ChIP assays of CTGF , CYR61 α - SMA promoters using anti - FLAG - tag , anti - Myc - tag , anti - TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc - tagged YAP - 5SA / or FLAG - tagged HOXA4 . * P < 0 . 05 , * * P < 0 . 01 vs FLAG - GFP , by unpaired two - Student ' s t - . Data are expressed as means ± of three independent experiments .

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

ChIP assays of CTGF, CYR61 and α-SMA promoters using anti-FLAG-tag, anti-Myc-tag, anti-TEAD1 antibodies in human VSMCs transfected with plasmids expressing Myc-tagged YAP-5SA and/or FLAG-tagged HOXA4. *P<0.05, **P<0.01 vs FLAG-GFP, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Quantitative real - time PCR of differentiated smooth muscle marker genes in left carotid artery of 8 - week - old WT Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) .

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

Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).


Quantitative real - time PCR of differentiated smooth muscle marker genes in ligated left carotid artery of WT Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at baseline was defined as 1 . 0 .

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

Quantitative real-time PCR analysis of differentiated smooth muscle marker genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.


Hoxa4 expression in ligated left carotid artery of WT mice at 1 week after ligation ( n = 6 ) .

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

Hoxa4 expression levels in the ligated left carotid artery of WT mice at 1 week after ligation (n=6).


Quantitative real - time PCR of Yap / Tead target genes in left carotid artery of 8 - week - old WT Hoxa4 KO mice ( WT : n = 7 , Hoxa4 KO : n = 5 ) .

Quantitative real-time PCR analysis of Yap/Tead target genes in the left carotid artery of 8-week-old WT and Hoxa4 KO mice (WT: n=7, Hoxa4 KO: n=5).


Quantitative real - time PCR of Yap / Tead target genes in ligated left carotid artery of WT Hoxa4 KO mice at 1 week after ligation ( n = 6 ) . Mean expression level of six WT mice at baseline was defined as 1 . 0 .

Quantitative real-time PCR analysis of Yap/Tead target genes in the ligated left carotid artery of WT and Hoxa4 KO mice at 1 week after ligation (n=6). Mean expression level of six WT mice at the baseline was defined as 1.0.


Representative western blotting densitometry of non - ligated ( right ; R ) ligated ( left ; L ) carotid arteries from WT Hoxa4 KO mice at 1 week after ligation ( WT : n = 6 , Hoxa4 KO : n = 7 ) . * P < 0 . 05 , * * P < 0 . 01 , by two - way ANOVA with Sidak ' s correction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 western blotting analysis and densitometry of non-ligated (right; R) and ligated (left; L) carotid arteries from WT and Hoxa4 KO mice at 1 week after ligation (WT: n=6, Hoxa4 KO: n=7). *P<0.05, **P<0.01, by two-way ANOVA with Sidak's correction.


Representative showing Elastica van Gieson ( G ) , vessel , neointima media ( H ) , neointima to media layer ( I ) of carotid arteries of WT Hoxa4 KO mice at 4 after ligation ( WT : n = 14 , Hoxa4 KO : n = 11 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images showing Elastica van Gieson staining (G), and vessel, neointima and media area (H), and neointima to media layer ratio (I) of carotid arteries of WT and Hoxa4 KO mice at 4 weeks after ligation (WT: n=14, Hoxa4 KO: n=11).


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in left rear footpad with 103 PFU of CHIKV . ( E ) Viral tissue burdens were analyzed at 1 day post inoculation ( dpi ) by FFA . Mean ± .

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of CHIKV. (E) Viral tissue burdens were analyzed at 1 day post inoculation (dpi) by FFA. Mean ± SEM.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in left rear footpad with 103 PFU of ONNV ( F ) Tissues serum were collected at 1 dpi analyzed by plaque .

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

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of ONNV (F) Tissues and serum were collected at 1 dpi and analyzed by plaque assay.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in left rear footpad with 103 PFU of RRV ( G ) . Tissues serum were collected at 1 dpi analyzed by plaque .

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 103 PFU of RRV (G). Tissues and serum were collected at 1 dpi and analyzed by plaque assay.


( A ) WT Clec4F - DTR + C57BL / 6 mice were treated with diphtheria toxin ( DT ) either intraperitoneally ( i . p . ) 24 h prior to inoculation ( n = 10 , data pooled from two experiments , black dots ) or intravenously ( i . v . ) 48 24 h prior to inoculation ( n = 4 , one experiment , grey dots ) to deplete KCs inoculated i . v . with 108 CHIKV particles . Viral genomes in inoculum serum at 45 minutes ( min ) - post inoculation were determined by RT - qPCR . Mean ± SD . Data are pooled from three experiments , n = 14 . Mann - Whitney ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT and Clec4F-DTR+ C57BL/6 mice were treated with diphtheria toxin (DT) either intraperitoneally (i.p.) 24 h prior to inoculation (n=10, data pooled from two experiments, black dots) or intravenously (i.v.) 48 and 24 h prior to inoculation (n=4, one experiment, grey dots) to deplete KCs and inoculated i.v. with 108 CHIKV particles. Viral genomes in the inoculum and serum at 45 minutes (min)-post inoculation were determined by RT-qPCR. Mean ± SD. Data are pooled from three experiments, n= 14. Mann-Whitney test; ****P < 0.0001.


( C ) WT or CD169 - DTR + mice were treated i . p . with DT prior to i . v . inoculation of 108 CHIKV particles . Viral genomes were quantified as in ( A ) . Mean ± SD . Data are pooled from two experiments , n = 7 - 8 . Mann - Whitney ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT or CD169-DTR+ mice were treated i.p. with DT prior to i.v. inoculation of 108 CHIKV particles. Viral genomes were quantified as in (A). Mean ± SD. Data are pooled from two experiments, n=7-8. Mann-Whitney test; ***P < 0.001.


( D ) Livers from WT , Clec4F - DTR + or CD169 - DTR + mice treated i . p . with DT , or WT mice treated i . v . with PLL or CLL were analyzed by to visualize F4 / 80 + macrophages ( brown ) . Two representative sections for each group are shown .

