( E ) Aβ was extracted by urea buffer from replicate slices of experiment shown in ( A ) total Aβ was identified by western blotting .

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(E) Aβ was extracted by urea buffer from replicate slices of the experiment shown in (A) and total Aβ was identified by western blotting.


( A ) Cryosections from unfixed brain of 6 - months old APP / PS1mice were pre - incubated with increasing concentrations of mAb11 ( 0 . 001 , 0 . 01 , 0 . 1 , 1 , 5 µg / ml ) . BMDM from wt or Trem2 ko mice were added for 24 h . Sections were stained with methoxy - X04 . Scale bar , 500 µm . ( B ) Methoxy - X04 signals were quantified from entire sagittal section . ( n = 5 , + / - ; 2 way ANOVA , interaction P = 0 . 0082 , genotype P < 0 . 0001 , treatment P < 0 . 0001 . Fisher ' s LSD post hoc comparisons ; * show statistics between wt ko under same experimental condition . # in black show wt compares to no antibody stimulation ; # in grey show ko compares to no antibody stimulation ; wt vs . ko for following conditions : no antibody P = 0 . 0053 , mAb11 0 . 001 µg / ml P = 0 . 0003 , mAb11 0 . 01 µg / ml P < 0 . 0001 , mAb11 0 . 1 µg / ml P = 0 . 0001 , mAb11 1 µg / ml P = 0 . 0007 , mAb11 5 µg / ml P = 0 . 0011 ; Following conditions compare to wt / no antibody : wt / mAb11 0 . 01 µg / ml P = 0 . 0166 , wt / mAb11 0 . 1 µg / ml P = 0 . 0002 , wt / mAb11 1 µg / ml P < 0 . 0001 , wt / mAb11 5 µg / ml P < 0 . 0001 ; Following conditions compare to ko / no antibody : ko / mAb11 0 . 1 µg / ml P = 0 . 0099 , ko / mAb11 1 µg / ml P < 0 . 0001 , ko / mAb11 5 µg / ml P < 0 . 0001 .

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(A) Cryosections from unfixed brain of 6-months old APP/PS1mice were pre-incubated with increasing concentrations of mAb11 (0.001, 0.01, 0.1, 1, 5 µg/ml). BMDM from wt or Trem2 ko mice were added for 24 h. Sections were stained with methoxy-X04. Scale bar, 500 µm.(B) Methoxy-X04 signals were quantified from the entire sagittal section. (n = 5, +/- SEM; 2 way ANOVA, interaction P=0.0082, genotype P<0.0001, treatment P<0.0001. Fisher's LSD post hoc comparisons; * show statistics between wt and ko under the same experimental condition. # in black show wt compares to no antibody stimulation; # in grey show ko compares to no antibody stimulation; wt vs. ko for the following conditions: no antibody P=0.0053, mAb11 0.001 µg/ml P=0.0003, mAb11 0.01 µg/ml P<0.0001, mAb11 0.1 µg/ml P=0.0001, mAb11 1 µg/ml P=0.0007, mAb11 5 µg/ml P=0.0011; Following conditions compare to wt / no antibody: wt / mAb11 0.01 µg/ml P=0.0166, wt / mAb11 0.1 µg/ml P=0.0002, wt / mAb11 1 µg/ml P<0.0001, wt / mAb11 5 µg/ml P<0.0001; Following conditions compare to ko / no antibody: ko / mAb11 0.1 µg/ml P=0.0099, ko / mAb11 1 µg/ml P<0.0001, ko / mAb11 5 µg/ml P<0.0001.


Dynamic patterns according to density in 8 × GTIIC - EmGFP 293T .

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Dynamic fluorescence patterns according to cell density in 8×GTIIC-EmGFP 293T cells.


EmGFP in LATS2 knockdown , or forced expression of a constitutively active form of YAP ( YAP - S127A ) . Scale bars indicate 100 μm .

