A , B Three independent C9 patient iPSC - MN lines ( Becker S6 , M211R2 , C90202 ) were treated with GELD , SPL , FSK , H89 or DMSO vehicle control and levels of poly - GA ( A ) and poly - GP ( B ) measured after 7 days . Values are displayed as percentages of mean DMSO signal . No significant changes in either DPR were observed ( linear mixed effects model , all not significant , P > 0 . 05 ; P = 0 . 1 DMSO vs FSK ) . Treatments were performed in four technical replicates .

O O O O O O B-ORGANISM B-CELL_TYPE O B-CELL_TYPE O O B-CELL_LINE I-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE 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 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 B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O

A, B Three independent C9 patient iPSC-MN lines (Becker S6, M211R2, C90202) were treated with GELD, SPL, FSK, H89 or DMSO vehicle control and levels of poly-GA (A) and poly-GP (B) measured after 7 days. Values are displayed as percentages of mean DMSO signal. No significant changes in either DPR were observed (linear mixed effects model, all not significant, P>0.05; P = 0.1 DMSO vs FSK). Treatments were performed in four technical replicates.


C , D Three independent C9 patient iPSC - MN lines were treated with 10 µM H89 and levels of poly - GA ( C ) and poly - GP ( D ) were measured after 14 days . Values are displayed as percentages of mean DMSO signal . H89 significantly reduced poly - GA * P = 0 . 039 and poly - GP levels * P = 0 . 018 ( linear mixed effects model ) . Treatments were performed in four technical replicates .

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

C, D Three independent C9 patient iPSC-MN lines were treated with 10 µM H89 and levels of poly-GA (C) and poly-GP (D) were measured after 14 days. Values are displayed as percentages of mean DMSO signal. H89 significantly reduced poly-GA *P = 0.039 and poly-GP levels *P = 0.018 (linear mixed effects model). Treatments were performed in four technical replicates.


E No increased LDH release was observed after 14 - day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC - MN lines ( unpaired t - tests , Becker S6 : significant decrease in LDH , * * P = 0 . 0064 ; M211R2 : not significant , P > 0 . 05 ) . In Becker S6 , mean LDH release was 22 % lower after H89 treatment compared to vehicle . Treatments were performed in eight technical replicates . Data information : Error bars reported as ± SD . Not significant = P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 .

O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM B-CELL_TYPE O B-CELL_TYPE O O O O O O O B-CELL_LINE I-CELL_LINE 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-CELL_LINE I-CELL_LINE O O B-GENEPROD 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 O O O O O O O O O O O O O O O O O O

E No increased LDH release was observed after 14-day 10 µM H89 treatment compared to vehicle treatment in two independent C9 patient iPSC-MN lines (unpaired t-tests, Becker S6: significant decrease in LDH, **P = 0.0064; M211R2: not significant, P>0.05). In Becker S6, mean LDH release was 22% lower after H89 treatment compared to vehicle. Treatments were performed in eight technical replicates. Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01.


F Relative fold changes of C9orf72 variant RNA levels normalised to RPL13A , after 14 - day 10 µM H89 treatment vs vehicle treatment ( N = 1 C9 patient line , four technical replicates ) . No significant changes were observed between H89 and vehicle for any of the isoforms ( 2 - way ANOVA with Šídák ' s multiple comparisons test , all not significant , P > 0 . 05 ) . Data information : Error bars reported as ± SD . Not significant = P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 . T

O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM 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 O O O O O O O O O O O O O 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 Relative fold changes of C9orf72 variant RNA levels normalised to RPL13A, after 14-day 10 µM H89 treatment vs vehicle treatment (N=1 C9 patient line, four technical replicates). No significant changes were observed between H89 and vehicle for any of the isoforms (2-way ANOVA with Šídák's multiple comparisons test, all not significant, P>0.05). Data information: Error bars reported as ± SD. Not significant = P>0.05, *P<0.05, **P<0.01. T


Human monocytic ( THP - 1 ) cells were induced to acquire an M2 - like phenotype ( see Methods ) and treated with different concentrations of TLR7 - 54 or FA - TLR7 - 54 for either 48h In the latter case , after the 2h incubation , culture medium was replaced with drug - free medium and incubation was continued for 46h . All treatment groups were then analyzed by qPCR for gene expression ( A ) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7 - 54 and FA - TLR7 - 54 ( n = 3 , technical replicates ) .

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

Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. All treatment groups were then analyzed by qPCR for gene expression (A) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7-54 and FA-TLR7-54 (n=3, technical replicates).


Human monocytic ( THP - 1 ) cells were induced to acquire an M2 - like phenotype ( see Methods ) and treated with different concentrations of TLR7 - 54 or FA - TLR7 - 54 for either 48h or 2h In the latter case , after the 2h incubation , culture medium was replaced with drug - free medium and incubation was continued for 46h . cell supernatants were analyzed for secreted cytokines by ELISA . ( B - C ) Changes in CCL18 and IL - 1β in the culture media induced upon treatment with TLR7 - 54 and FA - TLR7 - 54 for the treatment regimens ( n = 3 , technical replicates ) .

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

Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h or 2h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. cell supernatants were analyzed for secreted cytokines by ELISA. (B-C) Changes in CCL18 and IL-1β in the culture media induced upon treatment with TLR7-54 and FA-TLR7-54 for the treatment regimens (n=3, technical replicates).