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

(D) Livers from WT, Clec4F-DTR+ or CD169-DTR+ mice treated i.p. with DT, or WT mice treated i.v. with PLL or CLL were analyzed by IHC to visualize F4/80+ macrophages (brown). Two representative sections for each group are shown.


( A ) WT or Clec4F - DTR + C57BL / 6 mice were treated i . v . with DT prior to s . c . inoculation in left rear footpad with 103 PFU of CHIKV . Infectious was quantified at 24 hpi by FFA . Mean ± . Data are pooled from two experiments , n = 8 . Mann - Whitney ; P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT or Clec4F-DTR+ C57BL/6 mice were treated i.v. with DT prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus was quantified at 24 hpi by FFA. Mean ± SEM. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.


( B ) WT C57BL / 6 mice were treated i . v . with PLL or CLL 42 h prior to s . c . inoculation in left rear footpad with 103 PFU of CHIKV . Infectious at 24 hpi was quantified by FFA . Mean ± SD . Data are pooled from two experiments , n = 8 . Mann - Whitney ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT C57BL/6 mice were treated i.v. with PLL or CLL 42 h prior to s.c. inoculation in the left rear footpad with 103 PFU of CHIKV. Infectious virus at 24 hpi was quantified by FFA. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; ***P < 0.001.


( B ) WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R . Viral genomes in dLN serum at 2 hpi were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 10 . Two - way ANOVA with Bonferroni ' s multiple comparisons ; * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(B) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of WT CHIKV or CHIKV E2 K200R. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=10. Two-way ANOVA with Bonferroni's multiple comparisons test; ***P < 0.001, ****P < 0.0001.


( C ) WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in left rear footpad with 108 particles of RRV . Viral genomes in dLN serum were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 8 - 9 . Mann - Whitney ; * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(C) WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the left rear footpad with 108 particles of RRV. Viral genomes in the dLN and serum were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8-9. Mann-Whitney test; ***P < 0.001, ****P < 0.0001.


( A ) CD169 - DTR + mice were treated i . p . with PBS or DT prior to collection of popliteal LN . Frozen LN sections were stained for CD169 + macrophages ( green ) , B220 + B ( blue ) or ERTR - 7 + stromal ( red ) . Image shown is representative of three biological replicates . Scale bars : 200 μm .

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

(A) CD169-DTR+ mice were treated i.p. with PBS or DT prior to collection of the popliteal LN. Frozen LN sections were stained for CD169+ macrophages (green), B220+ B cells (blue) or ERTR-7+ stromal cells (red). Image shown is representative of three biological replicates. Scale bars: 200 μm.


( B ) WT or CD169 - DTR + C57BL / 6 mice were treated i . p . with DT prior to s . c . inoculation in left rear footpad with 108 particles of WT CHIKV . Viral genomes in dLN serum at 2 hpi were quantified by RT - qPCR . Mean ± SD . Data are pooled from two experiments , n = 8 . Mann - Whitney ; P > 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT or CD169-DTR+ C57BL/6 mice were treated i.p. with DT prior to s.c. inoculation in the left rear footpad with 108 particles of WT CHIKV. Viral genomes in the dLN and serum at 2 hpi were quantified by RT-qPCR. Mean ± SD. Data are pooled from two experiments, n=8. Mann-Whitney test; P > 0.05.


WT or MARCO - / - C57BL / 6 mice were inoculated s . c . in rear feet with 5 * 104 PFU of CHIKV - E2 - mCherry popliteal dLNs were collected at 2 hpi . ( A ) Frozen dLN sections were stained for Lyve - 1 + LECs ( ) , MARCO ( green ) , B220 ( blue ) mCherry + CHIKV particles ( red ) . Scale bars : 200 μm .

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

WT or MARCO-/- C57BL/6 mice were inoculated s.c. in the rear feet with 5*104 PFU of CHIKV-E2-mCherry and the popliteal dLNs were collected at 2 hpi. (A) Frozen dLN sections were stained for Lyve-1+ LECs (white), MARCO (green), B220 (blue) and mCherry+ CHIKV particles (red). Scale bars: 200 μm.


( H , I ) MARCO ( H ) Mxra8 ( I ) expression is shown for MARCO + LECs for mock - infected CHIKV - infected classified as either CHIKV - low or CHIKV - high . P values were calculated using a two - sided Wilcoxon rank - sum with Bonferroni correction . In boxplot , central lines , box limits , whiskers represent medians , interquartile range ( IQR ) , min / max values that are not outliers , respectively . Outliers are shown as points include any values that are more than 1 . 5x IQR away from box .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, I) MARCO (H) and Mxra8 (I) expression is shown for MARCO+ LECs for mock-infected cells and CHIKV-infected cells classified as either CHIKV-low or CHIKV-high. P values were calculated using a two-sided Wilcoxon rank-sum test with Bonferroni correction. In the boxplot, the central lines, the box limits, and the whiskers represent medians, the interquartile range (IQR), and min/max values that are not outliers, respectively. Outliers are shown as points and include any values that are more than 1.5x IQR away from the box.


( A , B ) Localization of oskar mRNA in wild - type ( A ) in Tm1eg9 / Tm1eg9 ( B ) egg - chambers .

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

(A, B) Localization of oskar mRNA in wild-type (A) and in Tm1eg9/Tm1eg9 (B) egg-chambers.


( C ) Kymograph of oskMS2 - GFP mRNPs ( cyan ) travelling along polarity marked MTs ( red , EB1 ) in an ex vivo ooplasmic preparation . dashed arrow shows growing plus tip of MT . Scale bars represent 1 s 1 µm , respectively . ( D ) Distribution of oskMS2 - GFP mRNP runs towards plus ( ) minus ends ( grey ) . Numbers within bars indicate number of runs . P value of Chi2 against wild - type is indicated above each bar .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Kymograph of oskMS2-GFP mRNPs (cyan) travelling along polarity marked MTs (red, EB1 protein) in an ex vivo ooplasmic preparation. White dashed arrow shows the growing plus tip of the MT. Scale bars represent 1 s and 1 µm, respectively.(D) Distribution of oskMS2-GFP mRNP runs towards plus (white) and minus ends (grey). Numbers within the bars indicate number of runs. P value of Chi2 test against wild-type is indicated above each bar.


