRF1 WT or Myc - UHRF1 - D4 mutants were incubated with 2 ug of purified GST or GST - PWWP2B - N for 1 h at 4 ° C . After pull - down with GST - beads , the bound complex was analyzed by Western blots .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 lysates of 293T expressing Myc-UHRF1 WT or Myc-UHRF1-D4 mutants were incubated with 2 ug of purified GST or GST-PWWP2B-N for 1 h at 4 °C. After pull-down with GST-beads, the bound complex was analyzed by Western blots.


E . Comparison of the translocation kinetics for GFP - UHRF1 and mCherry - PWWP2B to the sites of DNA damage . HeLa cells expressing both GFP - UHRF1 and mCherry - PWWP2B were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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E. Comparison of the translocation kinetics for GFP-UHRF1 and mCherry-PWWP2B to the sites of DNA damage. HeLa cells expressing both GFP-UHRF1 and mCherry-PWWP2B were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


F . HeLa cells were transfected with GFP - PWWP2B WT or deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( top panel ) . The cells with positive GFP expression on the laser stripes are presented in the bar graph ( bottom panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation .

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F. HeLa cells were transfected with GFP-PWWP2B WT or deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (top panel). The cells with positive GFP expression on the laser stripes are presented in the bar graph (bottom panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation.


H . GFP - PWWP2B - A2A3 deletion mutant translocates to the DNA damage sites . HeLa cells were transfected with GFP - tagged deletion mutant expression plasmids , and 24 h later , the cells were treated with laser microirradiation ( left panel ) . The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate the standard deviation for the cells transfected with each expression plasmid .

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H. GFP-PWWP2B-A2A3 deletion mutant translocates to the DNA damage sites. HeLa cells were transfected with GFP-tagged deletion mutant expression plasmids, and 24 h later, the cells were treated with laser microirradiation (left panel). The cells with positive GFP expression on the laser stripes after 10 minutes were counted and presented in the bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate the standard deviation for the cells transfected with each expression plasmid.


I - J . HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP - PWWP2B - A2A3 ( I ) and GFP - PWWP2B WT ( J ) expression plasmid . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points . The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel .

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I-J. HeLa cells depleted with either control or UHRF1 siRNAs were transfected with the GFP-PWWP2B-A2A3 (I) and GFP-PWWP2B WT (J) expression plasmid. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points. The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel.


The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel . K . HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP - UHRF1 expression plasmids . After 24 h , the cells were subjected to laser microirradiation . The laser stripes were examined at the indicated time points .

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The intensity of each laser stripes in each time point was measured by averaging values from indicated number of cells and graphed in the right panel. K. HeLa cells depleted with either control or PWWP2B siRNA were transfected with GFP-UHRF1 expression plasmids. After 24 h, the cells were subjected to laser microirradiation. The laser stripes were examined at the indicated time points.


D - G . Quantification of radiation - induced RAD51 , BRCA1 , and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs . All data were obtained from over cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 , BRCA1 , and 53BP1 foci formation . HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( D and E ) , BRCA1 ( F ) , or 53BP1 ( G ) antibodies . The cells in S phase were identified by Cyclin A staining . ( E ) The siPWWP2B - resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA - mediated PWWP2B depletion . IR - induced RAD51 foci formation was examined by immunofluorescence .

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D-G. Quantification of radiation-induced RAD51, BRCA1, and 53BP1 foci formation in HeLa cells treated with the indicated siRNAs. All data were obtained from over cells (open circles), and the mean values are shown as red bars (upper panel). Immunofluorescence analysis of irradiation-induced RAD51, BRCA1, and 53BP1 foci formation. HeLa cells transfected with either control or PWWP2B siRNAs were synchronized in S phase by a double thymidine block and exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (D and E), BRCA1 (F), or 53BP1 (G) antibodies. The cells in S phase were identified by Cyclin A staining. (E) The siPWWP2B-resistant PWWP2B WT expression vector was transfected into HeLa cells after siRNA-mediated PWWP2B depletion. IR-induced RAD51 foci formation was examined by immunofluorescence.