M2 - induced human monocyte - derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h , or initially for 2h in the presence or absence of FA - glucosamine ( competition ) followed by 46h in the absence of drug ( 2 + 46h ) , as described in Fig 1 . mRNA levels of profibrotic markers , Arg1 ( A ) , CD206 ( B ) and CD163 ( C ) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) .

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

M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. mRNA levels of profibrotic markers, Arg1 (A), CD206 (B) and CD163 (C) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).


M2 - induced human monocyte - derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h , or initially for 2h in the presence or absence of FA - glucosamine ( competition ) followed by 46h in the absence of drug ( 2 + 46h ) , as described in Fig 1 . protein levels of secreted profibrotic CCL18 ( D ) and antifibrotic cytokines , CXCL10 ( E ) and IL - 6 ( F ) ( n = 3 , technical replicates ) were then determined . Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) .

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

M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. protein levels of secreted profibrotic CCL18 (D) and antifibrotic cytokines, CXCL10 (E) and IL-6 (F) (n=3, technical replicates) were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).


( A ) Healthy mice or BLM - induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200 - fold excess FA - glucosamine to block all folate receptors . After 2h , mice were euthanized , resected and imaged for fluorescence intensity ( n = 5 ) .

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

(A) Healthy mice or BLM-induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200-fold excess FA-glucosamine to block all folate receptors. After 2h, mice were euthanized, resected and imaged for fluorescence intensity (n=5).


( B ) Alternatively , lungs from mice injected with 100 nmol OTL38 in the present or absence of 200 - fold excess FA - glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4 / 80 prior to FACS analysis ( n = 3 ) . Representative plots showing the gating strategy yielding live macrophages ( 7AAD - CD11b + F4 / 80 + ) and OTL38 positive macrophages are shown .

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

(B) Alternatively, lungs from mice injected with 100 nmol OTL38 in the present or absence of 200-fold excess FA-glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4/80 prior to FACS analysis (n=3). Representative plots showing the gating strategy yielding live macrophages (7AAD- CD11b+ F4/80+) and OTL38 positive macrophages are shown.


( C ) Percentages of live macrophages in BLM - induced mice ( n = 3 ) .

O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O

(C) Percentages of live macrophages in BLM-induced mice (n=3).


( D ) Percentages of lung macrophages that accumulated OTL38 in vivo ( n = 3 ) .

O O O O O B-TISSUE B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O O

(D) Percentages of lung macrophages that accumulated OTL38 in vivo (n=3).


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( A ) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR ( n = 3 ) .

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

Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (A) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR (n=3).


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( B ) BALF cells were pelleted and similarly analyzed for the indicated mRNAs ( n = 3 ) .

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

Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (B) BALF cells were pelleted and similarly analyzed for the indicated mRNAs (n=3).


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( C ) BALF supernatant was also collected and analyzed by ELISA for IL - 6 , IFNα and TNFα ( n = 3 ) .

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

Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (C) BALF supernatant was also collected and analyzed by ELISA for IL-6, IFNα and TNFα (n=3).


( B ) Survival analysis of the treatment groups ( Healthy , n = 5 ; others , n = 10 ) .

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

(B) Survival analysis of the treatment groups (Healthy, n=5; others, n=10).


BALF was collected on day 21 and centrifuged to isolate cell pellets . Cells ( primarily macrophages ) were analyzed by qPCR for Arg1 ( C ) , MMP9 ( D ) , TIMP3 ( E )

B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O

BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for Arg1 (C), MMP9 (D), TIMP3 (E)


BALF was collected on day 21 and centrifuged to isolate cell pellets . Cells ( primarily macrophages ) were analyzed by qPCR for CD86 ( F ) and IRAK4 ( G ) ( n = 5 ) .

B-TISSUE O O O O O O B-EXP_ASSAY O O O O O O O O B-CELL_TYPE O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O

BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for CD86 (F) and IRAK4 (G) (n=5).


BALF supernatant was analyzed by ELISA for IFNγ ( H ) ( n = 5 ) .

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

BALF supernatant was analyzed by ELISA for IFNγ (H) (n=5).


( I ) Lungs were resected and subjected to hematoxylin - eosin ( H & E ) , trichome ( collagen ) and α - SMA IHC staining ( scale bars , 200 μm ) ,

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

(I) Lungs were resected and subjected to hematoxylin-eosin (H&E), trichome (collagen) and α-SMA IHC staining (scale bars, 200 μm),


Lungs were ( J ) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content ( Healthy , n = 5 ; others , n = 10 ) ,

B-TISSUE O O O O O O O O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

Lungs were (J) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content (Healthy, n=5; others, n=10),


( K ) Ashcroft score quantitation of fibrosis ( n = 5 ) .

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

(K) Ashcroft score quantitation of fibrosis (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 and then sacrificed on day 21 for analysis . ( A ) Analysis of body weight 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 B-EXP_ASSAY I-EXP_ASSAY 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 and then sacrificed on day 21 for analysis . ( B ) Analysis of the number of cells 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 O O O O B-EXP_ASSAY I-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 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 ) .

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 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 and then sacrificed on day 21 for analysis . ( D ) H & E staining and trichrome staining 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 O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY 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 ) , 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 ) .

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 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 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 ) , 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 ) .

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

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 ) , 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 ) .

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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 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 .

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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 and p5 ) on VeroE6 cells , the mCherry reporter gene in ISA mCherry D614 supernatant medium was RT - PCR amplified and analysed using gel electrophoresis .

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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 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 ) .

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(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 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 .

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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 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 ) .

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

O 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 B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O 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 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.


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

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

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

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


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

O O O O O O O B-GENEPROD O 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 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 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