( E , F ) oskar mRNA ( green ) Oskar ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers expressing oskarMS2 ( 6x ) ( E ) or oskarMS2 ( 6x ) Khc401 - MCP ( F ) .

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

(E, F) oskar mRNA (green) and Oskar protein (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers expressing oskarMS2(6x) (E) or oskarMS2(6x) and Khc401-MCP (F).


( G ) oskar mRNA ( magenta ) distribution in Tm1eg1 / Tm1eg9 egg - chambers rescued with EmGFP - Tm1 - I ( green ) . Scale bars represent 50 µm .

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

(G) oskar mRNA (magenta) distribution in Tm1eg1/Tm1eg9 egg-chambers rescued with EmGFP-Tm1-I (green). Scale bars represent 50 µm.


( A - B " ) Colocalization of oskMS2 - mCherry ( red , A , B ) ( A ' , B ' ) with Khc - EGFP ( green , A ) ( A ' ) or with EmGFP - Tm1 - I ( green B ) ( B ' ) in ex vivo ooplasmic preparations . ( C ) Fraction of oskMS2 - MCP - mCherry mRNPs located non - randomly within a 200 nm of one of indicated GFP tagged particles in ex vivo ooplasmic preparations . MCP indicates MCP - EGFP which , like MCP - mCherry , can bind to MS2 loops . Staufen ( Stau ) is a dsRNA binding bona fide partner of oskar mRNA ( St Johnston et al . , 1991 ; St Johnston et al . , 1992 ) . values are significantly different from zero ( p < 10 - 3 , one sample t - ) . ( D ) Fraction of oskMS2 - mCherry mRNPs co - localizing ( max . 200 nm ) non - randomly with Khc - EGFP particles in wild - type in Tm1eg1 / Tm1eg9 ooplasms in presence of two ( ) or one ( grey ) copy of endogenous Khc . ( C , D ) P values of two sample t - are indicated above relevant bar pairs . Numbers indicate number of particle clusters ( 160 mRNPs in each ) number of preparations ( in brackets ) analysed . Error bars represent 95 % confidence intervals .

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

(A-B") Colocalization of oskMS2-mCherry (red, A, B) (A', B') with Khc-EGFP (green, A) (A') or with EmGFP-Tm1-I (green B) (B') in ex vivo ooplasmic preparations.(C) Fraction of oskMS2-MCP-mCherry mRNPs located non-randomly within a 200 nm distance of one of the indicated GFP tagged protein particles in ex vivo ooplasmic preparations. MCP indicates MCP-EGFP which, like MCP-mCherry, can bind to MS2 loops. Staufen (Stau) is a dsRNA binding protein and bona fide partner of oskar mRNA (St Johnston et al., 1991; St Johnston et al., 1992). All values are significantly different from zero (p<10-3, one sample t-test).(D) Fraction of oskMS2-mCherry mRNPs co-localizing (max. 200 nm) non-randomly with Khc-EGFP particles in wild-type and in Tm1eg1/Tm1eg9 ooplasms in presence of two (white) or one (grey) copy of endogenous Khc.(C, D) P values of two sample t-tests are indicated above the relevant bar pairs. Numbers indicate the number of particle clusters (160 mRNPs in each) and the number of preparations (in brackets) analysed. Error bars represent 95% confidence intervals.


( A , B ) Kymographs of oskMS2 - GFP mRNPs ( green ) associated with Khc - mKate2 ( A , red ) mCherry - Tm1 - I / C ( B , red ) ex vivo . Arrows indicate motile RNPs in stable with Khc ( A ) or Tm1 - I / C ( B ) , arrowheads point to non - motile oskMS2 - RNPs showing no obvious accumulation of tagged . Note that mCherry - Tm1 - I was exposed twice as long as Khc - mKate to obtain comparable red signals . Scale bars represent 1 µm 1 second , respectively . ( C - E ) Relative Khc - mKate2 ( C ) , mCherry - Tm1 - I / C ( D ) coverage of motile ( ) non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within boxes indicate number of trajectories number of ooplasms ( in brackets ) analysed . Percentages above plots show fraction of RNPs that were found stably reliably associating with indicated ( for at least half of duration of trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U are indicated above boxplots .

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

(A, B) Kymographs of oskMS2-GFP mRNPs (green) associated with Khc-mKate2 (A, red) and mCherry-Tm1-I/C (B, red) ex vivo. Arrows indicate motile RNPs in stable complex with Khc (A) or Tm1-I/C (B), the arrowheads point to non-motile oskMS2-RNPs showing no obvious accumulation of the tagged protein. Note that mCherry-Tm1-I was exposed twice as long as Khc-mKate to obtain comparable red fluorescence signals. Scale bars represent 1 µm and 1 second, respectively.(C-E) Relative Khc-mKate2 (C), mCherry-Tm1-I/C (D) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.


( C - E ) Relative GFP - Staufen ( E ) coverage of motile ( ) non - motile ( grey ) oskMS2 - GFP trajectories . Numbers within boxes indicate number of trajectories number of ooplasms ( in brackets ) analysed . Percentages above plots show fraction of RNPs that were found stably reliably associating with indicated ( for at least half of duration of trajectory , p < 0 . 01 , binominal distribution , see also Fig EV2B ) . P values of pairwise Mann - Whitney U are indicated above boxplots .

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

(C-E) Relative GFP-Staufen (E) coverage of motile (white) and non-motile (grey) oskMS2-GFP trajectories. Numbers within the boxes indicate the number of trajectories and the number of ooplasms (in brackets) analysed. Percentages above the plots show the fraction of RNPs that were found stably and reliably associating with the indicated protein (for at least half of the duration of the trajectory, p<0.01, binominal distribution, see also Fig EV2B). P values of pairwise Mann-Whitney U tests are indicated above the boxplots.