A . Immunoblot analysis of RAD51 , BRCA1 , or 53BP1 protein levels in PWWP2B - depleted HeLa cells .

A. Immunoblot analysis of RAD51, BRCA1, or 53BP1 protein levels in PWWP2B-depleted HeLa cells.


B . RPA phosphorylation at the indicated time points in response to IR exposure . HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4 / S8 phosphorylation and total RPA .

B. RPA phosphorylation at the indicated time points in response to IR exposure. HeLa cells treated with the indicated siRNAs were irradiated and the lysates prepared at the indicated time points were subjected to immunoblot analysis for the presence of S4/S8 phosphorylation and total RPA.


C - D . Quantification of radiation - induced RPA2 or S4 / S8 phosphorylated RPA foci formation in PWWP2B - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RPA2 or S4 / S8 phosphorylated RPA2 foci formation . HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RPA2 ( C ) , or S4 / S8 phosphorylation - specific RPA2 ( D ) antibodies ( bottom panel ) .

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C-D. Quantification of radiation-induced RPA2 or S4/S8 phosphorylated RPA foci formation in PWWP2B-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RPA2 or S4/S8 phosphorylated RPA2 foci formation. HeLa cells treated with either control or PWWP2B siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RPA2 (C), or S4/S8 phosphorylation-specific RPA2 (D) antibodies (bottom panel).


A . Immunofluorescence analysis for irradiation - induced MRE11 foci formation ( left panel ) . HeLa cells transfected with either control siRNA , siPWWP2B or siUHRF1 were irradiated , fixed and subjected to staining with MRE11 and γH2AX antibodies . More than 100 nuclei per condition were counted . The percentage of MRE11 - positive cells is presented as a bar graph ( right panel ) . The results represent the average of three independent experiments . The error bars indicate standard deviation .

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A. Immunofluorescence analysis for irradiation-induced MRE11 foci formation (left panel). HeLa cells transfected with either control siRNA, siPWWP2B or siUHRF1 were irradiated, fixed and subjected to staining with MRE11 and γH2AX antibodies. More than 100 nuclei per condition were counted.The percentage of MRE11-positive cells is presented as a bar graph (right panel). The results represent the average of three independent experiments. The error bars indicate standard deviation.


B . HeLa cells expressing GFP - MRE11 were treated with control siRNA or siRNAs against PWWP2B . After 48 h , the cells were subjected to laser microirradiation . Stripes on the laser were examined at the indicated time points ( upper panel ) . The intensity of the laser stripes at the time points was determined . The average intensity values from 10 cells were graphed in the bottom panel .

B. HeLa cells expressing GFP-MRE11 were treated with control siRNA or siRNAs against PWWP2B. After 48 h, the cells were subjected to laser microirradiation. Stripes on the laser were examined at the indicated time points (upper panel).The intensity of the laser stripes at the time points was determined. The average intensity values from 10 cells were graphed in the bottom panel.


C . The interaction between PWWP2B and MRE11 . HA - PWWP2B and Myc - MRE11 - expressing vectors were transfected into 293T cells . After 24 h , the cell lysates were immunoprecipitated with anti - HA antibody , followed by immunoblot analysis with the indicated antibodies .

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C. The interaction between PWWP2B and MRE11. HA-PWWP2B and Myc-MRE11-expressing vectors were transfected into 293T cells. After 24 h, the cell lysates were immunoprecipitated with anti-HA antibody, followed by immunoblot analysis with the indicated antibodies.


D . The interaction between endogenous PWWP2B and MRE11 . Immunoprecipitation reactions were performed using rabbit IgG or anti - MRE11 antibodies , followed by immunoblot analysis with the indicated antibodies . The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies .

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D. The interaction between endogenous PWWP2B and MRE11. Immunoprecipitation reactions were performed using rabbit IgG or anti-MRE11 antibodies, followed by immunoblot analysis with the indicated antibodies. The bottom panel shows equal volumes of cell lysates immunoblotted with the indicated antibodies.


E . HA - MRE11 and either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T cells . The cell lysates were immunoprecipitated with anti - HA antibody and then immunoblotted with the indicated antibodies .

