Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . RNA was collected for measuring gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates . NT , no treatment group ; HPC , human primary culture . ( C ) ZIKV infection induced a time - dependent increase in IFNβ mRNA expression in Sw . 71 and HPC . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA .

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

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. RNA was collected for measuring gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates. NT, no treatment group; HPC, human primary culture. (C) ZIKV infection induced a time-dependent increase in IFNβ mRNA expression in Sw.71 and HPC. *p < 0.05, **p < 0.01 by One-way ANOVA.


Human first - trimester trophoblast cell line Sw . 71 ( left panel ) and human primary culture ( right panel ) trophoblast cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media over time . ( D ) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay . Supernatants were collected from ZIKV - infected and control trophoblast cultures at different times , and IFNβ protein secretion was quantified by ELLA assay . Note the increase of secreted IFNβ in the ZIKV - infected supernatant in a time - dependent manner . * * * * p < 0 . 0001 by Student ' s t - test against individual time point NT control .

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

Human first-trimester trophoblast cell line Sw.71 (left panel) and human primary culture (right panel) trophoblast cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media over time. (D) Expression levels of secreted IFNβ detected in trophoblast supernatants by ELLA assay. Supernatants were collected from ZIKV-infected and control trophoblast cultures at different times, and IFNβ protein secretion was quantified by ELLA assay. Note the increase of secreted IFNβ in the ZIKV-infected supernatant in a time-dependent manner. ****p < 0.0001 by Student's t-test against individual time point NT control.


Sw . 71 ( left panel ) and HPC cells ( right panel ) were treated with IFNβ to detect ISG20 mRNA expression . Data represent as mean ± SEM ; n = 3 biological replicates . NT , no treatment group ; HPC , human primary culture . ( A ) Sw . 71 and HPC cells were treated with different doses of IFNβ ( 3 , 30 , 300 ng / ml ) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT - PCR . Note the increase of ISG20 mRNA expression in a dose - dependent manner . * p < 0 . 05 , * * p < 0 . 01 by One - way ANOVA . ( B ) Sw . 71 and HPC cells were treated with 300ng / ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT - PCR . Note the increase of ISG20 mRNA expression in a time - dependent manner . * p < 0 . 05 by One - way ANOVA . ( C )

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

Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 mRNA expression. Data represent as mean ± SEM; n=3 biological replicates. NT, no treatment group; HPC, human primary culture. (A) Sw.71 and HPC cells were treated with different doses of IFNβ (3, 30, 300 ng/ml) for 8 h and RNA were collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a dose-dependent manner. *p < 0.05, **p < 0.01 by One-way ANOVA. (B) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and RNA was collected for determining ISG20 mRNA expressions by qRT-PCR. Note the increase of ISG20 mRNA expression in a time-dependent manner. *p < 0.05 by One-way ANOVA. (C)


Sw . 71 ( left panel ) and HPC cells ( right panel ) were treated with IFNβ to detect ISG20 protein expression . ( D ) Sw . 71 and HPC cells were treated with 300ng / ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot . Note that there was no ISG20 protein expression in the no treatment group in both Sw . 71 and HPC , only after IFNβ treatment , ISG20 protein expression exhibited a time - dependent manner . β - actin served as a loading control .

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

Sw.71 (left panel) and HPC cells (right panel) were treated with IFNβ to detect ISG20 protein expression. (D) Sw.71 and HPC cells were treated with 300ng/ml IFNβ over time and protein was collected for determining ISG20 protein expressions by western blot. Note that there was no ISG20 protein expression in the no treatment group in both Sw.71 and HPC, only after IFNβ treatment, ISG20 protein expression exhibited a time-dependent manner. β-actin served as a loading control.


( B ) No ISG20 protein expression in ISG20 - / - Sw . 71 in ZIKV infection . Sw . 71 and ISG20 - / - Sw . 71 cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media for 48 h , and proteins were collected for western blot analysis . β - actin served as a loading control .

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(B) No ISG20 protein expression in ISG20-/- Sw.71 in ZIKV infection. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and proteins were collected for western blot analysis. β-actin served as a loading control.