( F ) Example kymograph of Khc molecules ( red ) associating with an oskMS2 - GFP mRNP ( green ) before during its run . arrowheads indicate two association events , yellow arrow indicates onset of motility . Scale bars represent 5 µm 1 second , respectively . ( G ) Frequency of Khc - mKate2 appearance on motile ( ) , before ( motility - primed , checked ) after ( dotted ) onset of motility , non - motile ( grey ) oskMS2 - GFP trajectories . Fractions within bars indicate number of association events that lasted longer than a single frame over total number of frames analysed . Indicated P values show results of pairwise Fisher ' s exact . Khc association frequency observed on RNPs before ( checked ) during ( dotted ) their motility is not significantly different from wild - type motile RNP controls ( p > 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Example kymograph of Khc molecules (red) associating with an oskMS2-GFP mRNP (green) before and during its run. White arrowheads indicate two association events, yellow arrow indicates the onset of motility. Scale bars represent 5 µm and 1 second, respectively.(G) Frequency of Khc-mKate2 appearance on motile (white), before (motility-primed, checked) and after (dotted) the onset of motility, and non-motile (grey) oskMS2-GFP trajectories. Fractions within the bars indicate number of association events that lasted longer than a single frame over the total number of frames analysed. Indicated P values show results of pairwise Fisher's exact test. The Khc association frequency observed on RNPs before (checked) and during (dotted) their motility is not significantly different from wild-type motile RNP controls (p>0.01).


( A ) Western blots of oskar mRNP components ( Staufen , Bruno Y14 ) motor associated ( Khc , BicD , Dic ) co - immunoprecipitated with EmGFP - Tm1 - I from ovarian lysates . marker bands their molecular in kDa are indicated .

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

(A) Western blots of oskar mRNP components (Staufen, Bruno and Y14) and motor associated proteins (Khc, BicD, Dic) co-immunoprecipitated with EmGFP-Tm1-I from ovarian lysates. Protein marker bands and their molecular weight in kDa are indicated.


( B ) Signal developed with oligo ( dT ) 25 - TexasRed probes on GFP - Trap _ M beads binding EmGFP - Tm1 - I or FlagMycGFP after denaturing washes . 594 nm reflection marks bead boundaries . ( B ' ) of mean oligo ( dT ) signal measured on beads .

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

(B) Signal developed with oligo(dT)25-TexasRed probes on GFP-Trap_M beads binding EmGFP-Tm1-I or FlagMycGFP after denaturing washes. 594 nm reflection marks bead boundaries.(B') Quantification of mean oligo(dT) signal measured on beads.


( C ) qRT - PCR of EmGFP - Tm1 - I control Flag - Myc - GFP bead alone ( w1118 ) bound oskar gapdh RNAs after stringent washes ( mean ± s . e . m . , N = 3 ) .

O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR of EmGFP-Tm1-I and control Flag-Myc-GFP and bead alone (w1118) bound oskar and gapdh RNAs after stringent washes (mean ± s.e.m., N = 3).


( D ) of beads binding to EmGFP - Tm1 - I ( green ) DIG labelled in vitro transcribed fragments ( magenta ) . ( E , F ) Mean DIG - Cy5 measured on beads capturing fragment , with or without UV cross - linking , indicated below charts . P values of pairwise Mann - Whitney U are indicated . In panel F , none of non - cross - linked samples differ significantly from no control ( p > 0 . 05 ) . Numbers below plots indicate number of beads analysed .

(D) Images of beads binding to EmGFP-Tm1-I (green) and DIG labelled in vitro transcribed RNA fragments (magenta).(E, F) Mean DIG-Cy5 fluorescence measured on beads capturing the RNA fragment, with or without UV cross-linking, indicated below the charts. P values of pairwise Mann-Whitney U tests are indicated. In panel F, none of the non-cross-linked samples differ significantly from the no RNA control (p>0.05). Numbers below the plots indicate the number of beads analysed.


( A , B , C ) Confocal image of a Tm1eg9 homozygous egg - chamber expressing EmGFP - Tm1 - I ( green ) Khc - mKate2 ( magenta ) . oskar mRNA labelled with osk1 - 5 FIT probes ( Hovelmann et al . , 2014 ) is in yellow . ( B ' , C ' ) oskar mRNPs co - localizing with both EmGFP - Tm1 - I Khc - mKate2 . Colors indicate maximal co - localization ( C ' ) . Panels B - C ' represent boxed regions in panel A .

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

(A, B, C) Confocal image of a Tm1eg9 homozygous egg-chamber expressing EmGFP-Tm1-I (green) and Khc-mKate2 (magenta). oskar mRNA labelled with osk1-5 FIT probes(Hovelmann et al., 2014) is in yellow.(B', C') oskar mRNPs co-localizing with both EmGFP-Tm1-I and Khc-mKate2. Colors indicate the maximal co-localization distance (C'). Panels B-C' represent the boxed regions in panel A.


( D , E ) Localization of Khc - mKate2 ( green ) oskar mRNA ( magenta ) in wild - type Tm1gs mutant nurse . ( F , G ) Fraction of oskar mRNPs co - localizing with Khc - mKate ( grey ) , EmGFP - Tm1 - I ( ) , or both of these ( grey ) in oocyte ( F ) or in nurse ( G ) ( max . colocalization is 250 nm ) . None of values are significantly different from each other ( one - way ANOVA , p > 10 - 3 ) . Horizontal dashed lines indicate expected value of observing both in an oskar mRNP if interactions are independent ( see Fig EV3C D ) . Significance of observed co - localization values versus expected values are shown . ( H ) Fraction of oskar mRNPs co - localizing with Khc - mKate in wild - type Tm1gs mutant nurse when half or Khc molecules are labelled ( as indicated above graph ) . P values of pair - wise t - are indicated . ( I ) Fraction of oskar mRNPs co - localizing with free mCherry in wild - type nurse used as negative control . measured fraction ( ~ 1 . 6 % ) is significantly different from zero ( one sample t - ) . other measured co - localization values are significantly different form this negative control ( p < 0 . 001 , one - way ANOVA ) . ( F - I ) Numbers indicate number of particle clusters ( 100 oskar mRNPs in each ) number of egg - chambers ( in brackets ) analysed . Error bars represent 95 % confidence intervals . values ( F - I ) are significantly different from zero ( p < 10 - 3 , one sample t - ) .