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E. HA-MRE11 and either GFP-PWWP2B WT or its serial deletion mutants were co-transfected into 293T cells. The cell lysates were immunoprecipitated with anti-HA antibody and then immunoblotted with the indicated antibodies.


B - C . Comparison of IR - induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs . Quantification of radiation - induced RAD51 or BRCA1 foci formation in PWWP2B alone , UHRF1 alone or PWWP2B and UHRF1 - depleted HeLa cells . All data were obtained from over 100 cells ( open circles ) , and the mean values are shown as red bars ( upper panel ) . Immunofluorescence analysis of irradiation - induced RAD51 or BRCA1 foci formation . HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR . The cells were incubated for 2 h , fixed , and subjected to staining with RAD51 ( B ) , or BRCA1 ( C ) antibodies ( bottom panel ) .

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B-C. Comparison of IR-induced foci formation for either RAD51 or BRCA1 in HeLa cells transfected with the indicated siRNAs. Quantification of radiation-induced RAD51 or BRCA1 foci formation in PWWP2B alone, UHRF1 alone or PWWP2B and UHRF1-depleted HeLa cells. All data were obtained from over 100 cells (open circles), and the mean values are shown as red bars (upper panel).Immunofluorescence analysis of irradiation-induced RAD51 or BRCA1 foci formation. HeLa cells treated with the indicated siRNAs were exposed to 0 or 2 Gy of IR. The cells were incubated for 2 h, fixed, and subjected to staining with RAD51 (B), or BRCA1 (C) antibodies (bottom panel).


A . CAF and WI - 38 cells pretreated with EVs from MDA - MB - 231 or MCF10A cells , or PBS for 48 h were unstarved ( U ) or starved ( S ) for 2 h in EBSS prior to addition of a mixture of leucine , arginine , and lysine ( LRK ) or a cocktail of essential AAs ( EAA ) . Cells were analyzed by Western blot analysis in a time course as indicated .

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A. CAF and WI-38 cells pretreated with EVs from MDA-MB-231 or MCF10A cells, or PBS for 48 h were unstarved (U) or starved (S) for 2 h in EBSS prior to addition of a mixture of leucine, arginine, and lysine (LRK) or a cocktail of essential AAs (EAA). Cells were analyzed by Western blot analysis in a time course as indicated.


B . Western blot analysis of puromycin ( Puro ) - labeled fibroblasts ( CAF , normal human mammary fibroblasts , WI - 38 , and NIH3T3 ) after 2 - h starvation and EAA add - back in the presence or absence of cycloheximide ( CHX ) . C . Western blot analysis of Puro - labeled fibroblasts cultured in growth medium .

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B. Western blot analysis of puromycin (Puro)-labeled fibroblasts (CAF, normal human mammary fibroblasts, WI-38, and NIH3T3) after 2-h starvation and EAA add-back in the presence or absence of cycloheximide (CHX). C. Western blot analysis of Puro-labeled fibroblasts cultured in growth medium.


B . TORCAR FRET reporter assay in WI - 38 or NIH3T3 cells after 2 - h starvation and EAA add - back at time 0 ( n = 3 biological replicates for each group ) . Data are presented as mean ± SD . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( one - way ANOVA ) .

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B. TORCAR FRET reporter assay in WI-38 or NIH3T3 cells after 2-h starvation and EAA add-back at time 0 (n=3 biological replicates for each group). Data are presented as mean ± SD. *P<0.05, **P<0.01, ***P<0.001 (one-way ANOVA).


A , B . Decreases of leucine , arginine , and lysine ( A ) as well as production of ammonium ( B ) in the conditioned medium ( CM ) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS ( n = 3 biological replicates for each group ) .

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A,B. Decreases of leucine, arginine, and lysine (A) as well as production of ammonium (B) in the conditioned medium (CM) of NIH3T3 cells at 30 min after the LRK mixture was added to starved cells that were pretreated with EVs or PBS (n=3 biological replicates for each group).


C . Intracellular levels of leucine , arginine , and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells ( n = 3 biological replicates for each group ) .