( C ) Higher ZIKA titer was shown in ISG20 - / - Sw . 71 cells . Sw . 71 and ISG20 - / - Sw . 71 cells were infected with ZIKV ( MOI = 2 ) for 1 h and refreshed with regular media for 48 h , and RNA was collected for determining the viral titer and gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O 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) Higher ZIKA titer was shown in ISG20-/- Sw.71 cells. Sw.71 and ISG20-/- Sw.71 cells were infected with ZIKV (MOI=2) for 1 h and refreshed with regular media for 48 h, and RNA was collected for determining the viral titer and gene expression by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.


( D ) More viral shedding in ZIKV - infected ISG20 - / - Sw . 71 cell culture supernatant . Supernatant from ZIKV - infected Sw . 71 and ISG20 - / - Sw . 71 cells were collected and plaque assay was performed using Vero cells . A representative plaque assay picture is presented . Note that more plaques were formed in Vero cells by incubating with ZIKV - infected ISG20 - / - Sw . 71 cell culture supernatant . Scale bar = 130μm . ( E ) Plaques were counted and plaque forming - unit ( pfu ) ratio was calculated for the comparison of the viral titers . Data represent as mean ± SEM ; n = 3 biological replicates . * p < 0 . 05 by Student ' s t - test .

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(D) More viral shedding in ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Supernatant from ZIKV-infected Sw.71 and ISG20-/- Sw.71 cells were collected and plaque assay was performed using Vero cells. A representative plaque assay picture is presented. Note that more plaques were formed in Vero cells by incubating with ZIKV-infected ISG20-/- Sw.71 cell culture supernatant. Scale bar=130μm. (E) Plaques were counted and plaque forming-unit (pfu) ratio was calculated for the comparison of the viral titers. Data represent as mean ± SEM; n=3 biological replicates. *p < 0.05 by Student's t-test.


( B ) ISGs mRNA expression stimulated by IFNβ in Sw . 71 and ISG20 - / - Sw . 71 trophoblast cells . Sw . 71 and ISG20 - / - Sw . 71 cells were treated with 30 ng / ml IFNβ for 24 h , and RNA was collected for determining the ISGs gene expression by qRT - PCR . Data represent as mean ± SEM ; n = 3 biological replicates ; * p < 0 . 05 , * * p < 0 . 01 by Student ' s t - test against control .

(B) ISGs mRNA expression stimulated by IFNβ in Sw.71 and ISG20-/- Sw.71 trophoblast cells. Sw.71 and ISG20-/- Sw.71 cells were treated with 30 ng/ml IFNβ for 24 h, and RNA was collected for determining the ISGs gene expression by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; *p < 0.05, **p < 0.01 by Student's t-test against control.


( D ) IFNβ pre - treatment significantly prevented trophoblast cells from ZIKV infection , however , this protection was evidently attenuated due to lack of ISG20 . Sw . 71 and ISG20 - / - Sw . 71 cells were pre - treated with or without 30 ng / ml IFNβ for 24 h , followed by ZIKV infection ( MOI = 2 ) for 1 h and refreshed with regular media for 24 h , and RNA was collected to determine the viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 4 biological replicates ; * p < 0 . 05 by Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O 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) IFNβ pre-treatment significantly prevented trophoblast cells from ZIKV infection, however, this protection was evidently attenuated due to lack of ISG20. Sw.71 and ISG20-/- Sw.71 cells were pre-treated with or without 30 ng/ml IFNβ for 24 h, followed by ZIKV infection (MOI=2) for 1 h and refreshed with regular media for 24 h, and RNA was collected to determine the viral titers by qRT-PCR. Data represent as mean ± SEM; n=4 biological replicates; *p < 0.05 by Student's t-test.


