Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 then sacrificed on day 21 for . ( A ) of change versus time ( n = 10 ) .

O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (A) Analysis of body weight change versus time (n=10).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 then sacrificed on day 21 for . ( B ) of number of per milliliter of BALF ( n = 5 ) .

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

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (B) Analysis of the number of cells per milliliter of BALF (n=5).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 then sacrificed on day 21 for . ( C ) Quantitation of total hydroxyproline content per right lung ( Healthy control , n = 5 ; others , n = 7 - 9 ) .

O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O B-TISSUE O O O O O O O O O O O O O O O O O

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (C) Quantitation of total hydroxyproline content per right lung (Healthy control, n=5; others, n=7-9).


Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 then sacrificed on day 21 for . ( D ) H & E trichrome of lung tissue ( scale bars , 200 μm ) .

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

Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (D) H&E staining and trichrome staining of lung tissue (scale bars, 200 μm).


Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , peripheral blood was collected at indicated time points after drug injection . ( A - C ) Measurement of plasma IL - 6 ( A ) , IFNα ( B ) TNFα ( C ) ( n = 3 ) .

O B-ORGANISM O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O

Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (A-C) Measurement of plasma IL-6 (A), IFNα (B) and TNFα (C) (n=3).


Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , peripheral blood was collected at indicated time points after drug injection . ( D - F ) Effect of drug on plasma of IL - 6 ( D ) , IFNα ( E ) , TNFα ( F ) at 1 . 5h , 1h , or 1h after treatment , respectively ( n = 2 ) .

O B-ORGANISM O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-TISSUE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (D-F) Effect of drug concentration on plasma levels of IL-6 (D), IFNα (E), and TNFα (F) at 1.5h, 1h, or 1h after treatment, respectively (n=2).


Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , peripheral blood was collected at indicated time points after drug injection . ( G ) Change in as a measure of systemic toxicity during alternate day dosing ( n = 2 ) .

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

Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (G) Change in body weight as a measure of systemic toxicity during alternate day dosing (n=2).


a . Vero E6 were infected with an moi of 0 . 05 with mCherry D614 strains , wild - type ISA D614 or mock infected . Pictures were taken at 48h pi ( 20x ) . Scale bar , 100 µM .

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

a.Vero E6 cells were infected with an moi of 0.05 with the fluorescent mCherry D614 strains, wild-type ISA D614 or mock infected. Pictures were taken at 48h pi (20x). Scale bar, 100 µM.


b . After 5 passages ( p2 , p3 , p4 p5 ) on VeroE6 , mCherry reporter gene in ISA mCherry D614 supernatant medium was RT - PCR amplified analysed using gel electrophoresis .

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

b. After 5 passages (p2, p3, p4 and p5) on VeroE6 cells, the mCherry reporter gene in ISA mCherry D614 supernatant medium was RT-PCR amplified and analysed using gel electrophoresis.


( d ) Representative neutralizing curves of nAb - . four - parameter dose - response curve was fitted using nonlinear regression method nAbs were calculated in software Prism 7 . 0 . For negative serum samples , an arbitrary value of 10 was assigned ( detection threshold for both methods ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O

(d) Representative neutralizing curves of the nAb fluorescence-based assay. The four-parameter dose-response curve was fitted using the nonlinear regression method and nAbs were calculated in the software Prism 7.0. For negative serum samples, an arbitrary value of 10 was assigned (detection threshold for both methods).


a : Dose response curve for ISA D614 for mCherry D614 strains obtained by or viral measurement in VeroE6 from one representative experiment . Data are represented as mean ± SD ( indicated by error bars ) . Each experiment was performed in technical triplicates ( N = 3 ) . b : Table of EC50 values obtained for two different strains from two technical replicates . their respective mean ± SD .

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

a: Dose response curve for the ISA D614 and for the mCherry D614 strains obtained by fluorescence or viral RNA measurement in VeroE6 cells from one representative experiment. Data are represented as mean ± SD (indicated by the error bars). Each experiment was performed in technical triplicates (N=3). b: Table of EC50 values obtained for the two different strains from two technical replicates. and their respective mean ± SD. c


b In HEK293 , recombinantly expressed GluN1 / GluN2A GluN1 / GluN2A ( N615S ) channels were activated by fast glutamate application ( 1 mM ; in continuous presence of co - agonist glycine , 10 µM ) at holding potentials from - 100 to + 40 mV in different extracellular Mg2 + concentrations . NMDAR - mediated peak currents were normalized to those obtained at + 40 mV . Data points represent mean ± for n = 4 - 7 different HEK293 . Representative current traces evoked in 0 mM Mg2 + at - 60 mV , with 20 600 ms applications , are shown below IV plots were used to determine current kinetics ( Supplementary Table 1 ) .