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

(D, E) Localization of Khc-mKate2 (green) and oskar mRNA (magenta) in wild-type and Tm1gs mutant nurse cells.(F, G) Fraction of oskar mRNPs co-localizing with Khc-mKate (dark grey), EmGFP-Tm1-I (white), or both of these proteins (light grey) in the oocyte (F) or in the nurse cells (G) (max. colocalization distance is 250 nm). None of the values are significantly different from each other (one-way ANOVA, p>10-3). Horizontal dashed lines indicate the expected value of observing both proteins in an oskar mRNP if the interactions are independent (see Fig EV3C and D). Significance of the observed co-localization values versus the expected values are shown.(H) Fraction of oskar mRNPs co-localizing with Khc-mKate in wild-type and Tm1gs mutant nurse cells when half or all Khc molecules are labelled (as indicated above the graph). P values of pair-wise t-tests are indicated.(I) Fraction of oskar mRNPs co-localizing with free mCherry in wild-type nurse cells used as negative control. The measured fraction (~1.6%) is significantly different from zero (one sample t-test). All other measured co-localization values are significantly different form this negative control (p<0.001, one-way ANOVA).(F-I) Numbers indicate the number of particle clusters (100 oskar mRNPs in each) and the number of egg-chambers (in brackets) analysed. Error bars represent 95% confidence intervals. All values (F-I) are significantly different from zero (p<10-3, one sample t-test).


( A - C ) Localization of EmGFP - Tm1 - I ( A ) oskar mRNA ( B ) Khc ( C ) around nurse envelope ( magenta , WGA ) . ( D - F ) Mean distribution profile of Khc ( D ) mKate2 - Tm1 - I , EmGFP - Tm1 - I ( E ) oskar mRNA ( F ) around NE of nurse ( marked with a dotted vertical ) . Genotypes are indicated as follows : wild - type control with solid black ( D - F ) , oskar null with solid grey ( D , E ) , Tm1eg9 / Tm1eg9 with dotted black ( D , F ) Lines indicate mean 95 % confidence intervals . We analysed accumulation of mKate2 - Tm1 - I instead of EmGFP - Tm1 - I due to unexpected GFP expression from oskar0 allele .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Localization of EmGFP-Tm1-I (A) oskar mRNA (B) and Khc (C) around the nurse cell nuclear envelope (magenta, WGA staining).(D-F) Mean distribution profile of Khc (D) mKate2-Tm1-I, EmGFP-Tm1-I (E) and oskar mRNA (F) around the NE of nurse cells (marked with a dotted vertical line). Genotypes are indicated as follows: wild-type control with solid black line (D-F), oskar RNA null with solid grey line (D, E), Tm1eg9/Tm1eg9 with dotted black line (D, F) Lines indicate mean and 95% confidence intervals. We analysed the accumulation of mKate2-Tm1-I instead of EmGFP-Tm1-I due to unexpected GFP expression from the oskar0 allele.


( G ) Khc accumulation around NE . To calculate accumulation signal measured at position of peak observed in wild - type control ( D , arrowhead , 356 ± 17 . 6 nm away from NE ) was divided by signal 2 s . d . away ( d , arrow , at 356 + 2 * 410 nm ) . P values of pairwise Mann - Whitney U against wild - type control or Tm1gs rescued with EmGFP - Tm1 - I are indicated above boxplots . Numbers indicate number of nuclei number of egg - chambers ( in brackets ) analysed . ( H - K ) Khc accumulation around NE of nurse over - expressing EmGFP - Tm1 - I within Tm1eg9 / Tm1eg9 ( H ) , oskar null egg - chambers ( I ) expressing either oskar 3 ' UTR ( J ) or oskar Δi ( 1 , 2 , 3 ) ( K ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Khc accumulation around the NE. To calculate accumulation the signal intensity measured at the position of the peak observed in the wild-type control (D, arrowhead, 356±17.6 nm away from NE) was divided by the signal intensity 2 s.d. away (d, arrow, at 356+2*410 nm). P values of pairwise Mann-Whitney U tests against wild-type control or Tm1gs rescued with EmGFP-Tm1-I are indicated above the boxplots. Numbers indicate the number of nuclei and the number of egg-chambers (in brackets) analysed.(H-K) Khc accumulation around the NE of nurse cells over-expressing EmGFP-Tm1-I within Tm1eg9/Tm1eg9 (H), and oskar RNA null egg-chambers (I) expressing either the oskar 3' UTR (J) or oskar Δi(1,2,3) (K).


( A - E ) Mean oskar mRNA distribution ( green , detected by conventional FISH ) within oocytes in which oskar mRNA is substituted by oskar Δi ( 1 , 2 , 3 ) mRNA ( A , B ) , oskar 3 ' UTR mRNA ( D ) that ( in addition ) over - express EmGFP - Tm1 - I ( B , D E ) . Wild - type control ( C ) oocytes expressing Khc RNAi EmGFP - Tm1 - I ( E ) . Magenta indicates standard deviation of distribution of oskar mRNA . Scale bar is 10 % of total oocyte ( C ) . ( F ) Position of oskar mRNA center of mass relative to geometric center of oocyte ( dotted horizontal ) along anteroposterior ( AP ) axis . Posterior pole is top of chart . P values of pairwise Mann - Whitney U are indicated . Numbers indicate number of oocytes analysed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Mean oskar mRNA distribution (green, detected by conventional FISH) within oocytes in which oskar mRNA is substituted by oskar Δi(1,2,3) mRNA (A, B), oskar 3'UTR mRNA (D) and that (in addition) over-express EmGFP-Tm1-I (B, D and E). Wild-type control (C) and oocytes expressing Khc RNAi and EmGFP-Tm1-I (E). Magenta indicates standard deviation of the distribution of oskar mRNA. Scale bar is 10% of total oocyte length (C).(F) Position of the oskar mRNA center of mass relative to the geometric center of the oocyte (dotted horizontal line) along the anteroposterior (AP) axis. Posterior pole is the top of the chart. P values of pairwise Mann-Whitney U tests are indicated. Numbers indicate number of oocytes analysed.


