d Reverse transcription PCR ( RT - PCR ) - sequence analyses of total brain mRNA show the A - to - G mutation in pos . 0 and two diagnostic silent mutations at pos . - 11 and - 14 in the pore loop encoding gene segment in Grin2a + / + , Grin2aS / S , Grin2a + / S mice . In Grin2a + / S mice , the overlay of two different colored " nucleotide " peaks , at position 0 , - 11 , and - 14 indicate equimolar amounts of mRNA from the Grin2a + and the targeted Grin2aS alleles .

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d Reverse transcription PCR (RT-PCR)-sequence analyses of total brain mRNA show the A-to-G mutation in pos. 0 and two diagnostic silent mutations at pos. -11 and -14 in the pore loop encoding gene segment in Grin2a+/+, Grin2aS/S, Grin2a+/S mice. In Grin2a+/S mice, the overlay of two different colored "nucleotide" peaks, at position 0, -11, and -14 indicate equimolar amounts of mRNA from the Grin2a+ and the targeted Grin2aS alleles.


e Immunoblots of forebrain protein lysates of 4 - week - old mice ( Supplementary Fig . 2 ) indicate no genotype - specific differences of GluN1 , GluN2A , and the AMPAR subunit GluA1 expression relative to the β - actin levels ( p > 0 . 05 ) . The levels of PSD95 and αCaMKII ( in its phosphorylated state , pCaMKII ) are also comparable between genotypes relative to the GAPDH expression . The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS / S mice when compared to Grin2a + / + and Grin2a + / S mice but not in the levels of total protein lysates ( for statistics : Supplementary Statistics to Fig . 1 ) . The number of mice is given in brackets . Error bars represent mean ± SEM .

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e Immunoblots of forebrain protein lysates of 4-week-old mice (Supplementary Fig. 2) indicate no genotype-specific differences of GluN1, GluN2A, and the AMPAR subunit GluA1 expression relative to the β-actin levels (p > 0.05). The levels of PSD95 and αCaMKII (in its phosphorylated state, pCaMKII) are also comparable between genotypes relative to the GAPDH expression. The GluN2B expression level in the membrane fraction was significantly increased in Grin2aS/S mice when compared to Grin2a+/+ and Grin2a+/S mice but not in the levels of total protein lysates (for statistics: Supplementary Statistics to Fig. 1). The number of mice is given in brackets. Error bars represent mean ± SEM.


a ( left ) In the absence of extracellular Mg2 + the synaptic AMPA / NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a + / S and Grin2aS / S mice compared to control littermates . ( right ) In the presence of extracellular Mg2 + the strong reduction of the AMPA / NMDA ratio in Grin2aS / S mice relates to the increased NMDA currents at CA1 synapses ( Tukey ' s test ) . Example traces are depicted to the right of each bar graph . Data from the same experimental group were pooled across animals and are presented as mean ± SEM ( see also ref . 35 ) with p < 0 . 05 being designated as statistically significant . Numbers in bar graphs indicate the number of slices .

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a (left) In the absence of extracellular Mg2+ the synaptic AMPA/NMDA ratio of CA1 pyramidal cells in acute hippocampal slices is not altered in Grin2a+/S and Grin2aS/S mice compared to control littermates. (right) In the presence of extracellular Mg2+ the strong reduction of the AMPA/NMDA ratio in Grin2aS/S mice relates to the increased NMDA currents at CA1 synapses (Tukey's test). Example traces are depicted to the right of each bar graph. Data from the same experimental group were pooled across animals and are presented as mean ± SEM (see also ref. 35) with p < 0.05 being designated as statistically significant. Numbers in bar graphs indicate the number of slices.


b Paired - pulse facilitation at CA3 - to - CA1 synapses excluded strong alterations of presynaptic function in mutant mice . The stimulation strengths ( in nC ) necessary to elicit a pre - volley of 1 . 0 and 1 . 5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1 . 5 mV pre - volley amplitudes in Grin2aS / S mice . The fEPSP amplitudes necessary to elicit a just detectable population spike ( 1 ) and a population spike of 2 mV amplitude ( 2 ) and the paired - pulse facilitation ratio ( PPF ) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS / S and Grin2a + / S mice compared to WT littermates . The number of slices is indicated in the bar graphs or in brackets .