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C. Intracellular levels of leucine, arginine, and lysine in NIH3T3 cells collected 30 min after the LRK mixture was added to starved cells (n=3 biological replicates for each group).


A . Western blot analysis of WI - 38 and NIH3T3 cells treated with EVs or PBS for 48 h .

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A. Western blot analysis of WI-38 and NIH3T3 cells treated with EVs or PBS for 48 h.


B . Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h .

B. Western blot analysis of NIH3T3 cells transfected with indicated siRNA for 48 h.


C . Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX .

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C. Western blot analysis of NIH3T3 cells that had been transfected with indicated siRNA and labeled with Puro in the presence or absence of CHX.


D . Western blot analysis of NIH3T3 cells that had been transfected with a pRK5 - RAGC - HA - GST expression plasmid or control vector and treated with EVs or PBS for 48 h , and then labeled with Puro .

D. Western blot analysis of NIH3T3 cells that had been transfected with a pRK5-RAGC-HA-GST expression plasmid or control vector and treated with EVs or PBS for 48 h, and then labeled with Puro.


E . Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h .

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E. Western blot analysis of CAF that had been treated with PBS or EVs from indicated cells for 48 h.


A . Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti - miRNAs or control for 24 h and then treated with EVs or PBS for 48 h .

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A. Western blot analysis of CAF and NIH3T3 cells transfected with indicated anti-miRNAs or control for 24 h and then treated with EVs or PBS for 48 h.


C . Dual luciferase assay of the psiCHECK reporters containing the 3 ' UTR of RRAGC that was wild - type , with all three miR - 105 sites mutated , or with the miR - 204 site mutated . NIH3T3 cells were co - transfected with the reporter and indicated miRNA mimic ( n = 3 biological replicates for each group ) .

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C. Dual luciferase assay of the psiCHECK reporters containing the 3'UTR of RRAGC that was wild-type, with all three miR-105 sites mutated, or with the miR-204 site mutated. NIH3T3 cells were co-transfected with the reporter and indicated miRNA mimic (n=3 biological replicates for each group).


D . Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h ( n = 3 biological replicates for each group ) .

D. Dual luciferase assay of the indicated psiCHECK reporters in NIH3T3 cells treated with EVs or PBS for 48 h (n=3 biological replicates for each group).


D . A heatmap showing the comparison of log2 fold change ( FC ) of the abundance of each identified 13C6 - labeled protein and its corresponding mRNA level determined by RNA - seq .

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D. A heatmap showing the comparison of log2 fold change (FC) of the abundance of each identified 13C6-labeled protein and its corresponding mRNA level determined by RNA-seq.


A , B . A breast cancer tissue array was analyzed for correlations among RagC , miR - 105 , and miR - 204 in tumor stroma by IHC / ISH - determined scores . Representative IHC and ISH images ( A ) and correlations determined by Kendall ' s tau correlation tests ( N = 24 independent tumors ; Kendall ' s tau and two - sided P value as indicated ) ( B ) are shown . Bar = 200 μm .

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A,B. A breast cancer tissue array was analyzed for correlations among RagC, miR-105, and miR-204 in tumor stroma by IHC/ISH-determined scores. Representative IHC and ISH images (A) and correlations determined by Kendall's tau correlation tests (N=24 independent tumors; Kendall's tau and two-sided P value as indicated) (B) are shown. Bar=200 μm.


C , D . Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR - 105 or empty vector were collected from NSG mice for RagC IHC ( n = 5 tumors for each group ) . The relative intensity of RagC stain in tumor stroma was quantified by ImageJ . Bar = 50 μm . In ( D ) , data are presented as mean ± SD . * P < 0 . 05 ( Student ' s t - test ) .

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C,D. Xenograft mammary tumors formed from MCFDCIS cells overexpressing miR-105 or empty vector were collected from NSG mice for RagC IHC (n=5 tumors for each group). The relative intensity of RagC stain in tumor stroma was quantified by ImageJ. Bar=50 μm. In (D), data are presented as mean ± SD. *P<0.05 (Student's t-test).