( D ) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells . Supernatants from negative and positive clones were collected and added to ISG20 - / - Sw . 71 trophoblast cells together with ZIKA virus ( MOI = 2 ) for 1 h , followed by refreshing with new growth media for 48 h . RNA was then collected to determine viral titers by qRT - PCR . Data represent as mean ± SEM ; n = 3 biological replicates ; * * p < 0 . 01 by Student ' s t - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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) ISG20 secretion in the positive clone supernatant significantly decreased ZIKV infection in trophoblast cells. Supernatants from negative and positive clones were collected and added to ISG20-/- Sw.71 trophoblast cells together with ZIKA virus (MOI=2) for 1 h, followed by refreshing with new growth media for 48 h. RNA was then collected to determine viral titers by qRT-PCR. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01 by Student's t-test.


( E ) Recombinant ISG20 - Fc degrades ZIKA viral RNA and HSV2 viral DNA . 50ng purified viral RNA ( ZIKV ) or DNA ( HSV - 2 ) were incubated with increasing concentrations of rISG20 - Fc ( 5 , 50 , 500 ng ) in the presence of RNase inhibitor for 90min at 37 ° C followed by quantification of viral titers by qRT - PCR , and agarose gel was used to evaluate the RNA degradation by electrophoresis . The representative picture of agarose gel is presented . rISG20 - Fc was able to degrade both ZIKV RNA and HSV - 2 DNA in a dose dependent manner ; however , rISG20 - Fc was more efficient in degrading viral RNA than DNA . Data represent as mean ± SEM ; n = 3 biological replicates ; * * p < 0 . 01 . * * * p < 0 . 001 and * * * * p < 0 . 0001 by One - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Recombinant ISG20-Fc degrades ZIKA viral RNA and HSV2 viral DNA. 50ng purified viral RNA (ZIKV) or DNA (HSV-2) were incubated with increasing concentrations of rISG20-Fc (5, 50, 500 ng) in the presence of RNase inhibitor for 90min at 37°C followed by quantification of viral titers by qRT-PCR, and agarose gel was used to evaluate the RNA degradation by electrophoresis. The representative picture of agarose gel is presented. rISG20-Fc was able to degrade both ZIKV RNA and HSV-2 DNA in a dose dependent manner; however, rISG20-Fc was more efficient in degrading viral RNA than DNA. Data represent as mean ± SEM; n=3 biological replicates; **p < 0.01. ***p < 0.001 and ****p < 0.0001 by One-way ANOVA.


( B ) Macroscopic evaluation of the pregnant uterus and fetus . Compare to the fetal death and reabsorptions in ZIKV - infected mice , rISG20 - Fc treatment rescued this phenotype by decreasing pregnancy loss ( resorption ) .

O O O O O O O O O O O O O O O O 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) Macroscopic evaluation of the pregnant uterus and fetus. Compare to the fetal death and reabsorptions in ZIKV-infected mice, rISG20-Fc treatment rescued this phenotype by decreasing pregnancy loss (resorption).


( A ) rISG20 - Fc treatment alleviates maternal viral burden . Note that rISG20 - Fc treatment significantly decreased the maternal serum viral titers . Data represent as mean ± SEM ; n = 3 mice for control PBS group , and n = 4 mice for treatment groups ; * p < 0 . 05 by Student ' s t - test . ( B ) rISG20 - Fc treatment reduces ZIKV titers in the fetal brain . Although there was no difference of ZIKV titer in the placenta , at the fetal side , there was a significant decrease of viral titer in the fetal brain , suggesting rISG20 - Fc can block viral transmission from placenta to fetus . Data represent as mean ± SEM ; n = 4 for each group , and 3 - 4 placentas / fetal brains from every mouse were analyzed ; * p < 0 . 05 by Student ' s t - test . (

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) rISG20-Fc treatment alleviates maternal viral burden. Note that rISG20-Fc treatment significantly decreased the maternal serum viral titers. Data represent as mean ± SEM; n=3 mice for control PBS group, and n=4 mice for treatment groups; *p < 0.05 by Student's t-test. (B) rISG20-Fc treatment reduces ZIKV titers in the fetal brain. Although there was no difference of ZIKV titer in the placenta, at the fetal side, there was a significant decrease of viral titer in the fetal brain, suggesting rISG20-Fc can block viral transmission from placenta to fetus. Data represent as mean ± SEM; n=4 for each group, and 3-4 placentas/fetal brains from every mouse were analyzed; *p < 0.05 by Student's t-test. (