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

b Paired-pulse facilitation at CA3-to-CA1 synapses excluded strong alterations of presynaptic function in mutant mice. The stimulation strengths (in nC) necessary to elicit a pre-volley of 1.0 and 1.5 mV and the resulting fEPSP amplitudes were comparable in all genotypes but showed only a trend towards lower fEPSP amplitudes recorded at 1.5 mV pre-volley amplitudes in Grin2aS/S mice. The fEPSP amplitudes necessary to elicit a just detectable population spike (1) and a population spike of 2 mV amplitude (2) and the paired-pulse facilitation ratio (PPF) at an interstimulus interval of 50 ms did not indicate any synaptic impairments in Grin2aS/S and Grin2a+/S mice compared to WT littermates. The number of slices is indicated in the bar graphs or in brackets.


c Field LTP ( fLTP ) at CA3 - to - CA1 synapses , induced by tetanic stimulation in slices ( 1 s ; 100 Hz ; arrow ) , was comparable in all three genotypes . The inset in c gives a schematic view of the stimulating ( white arrow ) and the recording ( green arrow ) electrode positions in str . radiatum ( r ) and str . oriens ( o ) . d Similarly , CA1 - to - CA3 fLTP ( induction : 2 × 1 s ; 100 Hz ; arrow ) in freely moving mice was comparable between Grin2aS / S and Grin2a + / + mice . The inset shows a Nissl - stained slice of one recorded mouse post mortem . e In mutant mice , but not in control littermates , the GluN2B - containing NMDAR contributes significantly to the magnitude of LTP , since LTP was significantly reduced by the GluN2B - specific antagonist CP101 , 106 ( CP ) . Recordings of the non - tetanized control pathway in c and e are given as dashed lines . Error bars represent mean ± SEM ( for statistics : Supplementary Statistics to Fig . 2 ) .

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c Field LTP (fLTP) at CA3-to-CA1 synapses, induced by tetanic stimulation in slices (1 s; 100 Hz; arrow), was comparable in all three genotypes. The inset in c gives a schematic view of the stimulating (white arrow) and the recording (green arrow) electrode positions in str. radiatum (r) and str. oriens (o). d Similarly, CA1-to-CA3 fLTP (induction: 2 × 1 s; 100 Hz; arrow) in freely moving mice was comparable between Grin2aS/S and Grin2a+/+ mice. The inset shows a Nissl-stained slice of one recorded mouse post mortem. e In mutant mice, but not in control littermates, the GluN2B-containing NMDAR contributes significantly to the magnitude of LTP, since LTP was significantly reduced by the GluN2B-specific antagonist CP101,106 (CP). Recordings of the non-tetanized control pathway in c and e are given as dashed lines. Error bars represent mean ± SEM (for statistics: Supplementary Statistics to Fig. 2).


aGrin2aS / S mice had a reduced body weight ( by 18 % in adults ) , significantly lower score in nesting and in overnight burrowing activity ( filled circles : 30 - week - old mice , white dots : 18 - week - old mice ) .

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aGrin2aS/S mice had a reduced body weight (by 18% in adults), significantly lower score in nesting and in overnight burrowing activity (filled circles: 30-week-old mice, white dots: 18-week-old mice).


b Grin2aS / S mice showed the clasping reflex . c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS / S mice .

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b Grin2aS/S mice showed the clasping reflex. c During the 12 h dark phase on the Lafayette running wheel the running distance is reduced in Grin2aS/S mice.


d Nissl staining revealed unaltered cell density throughout the brain ( top ) and layering of hippocampal subfields , including CA1 ( bottom ) , in Grin2aS / S mice compared to Grin2a + / + . Error bars represent mean ± SEM . e In the TUNEL assay no apoptotic cells could be detected in the hippocampus of Grin2aS / S mice , compared to the staining of the nuclease - treated positive control slice . f No aberrant mossy fiber sprouting in either genotype in the dentate gyrus inner ( IML ) and outer molecular layer ( OML ) can be found by Timm staining of coronal sections .