Wild - type ( WT ) RAD51 SPR curves for ssDNA dN - 50 and dsDNA dN - 50p . RAD51 was injected at 150 nM in the presence of 2 . 5 mM ATP ( pH 7 . 5 ) and 10 mM CaCl2 . The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides ( N ) are plotted versus time , as measured by SPR , following the equation N = S / ( L * ( MRAD51 / MDNA ) ) , where S is the signal in RU units ( 1 RU ~ 50 pg / mm2 ) , L is the amount of DNA ligand immobilised onto the experimental flow cell ( RU ) , MRAD51 is the molecular weight ( kDa ) of RAD51 ( ~ 37 kDa ) and MDNA is the molecular weight ( kDa ) of the immobilised DNA molecule .

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Wild-type (WT) RAD51 SPR curves for ssDNA dN-50 and dsDNA dN-50p. RAD51 was injected at 150 nM in the presence of 2.5 mM ATP (pH 7.5) and 10 mM CaCl2. The normalised mean number of WT RAD51 bound to respective DNA oligonucleotides (N) are plotted versus time, as measured by SPR, following the equation N = S / (L * (MRAD51 / MDNA)), where S is the signal in RU units (1 RU ~ 50 pg/mm2), L is the amount of DNA ligand immobilised onto the experimental flow cell (RU), MRAD51 is the molecular weight (kDa) of RAD51 (~ 37 kDa) and MDNA is the molecular weight (kDa) of the immobilised DNA molecule.


Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin - streptavidin interaction . Wild - type ( WT ) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics .

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Biotinylated ssDNA oligonucleotides of indicated lengths were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction. Wild-type (WT) RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics.


The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides , as measured by SPR ( see Fig . 1 for equation ) and the ODE model fits ( see Fig . 3A for model description ) , respectively .

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The dotted and solid curves show the normalised mean number of RAD51 bound to the ssDNA oligonucleotides, as measured by SPR (see Fig. 1 for equation) and the ODE model fits (see Fig. 3A for model description), respectively.


As in A , except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics .

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As in A, except biotinylated dsDNA molecules of indicated lengths were used to measure association and dissociation kinetics.


As in B , the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides , as measured by SPR and the ODE model

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As in B, the dotted and solid curves show the mean number of RAD51 bound to the dsDNA oligonucleotides, as measured by SPR and the ODE model fits


Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . In panels C and D , the KD was fixed to the mean value identified in Figs . 3 A and B ( i . e . KD = 1 . 14 nM ) , and the concentration of WT RAD51 monomer concentration was varied accordingly . In panel D , the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length , and dividing each value by the total RAD51 monomer concentration ( i . e . % WT RAD51 = [ n - mer ] * n / [ WT RAD51monomer ] ) . [ WT RAD51monomer ] was calculated via coomassie staining image quantification using a BSA standard curve . It is important to note that the assumed maximum polymer length in solution ( 16 - mer ) leads to an overestimation of the concentration of each n - mer in solution , given that RAD51 is likely to form polymers of length greater than 16 at 150 nM , 3 µM and 30 µM WT RAD51 monomer concentration . However , this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration .

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

Predicted concentrations of WT RAD51 polymers in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % WT RAD51 within each polymer state in solution at equilibrium as a function of WT RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels C and D, the KD was fixed to the mean value identified in Figs. 3 A and B (i.e. KD = 1.14 nM), and the concentration of WT RAD51 monomer concentration was varied accordingly. In panel D, the % WT RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % WT RAD51 = [n-mer] * n / [WT RAD51monomer]). [WT RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. It is important to note that the assumed maximum polymer length in solution (16-mer) leads to an overestimation of the concentration of each n-mer in solution, given that RAD51 is likely to form polymers of length greater than 16 at 150 nM, 3 µM and 30 µM WT RAD51 monomer concentration. However, this overestimation is likely to not alter the conclusion that WT RAD51 forms long polymers in solution at 150 nM - 30 µM concentration.


Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin - streptavidin interaction , and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics . The flexibility of respective DNA , the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer - protomer binding energy contribution of WT RAD51 are indicated in gray , green and blue boxes . H and L in each box denote high and low , respectively .