C Confirmatory screening performed as described above . Schematic distribution of compounds on Z - score values of expressing polyGP - GFP AUG - RFP ( above ) on Z - score values of of two reporters per each compound ( below ) . Data are from four technical replicates , boxes are Z - score mean value whiskers represent ± SD . Baseline of Z - score = 0 indicates that Z - score is identical to mean score . Asterisk ( * ) represents selected compounds : 1 Forskolin ( FSK ) , 2 Erysolin ( ERY ) , 3 Geldanamycin ( GELD ) , 4 Helenin ( HLN ) 5 Spironolactone ( SPL ) . Number sign ( # ) represents stress inducers : # 1 Thapsigargin # 2 Tunicamycin .

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

C Confirmatory screening performed as described above. Schematic distribution of compounds based on Z-score values of cells expressing polyGP-GFP and AUG-RFP (above) and on Z-score values of the fluorescent intensity of the two reporters per each compound (below). Data are from four technical replicates, boxes are the Z-score mean value and whiskers represent ±SD. Baseline of Z-score = 0 indicates that Z-score is identical to the mean score. Asterisk (*) represents the selected compounds: 1 Forskolin (FSK), 2 Erysolin (ERY), 3 Geldanamycin (GELD), 4 Helenin (HLN) and 5 Spironolactone (SPL). Number sign (#) represents cellular stress inducers: #1 Thapsigargin and #2 Tunicamycin.


D , E Dose - response of ERY , HLN , SPL , FSK GELD . were co - transfected with AUG - RFP polyGP - GFP plasmids treated with two ranges 0 . 5 , 1 , 5 10 µM ( D ) 20 , 40 60 µM ( E ) for 24 h . CHX used only low dosages . Data are mean ± SD from three biological replicates . 1 - way ANOVA followed by Dunnett ' s multiple comparison : * P < 0 . 5 ; * * P < 0 . 01 ; * * * P < 0 .

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

D, E Dose-response analysis of ERY, HLN, SPL, FSK and GELD. Cells were co-transfected with AUG-RFP and polyGP-GFP plasmids and treated with two concentration ranges 0.5, 1, 5 and 10 µM (D) and 20, 40 and 60 µM (E) for 24 h. CHX used only low dosages. Data are mean ± SD from three biological replicates. 1-way ANOVA followed by Dunnett's multiple comparison tests: *P<0.5; **P<0.01; ***P<0.001


B , C HEK293T were treated for 24 h general synthesis were monitored by incorporation of 5 - ethynyl uridine ( EU ) O - propargyl - puromycin ( OPP ) , respectively . ActD ( 5 μM ) CHX ( 350 μM ) were used for 3 h as positive control to block synthesis , respectively . Data are from three biological replicates represented as mean ± . ( B ) Two - , unpaired t - ; * * * P < 0 . 001 , * * P = 0 . 0047 . used : 1 μM GELD , 10 μM ERY , SPL FSK .

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

B, C HEK293T cells were treated for 24 h and general RNA and protein synthesis were monitored by the incorporation of 5-ethynyl uridine (EU) and O-propargyl-puromycin (OPP), respectively. ActD (5 μM) and CHX (350 μM) were used for 3 h as positive control to block RNA and protein synthesis, respectively. Data are from three biological replicates and represented as mean ± SEM. (B) Two-tailed, unpaired t-test; ***P<0.001, **P = 0.0047. Concentration used: 1 μM GELD, 10 μM ERY, SPL and FSK.


G Luminescence derived from HEK293T transfected with GA - NLuc reporters WT or mutated treated for 24 h . Mutational of near - cognate CUG start codon from three biological replicates four technical replicates , n = 12 . Data are mean ± . 2 - way ANOVA followed by Dunnett ' s multiple comparisons , * * * * P < 0 . 0001 . used : 1 μM GELD , 10 μM SPL FSK .

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

G Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, ****P<0.0001. Concentration used: 1 μM GELD, 10 μM SPL and FSK.


C Relative expression level of Sqstm1 / p62 , Lc3 , Tfeb , Hspb8 Bag3 mRNAs in NSC34 treated for 24 h . Data are mean ± from three biological replicates . 2 - way ANOVA followed by Dunnett ' s multiple comparisons , * * * P < 0 . 001 . Dotted lines represent expression in DMSO control . used : 1 μM GELD , 10 μM ERY , SPL , FSK .

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

C Relative expression level of Sqstm1/p62, Lc3, Tfeb, Hspb8 and Bag3 mRNAs in NSC34 cells treated for 24 h. Data are mean ± SEM from three biological replicates. 2-way ANOVA followed by Dunnett's multiple comparisons test, ***P<0.001. Dotted lines represent RNA expression in the DMSO control. Concentration used: 1 μM GELD, 10 μM ERY, SPL, FSK.


G Lysates from NSC34 transfected with scrambled - GPx100 , treated for 24 h with SPL / or 10 mM 3MA immunoblotted for scrambled - GP expression . from three biological replicates . Data are mean ± . Two - , unpaired t - , * * * * P < 0 . 0001 for SPL vs DMSO 3MA vs DMSO , * * * P = 0 . 0008 SPL + 3MA vs SPL . used : 1 μM GELD , 10 μM

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

G Lysates from NSC34 cells transfected with scrambled-GPx100, treated for 24 h with SPL and/or 10 mM 3MA and immunoblotted for scrambled-GP expression. Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001 for SPL vs DMSO and 3MA vs DMSO, ***P = 0.0008 SPL + 3MA vs SPL. Concentration used: 1 μM GELD, 10 μM SPL


B Lysates from HEK293T , transfected with 2R or 66R treated for 24 h , immunoblotted using antibody for poly - GA expression ( HA - tagged ) . from three biological replicates . Data are mean ± . Two - , unpaired t - , * * * * P < 0 . 0001 , * * P = 0 . 0027 H89 vs DMSO , * * * P = 0 . 0004 FSK + H89 vs DMSO , * P = 0 . 0234 CLZ vs DMSO , * P = 0 . 0209 FSK + H89 vs FSK . used : 2 . 5 μM H89 , 10 μM FSK , CLZ .