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d Nissl staining revealed unaltered cell density throughout the brain (top) and layering of hippocampal subfields, including CA1 (bottom), in Grin2aS/S mice compared to Grin2a+/+. Error bars represent mean ± SEM. e In the TUNEL assay no apoptotic cells could be detected in the hippocampus of Grin2aS/S mice, compared to the staining of the nuclease-treated positive control slice. f No aberrant mossy fiber sprouting in either genotype in the dentate gyrus inner (IML) and outer molecular layer (OML) can be found by Timm staining of coronal sections.


g The distribution of NeuN - positive neurons and of GFAP - positive glial cells are indistinguishable between Grin2aS / S and Grin2a + / + mice . Mossy fiber projections visualized by anti - calbindin ( CB ) staining , as well as numbers of parvalbumin ( PV ) - positive interneurons are similar between both genotypes . Anti - GluA1 immunosignal in all hippocampal layers is comparable between brain sections of Grin2aS / S mice and control littermates . CA1 cornu ammonis region 1 , CA3 cornu ammonis region 3 , DG dentate gyrus , mol stratum moleculare , ori stratum oriens , rad , stratum radiatum . Scale bars in d - g are in mm . The number of animals is given below the bars . For the Nissl stain , Tunnel test and Timm stain 3 mice were used per genotype . For the immunohistological analysis of glial fibrillary acidic protein ( GFAP ) , neuronal nuclear antigen ( NeuN ) , Calbindin ( CB ) , and Parvalbumin ( PV ) five mice and for the GluA1 immunofluorescence stain , three mice were used ( for statistics : Supplementary Statistics to Fig . 3 ) .

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g The distribution of NeuN-positive neurons and of GFAP-positive glial cells are indistinguishable between Grin2aS/S and Grin2a+/+ mice. Mossy fiber projections visualized by anti-calbindin (CB) staining, as well as numbers of parvalbumin (PV)-positive interneurons are similar between both genotypes. Anti-GluA1 immunosignal in all hippocampal layers is comparable between brain sections of Grin2aS/S mice and control littermates. CA1 cornu ammonis region 1, CA3 cornu ammonis region 3, DG dentate gyrus, mol stratum moleculare, ori stratum oriens, rad, stratum radiatum. Scale bars in d-g are in mm. The number of animals is given below the bars. For the Nissl stain, Tunnel test and Timm stain 3 mice were used per genotype. For the immunohistological analysis of glial fibrillary acidic protein (GFAP), neuronal nuclear antigen (NeuN), Calbindin (CB), and Parvalbumin (PV) five mice and for the GluA1 immunofluorescence stain, three mice were used (for statistics: Supplementary Statistics to Fig. 3).


a Mortality curve showing that none of the Grin2a + / + controls were affected , while audiogenic seizures ( AGSs ) followed by respiratory arrest ( RA ) were induced in all Grin2aS / S mice and in a subset of heterozygous Grin2a + / S mutants during the 11 kHz tone exposure [ 4 repetitions × 20 s tone , 2 s brake ; pink squares ; p < 0 . 0001 by Log - rank ( Mantel - Cox ) test ] .

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a Mortality curve showing that none of the Grin2a+/+ controls were affected, while audiogenic seizures (AGSs) followed by respiratory arrest (RA) were induced in all Grin2aS/S mice and in a subset of heterozygous Grin2a+/S mutants during the 11 kHz tone exposure [4 repetitions × 20 s tone, 2 s brake; pink squares; p < 0.0001 by Log-rank (Mantel-Cox) test].


b The c - Fos immunoreactivity was specifically increased in the hypothalamus ( VMH ) , the inferior colliculus ( IC ) , and periaqueductal gray ( PAG ) but not in the hippocampus ( HPC ) of a resuscitated Grin2aS / S mouse 90 min after AGS when compared to tone - exposed Grin2a + / + littermates .

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b The c-Fos immunoreactivity was specifically increased in the hypothalamus (VMH), the inferior colliculus (IC), and periaqueductal gray (PAG) but not in the hippocampus (HPC) of a resuscitated Grin2aS/S mouse 90 min after AGS when compared to tone-exposed Grin2a+/+ littermates.


c Memantine i . p . injection in Grin2aS / S mice , 3 h before tone exposure , rescued AGS susceptibility in Grin2aS / S mice . The rescue effect could still be observed in four out of eight , and in two out of eight , mice 27 and 51 h after memantine treatment , respectively .