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

Biotinylated DNA molecules of varying flexibility were separately immobilised onto SPR CM5 chips via biotin-streptavidin interaction, and WT RAD51 was injected at the indicated concentrations to measure association and dissociation kinetics. The flexibility of respective DNA, the expected entropic penalties upon RAD51 binding to corresponding DNA and protomer-protomer binding energy contribution of WT RAD51 are indicated in gray, green and blue boxes. H and L in each box denote high and low, respectively.


The mean number of WT RAD51 bound to respective DNA oligonucleotides , as measured by SPR ( dotted lines ) and ODE model fits ( solid lines ) .

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

The mean number of WT RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and ODE model fits (solid lines).


Bar plots of fitted kp values ( D ) and ks values ( E ) for each SPR curves . In panel D , all parameters except kp were fixed to the mean values as identified in Fig . 3 A and B , and kp was fitted using lsqcurvefit ( MATLAB ) . In panel E , all parameters except ks and kp were fixed to the mean values as identified in Fig . 3 A and B , and ks and kp were fitted using lsqcurvefit ( MATLAB ) . Only the ks values are reported here . Mean ± 1 SD of 3 ODE model fits ( n = 3 ) . D : un - paired , one - tailed Mann - Whitney - Wilcoxon tests . E : un - paired , two - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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

Bar plots of fitted kp values (D) and ks values (E) for each SPR curves. In panel D, all parameters except kp were fixed to the mean values as identified in Fig. 3 A and B, and kp was fitted using lsqcurvefit (MATLAB). In panel E, all parameters except ks and kp were fixed to the mean values as identified in Fig. 3 A and B, and ks and kp were fitted using lsqcurvefit (MATLAB). Only the ks values are reported here. Mean ± 1 SD of 3 ODE model fits (n=3). D: un-paired, one-tailed Mann-Whitney-Wilcoxon tests. E: un-paired, two-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


As in Fig . 4B , but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics . The flexibility of respective DNA , the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer - protomer binding enegy contribution of F86 RAD51 are indicated in gray , green and blue boxes . H and L shown in each box indicate high and low , respectively .

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

As in Fig. 4B, but F86E RAD51 was injected at the indicated concentration to measure association and dissociation kinetics. The flexibility of respective DNA, the expected relative entropic penalties upon F86E RAD51 binding to each DNA molecule and protomer-protomer binding enegy contribution of F86 RAD51 are indicated in gray, green and blue boxes. H and L shown in each box indicate high and low, respectively.


The mean number of F86E RAD51 bound to respective DNA oligonucleotides , as measured by SPR ( dotted lines ) and corresponding model fits ( solid lines ) using the model shown in panel D . Using ABC - SMC , individual forward rates ( kp ) and reverse rates ( kpr ) were fitted to each experimental curve , while a single KD was fitted to all experimental curves .

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

The mean number of F86E RAD51 bound to respective DNA oligonucleotides, as measured by SPR (dotted lines) and corresponding model fits (solid lines) using the model shown in panel D. Using ABC-SMC, individual forward rates (kp) and reverse rates (kpr) were fitted to each experimental curve, while a single KD was fitted to all experimental curves.


Bar plots of fitted kp values for the SPR curves . Mean values of mode particles ± 1 SD derived from the ABC - SMC fits ( n = 3 ) . Un - paired , one - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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

Bar plots of fitted kp values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration ( 150 nM , 3 µM , 30 µM ) , prior to RAD51 injection onto DNA - coated SPR CM5 chips . In panels F and G , the KD was fixed to the mean value identified in Fig . 5D ( i . e . KD = 10 . 2 µM ) , and the concentration of F86E RAD51 monomer concentration was varied accordingly . In panel G , the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length , and dividing each value by the total RAD51 monomer concentration ( i . e . % F86E RAD51 = [ n - mer ] * n / [ F86E RAD51monomer ] ) . [ F86E RAD51monomer ] was calculated via coomassie staining image quantification using a BSA standard curve . Bar plots of fitted kpr values for the SPR curves . Mean values of mode particles ± 1 SD derived from the ABC - SMC fits ( n = 3 ) . Un - paired , one - tailed Mann - Whitney - Wilcoxon tests . * p ≤ 0 . 05 ; ns = non - significant .