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EmGFP intensity in LATS2 knockdown, or forced expression of a constitutively active form of YAP (YAP-S127A). Scale bars indicate 100 μm.


Scatter plots of mean frequency of each shRNA per gene . Average of v HOXA4 is indicated as a red dot .

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Scatter plots of mean frequency of each shRNA per gene. Average of v HOXA4 is indicated as a red dot.


Augmented 8 × GTIIC - luciferase reporter activity by loss of HOXA4 . ShSAV1 was used as an experimental control . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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Augmented 8×GTIIC-luciferase reporter activity by loss of HOXA4. ShSAV1 was used as an experimental control. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Quantitative real - time PCR of ANKRD1 expression in HEK 293T transfected with

Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with shHOXA4


Quantitative real - time PCR of ANKRD1 expression in HEK 293T transfected with HOXA4 expression

Quantitative real-time PCR analysis of ANKRD1 expression in HEK 293T cells transfected with HOXA4 expression vector


Quantitative real - time PCR of CTGF , CYR61 , BIRC5 in HEK 293T with knockdown of endogenous HOXA4 . ShLATS1 / 2 shYAP were used as an experimental control . * P < 0 . 05 , * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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Quantitative real-time PCR analysis of CTGF, CYR61, and BIRC5 in HEK 293T cells with knockdown of endogenous HOXA4. ShLATS1/2 and shYAP were used as an experimental control. *P<0.05, **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


8 × GTIIC - luciferase reporter assays in HEK 293T transfected HOXA4 / or shLATS1 / 2 . * * P < 0 . 01 vs shCtrl , by two - way ANOVA with Sidak ' s correction .

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8×GTIIC-luciferase reporter assays in HEK 293T cells transfected HOXA4 and/or shLATS1/2. **P<0.01 vs shCtrl, by two-way ANOVA with Sidak's correction.


Quantitative real - time PCR of HOXA4 , YAP , CTGF , CYR61 in HEK 293T with overexpression of HOXA4 / or constitutive active form of YAP ( YAP - 5SA ) . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with LacZ , by ANOVA with Sidak ' s correction .

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Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with overexpression of HOXA4 and/or constitutive active form of YAP (YAP -5SA). *P<0.05, **P<0.01 vs YAP-5SA with LacZ, by ANOVA with Sidak's correction.


Quantitative real - time PCR of HOXA4 , YAP , CTGF , CYR61 in HEK 293T with knockdown of endogenous HOXA4 / or overexpression of YAP - 5SA . * P < 0 . 05 , * * P < 0 . 01 vs YAP - 5SA with shCtrl , by ANOVA with Sidak ' s correction .

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Quantitative real-time PCR analysis of HOXA4, YAP, CTGF, and CYR61 in HEK 293T cells with knockdown of endogenous HOXA4 and/or overexpression of YAP-5SA. *P<0.05, **P<0.01 vs YAP-5SA with shCtrl, by ANOVA with Sidak's correction.


Association of HOXA4 with TEAD1 indicated by co - IP experiments . HEK 293T were co - transfected with FLAG - HOXA4 Myc -

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Association of HOXA4 with TEAD1 indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-TEAD1


Association of HOXA4 but not YAP indicated by co - IP experiments . HEK 293T were co - transfected with FLAG - HOXA4 Myc - YAP .

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Association of HOXA4 but not YAP indicated by co-IP experiments. HEK 293T cells were co-transfected with FLAG-HOXA4 and Myc-YAP.


Interaction between endogenous TEAD1 HOXA4 by co - IP experiments in HEK 293T . interaction between endogenous TEAD1 YAP was used as a positive control .

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Interaction between endogenous TEAD1 and HOXA4 by co-IP experiments in HEK 293T cells. The interaction between endogenous TEAD1 and YAP was used as a positive control.


Immunoprecipitation using FLAG antibody in lysates from HEK 293T transfected with plasmids expressing HOXA4 truncation mutants TEAD1 .