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c Memantine i.p. injection in Grin2aS/S mice, 3 h before tone exposure, rescued AGS susceptibility in Grin2aS/S mice. The rescue effect could still be observed in four out of eight, and in two out of eight, mice 27 and 51 h after memantine treatment, respectively.


d Increased c - Fos immunofluorescence ( in grayscale ) in the medial amygdala ( MeA ) and the VMH of a Grin2aS / S animal with AGS ( saline injection ) compared to a memantine rescued Grin2aS / S littermate . In the paraventricular nucleus of the thalamus ( PVT ) , the hippocampus ( HPC ) and cortex ( Cx ) there was no difference in c - Fos expression between memantine - injected and saline - injected animals ( for fluorescence images , see Supplementary Fig . 5 ) .

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d Increased c-Fos immunofluorescence (in grayscale) in the medial amygdala (MeA) and the VMH of a Grin2aS/S animal with AGS (saline injection) compared to a memantine rescued Grin2aS/S littermate. In the paraventricular nucleus of the thalamus (PVT), the hippocampus (HPC) and cortex (Cx) there was no difference in c-Fos expression between memantine-injected and saline-injected animals (for fluorescence images, see Supplementary Fig. 5).


e As in Fig . 4c but now MK - 801 and saline are used for pre - treatment . The MK - 801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK - 801 injection .

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e As in Fig. 4c but now MK-801 and saline are used for pre-treatment. The MK-801 effect was longer lasting compared to memantine and one out of four animals showed resistance to tone exposure even 4 days after MK-801 injection.


f Decreased c - Fos DAB immunosignal in an MK - 801 AGS - rescued Grin2aS / S animal ( bottom ) in the medial amygdala ( MeA ) , the VMH and the PVT when compared to saline - injected Grin2aS / S littermate ( top ) . Scale bars are in mm ( for statistics : Supplementary Statistics to Fig . 4 ) .

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f Decreased c-Fos DAB immunosignal in an MK-801 AGS-rescued Grin2aS/S animal (bottom) in the medial amygdala (MeA), the VMH and the PVT when compared to saline-injected Grin2aS/S littermate (top). Scale bars are in mm (for statistics: Supplementary Statistics to Fig. 4).


C - Fos expression 120 min after i . p . injection of MK - 801 . a C - Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated . The original images , including the cortical regions and results of PBS injected animals , are presented in Supplementary Fig . 6 . The number of mice analyzed is given in brackets . b For quantitative comparisons the total numbers of c - Fos - positive cells in the cellular layers of the hippocampus were counted . C - Fos - positive cells in extra - hippocampal regions are given per mm2 . Slices from six mice per genotype were used for the statistical analyses ( two - tailed t - tests ) and error bars represent mean ± SEM . CA1 and CA3 cornu ammonis regions 1 and 3 , DG dentate gyrus , PC piriform cortex , RSC retrosplenial cortex , Neo neocortex , MTN midline thalamic nuclei , BLA basolateral amygdala . All scale bars : 0 . 2 mm .

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C-Fos expression 120 min after i.p. injection of MK-801. a C-Fos DAB immunosignals are depicted for one representative brain section of the different genotypes as indicated. The original images, including the cortical regions and results of PBS injected animals, are presented in Supplementary Fig. 6. The number of mice analyzed is given in brackets. b For quantitative comparisons the total numbers of c-Fos-positive cells in the cellular layers of the hippocampus were counted. C-Fos-positive cells in extra-hippocampal regions are given per mm2. Slices from six mice per genotype were used for the statistical analyses (two-tailed t-tests) and error bars represent mean ± SEM. CA1 and CA3 cornu ammonis regions 1 and 3, DG dentate gyrus, PC piriform cortex, RSC retrosplenial cortex, Neo neocortex, MTN midline thalamic nuclei, BLA basolateral amygdala. All scale bars: 0.2 mm.


a ( left ) Peak frequencies during exploration and rapid eye movement ( REM ) sleep for theta ( 0 - 15 Hz , left diagrams ) and gamma oscillations ( 15 - 200 Hz , right diagrams ) in all genotypes . During exploration , the average theta range peak frequencies were at 8 . 61 ± 0 . 07 and 8 . 75 ± 0 . 28 Hz for Grin2a + / + mice and Grin2a + / S mice , respectively . In Grin2aS / S mice the theta range peak frequency was significantly slower , measuring 7 . 43 ± 0 . 24 Hz ( Kruskal Wallis test , p = 0 . 0159 ) . During REM sleep the average frequency ranges of Grin2a + / + and Grin2a + / S mice were around 7 . 00 ± 0 . 20 Hz whereas Grin2aS / S mice had a lower but not significantly different range 6 . 71 ± 0 . 22 Hz . a ( right ) No significant differences on the low and high gamma frequency bands - ranges between 20 - 100 Hz and 100 - 200 Hz , respectively111 - could be detected between genotypes , neither during exploration nor in REM sleep . The number of mice used in this experiment are given with the name of the genotypes .