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

B Lysates from HEK293T cells, transfected with 2R or 66R and treated for 24 h, immunoblotted using antibody for poly-GA expression (HA-tagged). Quantification from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test, ****P<0.0001, **P = 0.0027 H89 vs DMSO, ***P = 0.0004 FSK + H89 vs DMSO, *P = 0.0234 CLZ vs DMSO, *P = 0.0209 FSK + H89 vs FSK. Concentration used: 2.5 μM H89, 10 μM FSK, CLZ.


F Luminescence derived from HEK293T transfected with GA - NLuc reporters WT or mutated treated with H89 for 24 h . Mutational of near - cognate CUG start codon from three biological replicates four technical replicates , n = 12 . Data are mean ± . 2 - way ANOVA followed by Dunnett ' s multiple comparisons , * P = 0 . 0130 WT H89 vs WT DMSO , * * * * P < 0 . 0001 , CUG mutated H89 vs CUG mutated DMSO . used : 2 . 5 μM H89 ,

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

F Luminescence derived from HEK293T transfected with GA-NLuc reporters WT or mutated and treated with H89 for 24 h. Mutational analysis of near-cognate CUG start codon from three biological replicates and four technical replicates, n = 12. Data are mean ± SEM. 2-way ANOVA followed by Dunnett's multiple comparisons test, *P = 0.0130 WT H89 vs WT DMSO, ****P<0.0001, CUG mutated H89 vs CUG mutated DMSO. Concentration used: 2.5 μM H89,


A , B Lysates from HEK293T , transfected with RNAi scrambled or RNAi PRKACA ( 72 h ) 2R or 66R ( 24 h ) , immunoblotted for PKA - Cα poly - GA ( HA - tag ) expression . α - Tubulin used as loading control . ( B ) of PKA - Cα poly - GA expression from three biological replicates . Data are mean ± . Two - , unpaired t - . PKA - Cα : ns siScr 66R vs siScr 2R , * * * P = 0 . 0002 siPRKACA 66R vs siScr 2R , * * * P = 0 . 0003 siPRKACA 66R vs siScr 66R . poly - GA expression : * * * * P < 0 . 0001 siScr 66R vs siScr 2R , * * * * P < 0 . 0001 siPRKACA 66R vs siScr 2R , * * P = 0 . 0062 siPRKACA 66R vs siScr 66R . C , D Lysates from HEK293T , transfected with RNAi scrambled or RNAi PRKACB ( 72 h ) 2R or 66R ( 24 h ) , immunoblotted PKA - Cβ poly - GA ( HA tag ) expression . α - Tubulin used as loading control . ( D ) of PKA - Cβ poly - GA expression from three biological replicates . Data are mean ± . Two - , unpaired t - . PKA - Cβ : ns siScr 66R vs siScr 2R , * * P = 0 . 0049 siPRKACB 66R vs siScr 2R , * * P = 0 . 0091 siPRKACB 66R vs siScr 66R . poly - GA expression : * * * * P < 0 . 0001 siScr 66R vs siScr 2R , * * * * P < 0 . 0001 siPRKACB 66R vs siScr 2R , * * * P = 0 . 0007 siPRKACB 66R vs siScr 66R . E

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

A, B Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACA (72 h) and 2R or 66R (24 h), immunoblotted for PKA-Cα and poly-GA (HA-tag) expression. α-Tubulin used as loading control. (B) Quantification of PKA-Cα and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cα: ns siScr 66R vs siScr 2R, ***P = 0.0002 siPRKACA 66R vs siScr 2R, ***P = 0.0003 siPRKACA 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACA 66R vs siScr 2R, **P = 0.0062 siPRKACA 66R vs siScr 66R . C, D Lysates from HEK293T cells, transfected with RNAi scrambled or RNAi PRKACB (72 h) and 2R or 66R (24 h), immunoblotted PKA-Cβ and poly-GA (HA tag) expression. α-Tubulin used as loading control. (D) Quantification of PKA-Cβ and poly-GA expression from three biological replicates. Data are mean ± SEM. Two-tailed, unpaired t-test. PKA-Cβ: ns siScr 66R vs siScr 2R, **P = 0.0049 siPRKACB 66R vs siScr 2R, **P = 0.0091 siPRKACB 66R vs siScr 66R. poly-GA expression: ****P<0.0001 siScr 66R vs siScr 2R, ****P<0.0001 siPRKACB 66R vs siScr 2R, ***P = 0.0007 siPRKACB 66R vs siScr 66R. E


A , B Representative graphs showing climbing ability flies carrying UAS - ( G4C2 ) x36 construct in neurons using Elav - gal4 promoter upon treatment with H89 10 µM diluted with 0 . 1 % of DMSO 5 % sucrose in PBS . wild - type ( w1118 ) was used as control . ( A ) flies ( ♀ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for 6th day of treatment * * * * P < 0 . 0001 ( G4C2 ) x36 vs ( G4C2 ) x36 + H89 . ( B ) flies ( ♂ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for 6th day of treatment . * * * * P < 0 . 0001 ( G4C2 ) x36 vs ( G4C2 ) x36 + H89 . Experiments were performed three times , N = 15 adult flies were used for each genotype , sex treatment ( N tot = 45 ) . C , D Representative graphs showing climbing activity of flies co - expressing UAS - Pka - RNAi construct ( 1 , v330111 ) in combination with UAS - ( G4C2 ) x36 in neurons using Elav - gal4 promoter . wild - type ( w1118 ) was used as control . ( C ) flies ( ♀ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for 9th day * * * * P < 0 . 0001 ( G4C2 ) x36 vs Pka - RNAi ( 1 ) ; ( G4C2 ) x36 . ( D ) flies ( ♂ ) . Data are mean ± SD . 2 - way ANOVA followed by Tukey ' s multiple comparisons , significance is reported for 7th day * * * * P < 0 . 0001 ( G4C2 ) x36 vs Pka - RNAi ( 1 ) ; ( G4C2 ) x36 . Experiments were performed twice with different interfering , N = 15 adult flies were used for each genotype sex ( N tot = 30 ) .