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Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing HOXA4 truncation mutants and TEAD1.


Immunoprecipitation using FLAG antibody in lysates from HEK 293T transfected with plasmids expressing TEAD1 truncation mutants HOXA4 . Arrowheads indicate IgG heavy chain . Arrows indicate TEAD1 ( a . a . 1 - 426 ) .

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Immunoprecipitation using FLAG antibody in lysates from HEK 293T cells transfected with plasmids expressing TEAD1 truncation mutants and HOXA4. Arrowheads indicate IgG heavy chain. Arrows indicate TEAD1 (a.a. 1-426).


Requirement of TEAD1 ( a . a . 30 - 135 ) for its interaction with HOXA4 via its homeodomain ( a . a . 216 - 274 ) .

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Requirement of TEAD1 (a.a. 30-135) for its interaction with HOXA4 via its homeodomain (a.a. 216-274).


Failure of TEAD1 ( a . a . 30 - 102 ) , TEA domain alone , to interact with HOXA4 .

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Failure of TEAD1 (a.a. 30-102), TEA domain alone, to interact with HOXA4.


ChIP assays of CTGF CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T transfected with plasmids expressing HOXA4 . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - . Data are expressed as means ± of three independent experiments .

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ChIP assays of CTGF and CYR61 promoters using TEAD4 or YAP antibodies in HEK 293T cells transfected with plasmids expressing HOXA4. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test. Data are expressed as the means ± SEM of three independent experiments.


Competitive co - IP densitometry showing significantly reduced TEAD - YAP interaction by overexpression of HOXA4 in a dose - dependent manner in HEK 293T . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction . Data are presented as mean ± of three independent experiments .

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Competitive co-IP assay and densitometry showing significantly reduced TEAD-YAP interaction by overexpression of HOXA4 in a dose-dependent manner in HEK 293T cells. *P<0.05, **P<0.01, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Expression of Hoxa4 in various mouse tissues using qRT - PCR . Expression of glyceraldehyde - 3 - phosphate dehydrogenase ( Gapdh ) mRNA was used as an internal control . WAT ; adipose tissue , BAT ; brown adipose tissue , Skm ; skeletal muscle .

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Expression of Hoxa4 in various mouse tissues using qRT-PCR. Expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA was used as an internal control. WAT; white adipose tissue, BAT; brown adipose tissue, Skm; skeletal muscle.


Quantitative real - time PCR of differentiated VSMC marker genes in human VSMCs with PDGF - BB stimulation for 24 hours . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - .

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Quantitative real-time PCR analysis of differentiated VSMC marker genes in primary human VSMCs with PDGF-BB stimulation for 24 hours. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Quantitative real - time PCR of smooth muscle specific genes in human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 YAP . infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - .

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Quantitative real-time PCR analysis of smooth muscle specific genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. Cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of YAP / TAZ Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 .

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Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of YAP/TAZ Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.


Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 .

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Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4 Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding knockdown of HOXA4.


Quantitative real - time PCR of YAP / TEAD target genes in human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 YAP . infected with lentiviral vectors encoding LacZ or shCtrl were used as controls . * P < 0 . 05 , * * P < 0 . 01 , by unpaired two - Student ' s t - .

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Quantitative real-time PCR analysis of YAP/TEAD target genes in primary human VSMCs transduced with lentiviral vectors encoding overexpression or knockdown of HOXA4 and YAP. The cells infected with lentiviral vectors encoding LacZ or shCtrl were used as controls. *P<0.05, **P<0.01, by unpaired two-tailed Student's t-test.


Proliferation in VSMCs transduced with indicated genes . - transfected human VSMCs were plated at equal density in growth medium , then were collected counted at each time point as indicated . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction .

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Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.


BrdU incorporation assays in VSMCs transduced with indicated genes . of BrdU positive in each group is shown . Nine to twelve were randomly acquired analyzed . * * P < 0 . 01 vs LacZ , by ANOVA with Sidak ' s correction .