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a (left) Peak frequencies during exploration and rapid eye movement (REM) sleep for theta (0-15 Hz, left diagrams) and gamma oscillations (15-200 Hz, right diagrams) in all genotypes. During exploration, the average theta range peak frequencies were at 8.61 ± 0.07 and 8.75 ± 0.28 Hz for Grin2a+/+ mice and Grin2a+/S mice, respectively. In Grin2aS/S mice the theta range peak frequency was significantly slower, measuring 7.43 ± 0.24 Hz (Kruskal Wallis test, p = 0.0159). During REM sleep the average frequency ranges of Grin2a+/+ and Grin2a+/S mice were around 7.00 ± 0.20 Hz whereas Grin2aS/S mice had a lower but not significantly different range 6.71 ± 0.22 Hz. a (right) No significant differences on the low and high gamma frequency bands-ranges between 20-100 Hz and 100-200 Hz, respectively111-could be detected between genotypes, neither during exploration nor in REM sleep. The number of mice used in this experiment are given with the name of the genotypes.


b ( left ) Grin2aS / S and Grin2a + / S mice show reduced theta - gamma phase - amplitude coupling as determined by the modulation index ( MI ) in the low gamma component in CA1 ( white arrowheads ) during awake and REM sleep states . b ( right ) For Grin2aS / S the MI value dropped significantly during exploration and REM sleep although the MI reduction reached significance only during exploration ( one - way ANOVA , Kruskal - Wallis test ) . Numbers below bars of bar graphs indicate the number of animals . Error bars represent mean ± SEM .

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b (left) Grin2aS/S and Grin2a+/S mice show reduced theta-gamma phase-amplitude coupling as determined by the modulation index (MI) in the low gamma component in CA1 (white arrowheads) during awake and REM sleep states. b (right) For Grin2aS/S the MI value dropped significantly during exploration and REM sleep although the MI reduction reached significance only during exploration (one-way ANOVA, Kruskal-Wallis test). Numbers below bars of bar graphs indicate the number of animals. Error bars represent mean ± SEM.


a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS / S mice .

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a The total locomotor activity during the entire 2 h session in a novel environment was enhanced in Grin2aS/S mice.


b Similarly , the exploratory behavior of Grin2aS / S mice remained high in all three successive 6 min exposure sessions to five novel objects ( ITI = 4 min ) , as exemplified by traces of the animal ' s movement on the right .

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b Similarly, the exploratory behavior of Grin2aS/S mice remained high in all three successive 6 min exposure sessions to five novel objects (ITI = 4 min), as exemplified by traces of the animal's movement on the right.


c ( left ) The time of first fall ( Cliff Avoidance Reflex , CAR ) was significantly reduced for Grin2aS / S and Grin2a + / S mice . c ( right ) In a 60 - min session Grin2aS / S mice showed a significantly increased number of falls compared to wild - type mice .

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c (left) The time of first fall (Cliff Avoidance Reflex, CAR) was significantly reduced for Grin2aS/S and Grin2a+/S mice. c (right) In a 60-min session Grin2aS/S mice showed a significantly increased number of falls compared to wild-type mice.


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 ) .

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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 .

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(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 .

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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 .

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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 ) .

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 ) .

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

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 ) .

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

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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-MEASURED_VAR I-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

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 .

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

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 ) .

O O 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, 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

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

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

(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 B-MEASURED_VAR I-MEASURED_VAR I-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

(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 B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 .

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

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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 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

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 B-MEASURED_VAR I-MEASURED_VAR I-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

(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 B-MEASURED_VAR I-MEASURED_VAR I-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

(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 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 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

(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 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

(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 - / -

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

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

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 B-MEASURED_VAR 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 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

(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

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 B-CONTROLLED_VAR 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

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

(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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-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

(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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-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

(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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

(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 B-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-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 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

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 .

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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 .

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(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 ) .

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(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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.