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

b In HEK293 cells, recombinantly expressed GluN1/GluN2A and GluN1/GluN2A(N615S) channels were activated by fast glutamate application (1 mM; in the continuous presence of the co-agonist glycine, 10 µM) at holding potentials from -100 to +40 mV in different extracellular Mg2+ concentrations. NMDAR-mediated peak currents were normalized to those obtained at +40 mV. Data points represent mean ± SEM for n = 4-7 different HEK293 cells. Representative current traces evoked in 0 mM Mg2+ at - 60 mV, with 20 and 600 ms applications, are shown below the IV plots and were used to determine the current kinetics (Supplementary Table 1).


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

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

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

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

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

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

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

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

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 three genotypes . inset in c gives a schematic view of stimulating ( arrow ) recording ( green arrow ) electrode positions in str . radiatum ( r ) 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 Grin2a + / + mice . inset shows a Nissl - stained slice of one recorded mouse post mortem . e In mutant mice , but not in control , GluN2B - containing NMDAR contributes significantly to magnitude of LTP , since LTP was significantly reduced by GluN2B - specific antagonist CP101 , 106 ( CP ) . Recordings of non - tetanized control pathway in c e are given as dashed lines . Error bars represent mean ± ( for statistics : Supplementary Statistics to Fig . 2 ) .

O O O O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-TISSUE O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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 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 ( by 18 % in adults ) , significantly lower score in nesting in overnight burrowing activity ( filled circles : 30 - week - old mice , dots : 18 - week - old mice ) .

O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O

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 clasping reflex . c During 12 h phase on Lafayette running wheel running is reduced in Grin2aS / S mice .

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

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

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

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 distribution of NeuN - positive neurons of GFAP - positive glial are indistinguishable between Grin2aS / S Grin2a + / + mice . Mossy fiber projections visualized by anti - calbindin ( CB ) , as well as numbers of parvalbumin ( PV ) - positive interneurons are similar between both genotypes . Anti - GluA1 immunosignal in hippocampal layers is comparable between brain sections of Grin2aS / S mice control . 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 . number of is given below bars . For Nissl stain , Tunnel Timm stain 3 mice were used per genotype . For immunohistological of glial fibrillary acidic ( GFAP ) , neuronal antigen ( NeuN ) , Calbindin ( CB ) , Parvalbumin ( PV ) five mice for GluA1 immunofluorescence stain , three mice were used ( for statistics : Supplementary Statistics to Fig . 3 ) .

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

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 Grin2a + / + controls were affected , while audiogenic seizures ( AGSs ) followed by respiratory arrest ( RA ) were induced in Grin2aS / S mice in a subset of heterozygous Grin2a + / S mutants during 11 kHz tone exposure [ 4 repetitions × 20 s tone , 2 s brake ; pink squares ; p < 0 . 0001 by Log - rank ( Mantel - Cox ) ] .

O O O O O O O B-GENEPROD O O O O O O O O B-DISEASE I-DISEASE O B-DISEASE O O O B-DISEASE I-DISEASE O O O O O O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O O B-GENEPROD B-EXP_ASSAY O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O B-DISEASE O O O O O O B-GENEPROD O O O O

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 . rescue effect could still be observed in four out of eight , in two out of eight , mice 27 51 h after memantine treatment , respectively .

O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-DISEASE O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O

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

O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-TISSUE O O O O O O B-GENEPROD O O O B-DISEASE O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O B-TISSUE O O O O B-TISSUE O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

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 saline are used for pre - treatment . MK - 801 effect was longer lasting compared to memantine one out of four showed resistance to tone exposure even 4 days after MK - 801 injection .

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

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 ( bottom ) in medial amygdala ( MeA ) , VMH PVT when compared to saline - injected Grin2aS / S littermate ( top ) . Scale bars are in mm ( for statistics : Supplementary Statistics to Fig . 4 ) .

O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-DISEASE O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

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 different genotypes as indicated . original , including cortical regions results of PBS injected , are presented in Supplementary Fig . 6 . number of mice analyzed is given in brackets . b For quantitative comparisons total numbers of c - Fos - positive in layers of hippocampus were counted . C - Fos - positive in extra - hippocampal regions are given per mm2 . Slices from six mice per genotype were used for statistical analyses ( two - t - ) error bars represent mean ± . CA1 CA3 cornu ammonis regions 1 3 , DG dentate gyrus , PC piriform cortex , RSC retrosplenial cortex , Neo neocortex , MTN midline thalamic nuclei , BLA basolateral amygdala . scale bars : 0 . 2 mm .