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

A, B Representative graphs showing the climbing ability flies carrying the UAS-(G4C2)x36 construct in neurons using the Elav-gal4 promoter upon treatment with H89 10 µM diluted with 0.1 % of DMSO and 5% sucrose in PBS. The wild-type (w1118) line was used as control. (A) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. (B) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 6th day of treatment. ****P<0.0001 (G4C2)x36 vs (G4C2)x36 + H89. Experiments were performed three times, N=15 adult flies were used for each genotype, sex and treatment (N tot=45). C, D Representative graphs showing climbing activity of flies co-expressing the UAS-Pka-RNAi construct (1, v330111) in combination with UAS-(G4C2)x36 in neurons using the Elav-gal4 promoter. The wild-type (w1118) line was used as control. (C) Female flies (♀). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 9th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. (D) Male flies (♂). Data are mean ± SD. 2-way ANOVA followed by Tukey's multiple comparisons, significance is reported for the 7th day ****P<0.0001 (G4C2)x36 vs Pka-RNAi (1);(G4C2)x36. Experiments were performed twice with different RNA interfering sequence, N=15 adult flies were used for each genotype and sex (N tot=30).


A , B Three independent C9 iPSC - MN lines ( Becker S6 , M211R2 , C90202 ) were treated with GELD , SPL , FSK , H89 or DMSO vehicle control of poly - GA ( A ) 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 , not significant , P > 0 . 05 ; P = 0 . 1 DMSO vs FSK ) . Treatments were performed in four technical replicates .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 iPSC - MN lines were treated with 10 µM H89 of poly - GA ( C ) 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 poly - GP * P = 0 . 018 ( linear mixed effects model ) . Treatments were performed in four technical replicates .

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

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 iPSC - MN lines ( unpaired t - , 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 O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 normalised to RPL13A , after 14 - day 10 µM H89 treatment vs vehicle treatment ( N = 1 C9 , four technical replicates ) . No significant changes were observed between H89 vehicle for any of isoforms ( 2 - way ANOVA with Šídák ' s multiple comparisons , 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 O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O 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 indicated drug either continuously for 48h , or initially for 2h in presence or absence of FA - glucosamine ( competition ) followed by 46h in absence of drug ( 2 + 46h ) , as described in Fig 1 . mRNA of profibrotic markers , Arg1 ( A ) , CD206 ( B ) CD163 ( C ) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 indicated drug either continuously for 48h , or initially for 2h in presence or absence of FA - glucosamine ( competition ) followed by 46h in absence of drug ( 2 + 46h ) , as described in Fig 1 . of secreted profibrotic CCL18 ( D ) antifibrotic cytokines , CXCL10 ( E ) 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 absence or presence of 200 - fold excess FA - glucosamine to block folate receptors . After 2h , mice were euthanized , resected imaged for ( n = 5 ) .

O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O 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 present or absence of 200 - fold excess FA - glucosamine were collagenase digested stained with 7AAD plus antibodies to CD11b F4 / 80 prior to FACS ( n = 3 ) . Representative plots showing gating strategy yielding live macrophages ( 7AAD - CD11b + F4 / 80 + ) OTL38 positive macrophages are shown .

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

(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 O O O O B-CONTROLLED_VAR 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 , 1 or 4h later sacrificed to collect both lungs bronchioalveolar lavage fluid ( BALF ) . ( A ) Lungs were digested macrophages were isolated by flow cytometry prior to for expression of indicated mRNAs by qPCR ( n = 3 ) .

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

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 , 1 or 4h later sacrificed to collect both lungs bronchioalveolar lavage fluid ( BALF ) . ( B ) BALF were pelleted similarly analyzed for indicated mRNAs ( n = 3 ) .

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

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 , 1 or 4h later sacrificed to collect both lungs bronchioalveolar lavage fluid ( BALF ) . ( C ) BALF supernatant was also collected analyzed by ELISA for IL - 6 , IFNα TNFα ( n = 3 ) .

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

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 of treatment groups ( Healthy , n = 5 ; others , n = 10 ) .

O O 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 centrifuged to isolate pellets . ( primarily macrophages ) were analyzed by qPCR for Arg1 ( C ) , MMP9 ( D ) , TIMP3 ( E )

O O O O O O O O O O O O O O O O O O O O O O O O O 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 Arg1 (C), MMP9 (D), TIMP3 (E)


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

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 ) .

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


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

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

(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 analyzed for hydroxyproline content as a measure of collagen content ( Healthy , n = 5 ; others , n = 10 ) ,

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

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 ) .

(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 then sacrificed on day 21 for . ( A ) of change versus time ( n = 10 ) .

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

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

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 ) .

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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 ) .

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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 ) .

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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. (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 ) .

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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. (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 ) .

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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 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 .

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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 p5 ) on VeroE6 , mCherry reporter gene in ISA mCherry D614 supernatant medium was RT - PCR amplified 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 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 ) .

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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 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 .

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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 , 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 ) .

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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 ) - 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 .

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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 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 ± .

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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 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 .

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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 - 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 .

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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 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 ) .

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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 ( by 18 % in adults ) , significantly lower score in nesting in overnight burrowing activity ( filled circles : 30 - week - old mice , 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 clasping reflex . c During 12 h phase on Lafayette running wheel running 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 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 .

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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 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 ) .

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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 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 ) ] .

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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 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 + / + .

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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 . rescue effect could still be observed in four out of eight , in two out of eight , mice 27 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 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 ) .

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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 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 .

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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 ( 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 ) .

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