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BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs LacZ, by ANOVA with Sidak's correction.


Proliferation in VSMCs transduced with indicated genes . - transfected human VSMCs were plated at equal density in growth medium , then were collected counted at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction .

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Proliferation assay in VSMCs transduced with the indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cells were collected and counted at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction.


BrdU incorporation assays in VSMCs transduced with indicated genes . of BrdU positive in each group is shown . Nine to twelve were randomly acquired analyzed . * * P < 0 . 01 vs shCtrl , by ANOVA with Sidak ' s correction .

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BrdU incorporation assays in VSMCs transduced with the indicated genes. The percentage of BrdU positive cells in each group is shown. Nine to twelve images were randomly acquired and analyzed. **P<0.01 vs shCtrl, by ANOVA with Sidak's correction.


Representative western blotting of total lysates of human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 TEAD1 / 3 / 4 . of alpha smooth muscle actin calponin is expressed as means ± of three independent experiments . * P < 0 . 05 , * * P < 0 . 01 , by ANOVA with Sidak ' s correction .

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Representative western blotting analysis of total protein lysates of primary human VSMCs transduced with lentiviral vectors encoding shRNAs of HOXA4 and TEAD1/3/4. Quantification of alpha smooth muscle actin and calponin protein levels is expressed as the means ± SEM of three independent experiments. *P<0.05, **P<0.01, by ANOVA with Sidak's correction.


Proliferation in VSMCs transduced with indicated genes . - transfected human VSMCs were plated at equal density in growth medium , then viability was analyzed by Counting Kit - 8 at indicated days at each time point as indicated . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± of three independent experiments .

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Proliferation assay in VSMCs transduced with indicated genes. Virus-transfected human VSMCs were plated at equal density in growth medium, and then cell viability was analyzed by Cell Counting Kit-8 at indicated days at each time point as indicated. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


Representative immunoblotting of interaction between endogenous TEAD1 HOXA4 or YAP using co - IP experiments in human VSMCs with knockdown of HOXA4 . * P < 0 . 05 vs shCtrl , by ANOVA with Sidak ' s correction . Data are presented as mean ± of three independent experiments .

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Representative immunoblotting images and quantification of the interaction between endogenous TEAD1 and HOXA4 or YAP using co-IP experiments in human VSMCs with knockdown of HOXA4. *P<0.05 vs shCtrl, by ANOVA with Sidak's correction. Data are presented as mean ± SEM of three independent experiments.


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 .

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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 ) .

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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 .

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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 ) .

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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 ) .

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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 .

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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 .

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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 ) .

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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 ± .

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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 .

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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 .

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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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-TISSUE 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 .

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

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

(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 B-GENEPROD O B-TISSUE I-TISSUE I-TISSUE O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O

(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 B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-SUBCELLULAR O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

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


C , D Three independent C9 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 B-CELL_TYPE O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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


F Relative fold changes of C9orf72 variant 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 B-GENEPROD O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 ) .

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

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


Human monocytic ( THP - 1 ) 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 ) .

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

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

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


M2 - induced human monocyte - derived macrophages were treated with 100 nM of 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 ) .

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

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


( A ) Healthy mice or BLM - induced mice were tail vein injected on day 10 with 10 nmol OTL38 in 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 B-ORGANISM O B-SMALL_MOLECULE O O B-ORGANISM O B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O B-ORGANISM O O O O B-EXP_ASSAY 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 B-TISSUE O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O B-CELL_TYPE O O O

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


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

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

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


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

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

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


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , 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 B-ORGANISM O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O B-TISSUE O O B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O

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


Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , 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 B-ORGANISM O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE I-TISSUE I-TISSUE I-TISSUE O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O O

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

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


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

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O

(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 )

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

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


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

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

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


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

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

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


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

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

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

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


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

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

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