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

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 rapid eye movement ( REM ) sleep for theta ( 0 - 15 Hz , left diagrams ) gamma oscillations ( 15 - 200 Hz , right diagrams ) in genotypes . During exploration , average theta range peak frequencies were at 8 . 61 ± 0 . 07 8 . 75 ± 0 . 28 Hz for Grin2a + / + mice Grin2a + / S mice , respectively . In Grin2aS / S mice theta range peak frequency was significantly slower , measuring 7 . 43 ± 0 . 24 Hz ( Kruskal Wallis , p = 0 . 0159 ) . During REM sleep average frequency ranges of Grin2a + / + 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 low high gamma frequency bands - ranges between 20 - 100 Hz 100 - 200 Hz , respectively111 - could be detected between genotypes , neither during exploration nor in REM sleep . number of mice used in this experiment are given with name of genotypes .

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

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

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

b (left) 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 total locomotor activity during entire 2 h session in a novel environment was enhanced in Grin2aS / S mice .

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

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


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

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

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 ) time of first fall ( Cliff Avoidance Reflex , CAR ) was significantly reduced for Grin2aS / S 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 .

O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O B-ORGANISM O

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

O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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

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 T - maze rewarded alternation task ( right ) spatial working memory performance was substantially impaired in Grin2aS / S 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 B-GENEPROD O O B-ORGANISM O

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 Adcy9 - / - mice averaged over 5 min of non - active versus sustained activity on home cage running wheels . Student ' s t - P values for indicated comparisons are shown . Circles represent data from . boxplot central band is median while whiskers represent 10th 90th percentiles .

O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O 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 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 R - R interval of WT ( black bars ) Adcy9 - / - mice ( red bars ) before after ISO ( 1 μg / g )

O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O B-SMALL_MOLECULE O O O O O O

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 Adcy9 - / - mice , ~ 55 min post ISO - injection .

O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O

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


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

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

O O O B-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O O

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 ) of COS7 expressing BiFC constructs for AC9 , POPDC 1 - 3 . expressed tagged with VN ( top ) VC ( bottom ) are shown .

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

(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 - of indicated BiFC combinations in HEK293 ( n > 20 ; scale bar : 10 μm ) . of POPDC1 - VN : AC9 - VC is shown in Fig 3 7 .

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

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

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

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

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

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

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

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

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

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

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O 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) 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 expressing Flag - AC9 indicated Myc - tagged POPDC1 truncations . Western blotting with anti - AC9 anti - MYC ( POPDC1 ) of Co - IP lysates is shown . Kruskal - Wallis One Way ANOVA was performed ( n = 3 - 5 experiments , P = 0 . 006 ) , with multiple comparisons by Dunn ' s method ( * P = 0 . 021 , * * P = 0 . 01 ) .

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

(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 BiFC signal from AC9 - VN : AC9 - VC homodimer ( top ) POPDC1 - VN : AC9 - VC ( bottom ) in HEK293 .

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

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

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

B The 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 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 ) .

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

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 endogenous POPDC1 . AKAP79 is not pulled down in ( n = 3 experiments ) .

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

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 with IgG versus anti - TREK - 1 in heart homogenates from WT 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 ; # P < 0 . 001 for indicated comparison or to respective IgG controls . IgG versus anti - TREK - 1 in Adcy9 - / - was analyzed by Student ' s t - ( * * P = 0 . 009 , n = 3 ) . A portion of co - IP was subjected to western blotting with anti - TREK - 1 anti - AKAP150 .

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

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

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

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 300 nM calmodulin .

O O B-GENEPROD O O B-TISSUE O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-ORGANISM O O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O

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

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

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

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

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 for 10 min in presence of 10 µM ISO reduced amount of Flag - AC9 POPDC1 - Myc pulled down by TREK - 1 co - IP .

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

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

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

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

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

, 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 , TREK - 1 with a catalytically inactive AC9 ( TREK - 1 : AC9D ) .

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

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

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

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 indicated . Voltage was ramped from - 120 to + 40 mV . B Relative TREK - 1 current amplitudes at + 40 mV normalized to TREK - 1 injected alone . number of oocytes is given within bar graphs ( n = 37 - 97 ) . 3 - 5 batches of oocytes were used for each condition .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O B-CELL_TYPE 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 absence presence of theophylline . Data are plotted as mean ± .