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

Predicted concentrations of F86E RAD51 polymers in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. Predicted % F86E RAD51 within each polymer state in solution at equilibrium as a function of F86E RAD51 monomer concentration (150 nM, 3 µM, 30 µM), prior to RAD51 injection onto DNA-coated SPR CM5 chips. In panels F and G, the KD was fixed to the mean value identified in Fig. 5D (i.e. KD = 10.2 µM), and the concentration of F86E RAD51 monomer concentration was varied accordingly. In panel G, the % F86E RAD51 values were calculated by multiplying the polymer concentrations by their respective polymer length, and dividing each value by the total RAD51 monomer concentration (i.e. % F86E RAD51 = [n-mer] * n / [F86E RAD51monomer]). [F86E RAD51monomer] was calculated via coomassie staining image quantification using a BSA standard curve. Bar plots of fitted kpr values for the SPR curves. Mean values of mode particles ± 1 SD derived from the ABC-SMC fits (n=3). Un-paired, one-tailed Mann-Whitney-Wilcoxon tests. * p ≤ 0.05; ns = non-significant.


( B ) Southern Blot analysis of targeted ES cells . Positions of wild type ( WT - 14 . 7kb ) and targeted ( loxP - 12 . 8kb ) alleles are indicated .

(B) Southern Blot analysis of targeted ES cells. Positions of wild type (WT - 14.7kb) and targeted (loxP - 12.8kb) alleles are indicated.


( C ) Genotyping analysis of wild type ( Clpp + / + ) , heterozygous ( Clpp + / - ) and homozygous ( Clpp - / - ) Clpp knockout animals . DNA fragments corresponding to the wild type ( WT - 199 bp ) , and knockout ( KO - 273 bp ) Clpploci are 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

(C) Genotyping analysis of wild type (Clpp+/+), heterozygous (Clpp+/-) and homozygous (Clpp-/-) Clpp knockout animals. DNA fragments corresponding to the wild type (WT-199 bp), and knockout (KO - 273 bp) Clpploci are indicated.


( D ) Analysis of Clpp transcript levels in heart ( n = 4 ) . Bars represent mean ± standard deviation ( SD ) . Student ' s t - test was used to determine the level of statistical 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

(D) Analysis of Clpp transcript levels in heart (n=4). Bars represent mean ± standard deviation (SD). Student's t-test was used to determine the level of statistical difference.


( E ) Western blot analysis of CLPPprotein levels in heart ( He ) , quadriceps ( Quad ) , liver ( Li ) , kidney ( Ki ) , lung ( Lu ) , brain ( Br ) , spleen ( Sp ) and pancreas ( Pa ) . TUBULIN and ACTIN are used as a loading control . The arrowhead indicates positions of CLPP , and the star indicates position of cross - reacting bands .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of CLPPprotein levels in heart (He), quadriceps (Quad), liver (Li), kidney (Ki), lung (Lu), brain (Br), spleen (Sp) and pancreas (Pa). TUBULIN and ACTIN are used as a loading control. The arrowhead indicates positions of CLPP, and the star indicates position of cross-reacting bands.


( C ) Respiratory chain enzyme activities . Relative enzyme activities of respiratory chain enzyme C I ( NADH coenzyme Q reductase ) , C I / III ( NADH cytochrome c reductase ) , C II ( succinate dehydrogenase ) , C II / III ( succinate cytochrome c reductase ) and C IV ( cytochrome c oxidase ) in mitochondrial extracts from wild type and Clpp knockout hearts . ( n = 5 ) . Bars present mean levels ± SD . Statistical difference was calculated by Student ' s t - test and changes are presented in the figure ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Respiratory chain enzyme activities. Relative enzyme activities of respiratory chain enzyme C I (NADH coenzyme Q reductase), C I/III (NADH cytochrome c reductase), C II (succinate dehydrogenase), C II/III (succinate cytochrome c reductase) and C IV (cytochrome c oxidase) in mitochondrial extracts from wild type and Clpp knockout hearts. (n=5). Bars present mean levels ± SD. Statistical difference was calculated by Student's t-test and changes are presented in the figure (n=5).