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE 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 B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 . 5 . 2 vs 5 . 1 were finally discriminated within pDC gate

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

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 their expression pattern across samples shown as a . Red font , pDC ISG ( module A ) ; green font , pDC MSG ( module D ) ; mixed red green font , pDC ISG MSG ( module E ) ; black font , non - ISG , non - MSG ( module B ) . relative mRNA expression for each gene ( lanes ) across biological samples ( rows ) are represented by colors according to scale shown above . For legend of symbols depicting classes of biological samples above each column please refer to symbol key in Fig 1D .

O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O 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 + 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 shows isotype control . One histogram representative of 6 from 3 independent experiments is shown .

O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

(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 in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - infected MBMC . Isotype control ( IC ) was performed on a mixture of isolated from both types of MCMV - infected MBMC .

O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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.


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 of Mean ( MFI ) values for CD86 or of percentages of IFN - I - producing obtained from MCMV - infected Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red )

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


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 of Mean ( MFI ) values for CD86 or of percentages of IFN - I - producing obtained from CpG - stimulated mice . Black , CTR MBMC ; red , Ifnar1 - TST MBMC ( red ) Data shown ( mean ± ) are from 2 pooled independent experiments each with at least 3 mice per group .

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


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 of Mean ( MFI ) values for CD86 or of percentages of IFN - I - producing obtained from MCMV - infected Black , CTR MBMC pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± ) are from 2 pooled independent experiments each with at least 3 mice per group .

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


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 of Mean ( MFI ) values for CD86 or of percentages of IFN - I - producing obtained from CpG - stimulated mice . Black , CTR MBMC pale blue , Ap3b1 - TST MBMC . Data shown ( mean ± ) are from 2 pooled independent experiments each with at least 3 mice per group .

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


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

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


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

B-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 in 5 . 2 + vs 5 . 1 + pDC isolated from indicated MCMV - GFP - infected MBMC . One representative of 6 from 2 independent experiments is shown

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

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

O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O B-TISSUE O O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 ) 5 . 2 / 5 . 1 of cytokine - producing pDC ( F , G ) . Data ( mean ± ) are representative of 2 pooled independent experiments each with 2 - 3 mice per group

O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 percentages of cytokine - producing within pDC of each indicated mouse . Black , C57BL6 ; red , Ifnar1 - / - ; orange , Ifnar1 - / - x Myd88 - / -

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

B-GENEPROD O O O O O O O B-CELL_TYPE O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 expression in pDC isolated from Irf7 - / - ( pink , bottom ) , C57BL / 6 ( WT , black , top ) 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-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-ORGANISM O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O B-ORGANISM 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 expression in pDC ( left ) , cDC ( middle ) 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 MFI obtained using isotype control mAbs . Data shown ( mean ± ) are pooled from 2 independent experiments with 5 - 6 mice for each group

O O O B-GENEPROD B-EXP_ASSAY O B-CELL_TYPE O O O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 ) 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 ± ) are from 2 pooled independent experiments each with 2 - 3 mice per

O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 ) 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 B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM 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 ) 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 B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE 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 ) of IL - 12 - or TNF - producing in 5 . 2 + vs 5 . 1 + pDC of indicated MCMV - infected MBMC mice

O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O B-ORGANISM

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


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 ± ) are from 2 pooled independent experiments each with 3 mice per group .

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


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 ± ) are from 2 pooled independent experiments each with 3 mice per group .

O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-ORGANISM 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 . expression of indicated markers was analyzed on YFP + . Plots are representative of one out of 6 mice from 2 independent experiments .

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

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

(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 on whole - exome sequencing data of 25 STAD ' samples .

O O O O B-EXP_ASSAY O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-DISEASE 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 . Distribution of PWWP2B mutations identified in 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 B-GENEPROD B-EXP_ASSAY O O O B-DISEASE O O O B-GENEPROD B-EXP_ASSAY O O O O O B-DISEASE I-DISEASE O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-DISEASE I-DISEASE 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 with without PWWP2B mutations .

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

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


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

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

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

O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY 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 either GFP - PWWP2B WT or its serial deletion mutants were co - transfected into 293T . lysates were immunoprecipitated with anti - Myc antibody then immunoblotted with indicated antibodies .

O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-CELL_LINE O O O B-EXP_ASSAY O O O B-GENEPROD O O B-EXP_ASSAY 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 either Myc - UHRF1 WT or its serial deletion mutants were immunoprecipitated with anti - FLAG then immunoblotted with indicated antibodies .

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

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


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 , bound was analyzed by Western blots .

O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY 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 translocation kinetics for GFP - UHRF1 mCherry - PWWP2B to sites of damage . HeLa expressing both GFP - UHRF1 mCherry - PWWP2B were subjected to microirradiation . stripes were examined at indicated time points .

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

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

O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O 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-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.


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

O O O O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

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.