( D ) Substrate - dependent oxygen consumption rates in isolated heartmitochondria . Complex I substrates ( pyruvate , malate , glutamate ) ; complex II substrate ( succinate ) . State 3 - ADP stimulated oxygen consumption rate ; State 4 - respiration rate after addition of oligomycin ; Uncoupled - oxygen consumption rate in the presence of FCCP . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 7 ) .

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

(D) Substrate-dependent oxygen consumption rates in isolated heartmitochondria. Complex I substrates (pyruvate, malate, glutamate); complex II substrate (succinate). State 3 - ADP stimulated oxygen consumption rate; State 4 - respiration rate after addition of oligomycin; Uncoupled - oxygen consumption rate in the presence of FCCP. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=7).


( E ) Decrease of steady - state levels of respiratory chain subunits in Clpp - knockout hearts relative to wild type ( set to 1 ) . VDAC or TOMM20 were used as loading controls . Bars present mean levels ± SD . Student ' s t - test was used to determine the level of statistical difference ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Decrease of steady-state levels of respiratory chain subunits in Clpp-knockout hearts relative to wild type (set to 1). VDAC or TOMM20 were used as loading controls. Bars present mean levels ± SD. Student's t-test was used to determine the level of statistical difference (n=4).


( F ) Blue Native Page Analysis ( BN - PAGE ) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I ( anti - NDUFA9 ) ; C III ( anti - UQCRC1 ) and C IV ( anti - COX4A ) .

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

(F) Blue Native Page Analysis (BN-PAGE) of the respiratory chain supercomplexes followed by Western blot analysis using antibodies against C I (anti-NDUFA9); C III (anti-UQCRC1) and C IV (anti-COX4A).


( A ) Quantification of mtDNA levels after Southern blot analysis . Bars present mean levels ± SD ( n = 4 ) .

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) Quantification of mtDNA levels after Southern blot analysis. Bars present mean levels ± SD (n=4).


( B - C ) Northern blot analysis and corresponding quantification of mitochondrial ( B ) rRNAs and mRNAs or ( C ) tRNAs . 18S rRNA is used as loading control . Bars represent mean ± SD . Student ' s t - test was used to determine the level of statistical difference ( 12S n = 5 and the rest of samples n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Northern blot analysis and corresponding quantification of mitochondrial (B) rRNAs and mRNAs or (C) tRNAs. 18S rRNA is used as loading control. Bars represent mean ± SD. Student's t-test was used to determine the level of statistical difference (12S n=5 and the rest of samples n=4).


( D ) Western blot analysis of LRPPRC levels . VDAC was used as a loading control .

(D) Western blot analysis of LRPPRC levels. VDAC was used as a loading control.


( E ) In organello transcription analysis upon 60 minutes labelling with - 32P - UTP .

(E) In organello transcription analysis upon 60 minutes labelling with -32P-UTP.


( A ) De novo synthesized mitochondrial proteins were isolated after labelling with 35S - methionine for 1 hour . Positions of individual mitochondrial encoded proteins are 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

(A) De novo synthesized mitochondrial proteins were isolated after labelling with 35S-methionine for 1 hour. Positions of individual mitochondrial encoded proteins are indicated.


( B ) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin , doxycycline and chloramphenicol ( + antibiotic ) , followed by a 48 hours recovery period ( - antibiotic ) . During both periods , proteins are isolated at 12h , 24h and 48h . Antibodies against subunits of C I ( NDUFB6 ) , C III ( UQCRC1 ) , C IV ( COXI ) , MRPS35 and MRPL37 were used as indicated .

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

(B) Western blot analysis of RC subunits in MEFs upon 48 hours treatment with actinonin, doxycycline and chloramphenicol (+ antibiotic), followed by a 48 hours recovery period (- antibiotic). During both periods, proteins are isolated at 12h, 24h and 48h. Antibodies against subunits of C I (NDUFB6), C III (UQCRC1), C IV (COXI), MRPS35 and MRPL37 were used as indicated.