( C ) rISG20 - Fc treatment decreases Eotaxin significantly in maternal circulation . Data represent as mean ± SEM ; n = 2 for PBS control group , and n = 3 for every treatment group ; * p < 0 . 05 by One - way ANOVA ; difference not significant for IL - 9 and IFNγ by One - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) rISG20-Fc treatment decreases Eotaxin significantly in maternal circulation. Data represent as mean ± SEM; n=2 for PBS control group, and n=3 for every treatment group; *p < 0.05 by One-way ANOVA; difference not significant for IL-9 and IFNγ by One-way ANOVA.


A . Rabies virus infection kinetics in the central nervous system of infected mice . Tha - RABV load ( copies / µg of RNA ) detected in the thoracolumbar spinal cord , in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time - points post - infection ( n = 4 per time - point ) . nd : not detected . Horizontal lines indicate the median .

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A. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/µg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum and in the cerebral cortex of infected mice according to different time-points post-infection (n=4 per time-point). nd: not detected. Horizontal lines indicate the median.


B . Follow - up of the clinical signs of mice under different treatments . Heatmaps were established based on a progressive 0 - 7 clinical score scale ( 0 : no apparent changes | 1 : ruffled fur | 2 : slow movement , hind limb ataxia | 3 : apathy | 4 : monoplegia | 5 : hind limb paralysis , tremors | 6 : paralysis , conjunctivitis / keratitis , urine staining of the haircoat of the perineum | 7 : death ) . Each line represents one animal throughout time .

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B. Follow-up of the clinical signs of mice under different treatments. Heatmaps were established based on a progressive 0-7 clinical score scale (0: no apparent changes | 1: ruffled fur | 2: slow movement, hind limb ataxia | 3: apathy | 4: monoplegia | 5: hind limb paralysis, tremors | 6: paralysis, conjunctivitis/ keratitis, urine staining of the haircoat of the perineum | 7: death). Each line represents one animal throughout time.


C . Body weight progression of mice under different treatments . Mice were weighted on a daily basis during the treatment administration , and then twice a week up to 100 days post - infection . All the mice were equipped with iPrecio pumps weighting ca . 3 . 3 g . Each line represents one animal throughout time .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Body weight progression of mice under different treatments. Mice were weighted on a daily basis during the treatment administration, and then twice a week up to 100 days post-infection. All the mice were equipped with iPrecio pumps weighting ca. 3.3 g. Each line represents one animal throughout time.


D - E . Mouse behavioral testing . The tested animals were non - infected ( n = 3 mice with iPrecio pump + n = 4 age - related mice without iPrecio pump ) , infected non - treated ( n = 5 ) , infected and treated at 7 dpi ( n = 5 , mice # 9 to # 13 ) , and infected and treated at 8 dpi ( n = 5 , mice # 24 to # 28 ) . ( D ) Open field test performed at 7 dpi ( prodromal phase ) and after the remission of clinical signs for the survivors ( 80 dpi ) . ( E ) Rotarod motor performance at 7 dpi ( prodromal phase ) , 8 dpi ( symptomatic phase ) and after the remission of clinical signs for the survivors ( 20 , 30 and 60 dpi ) . Horizontal lines indicate the median . Kruskal - Wallis followed by the Dunn ' multiple comparisons test ( 7 and 8 days post - infection ) and Mann - Whitney test ( 20 , 30 and 60 days post - infection ) . The rotarod performance presented a negative correlation ( Spearman r = - 0 . 768 ) with the clinical score at 8 dpi . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = NI : non - infected ( n = 5 ) ; NT : infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Mouse behavioral testing. The tested animals were non-infected (n=3 mice with iPrecio pump + n=4 age-related mice without iPrecio pump), infected non-treated (n=5), infected and treated at 7 dpi (n=5, mice #9 to #13), and infected and treated at 8 dpi (n=5, mice #24 to #28). (D) Open field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase) and after the remission of clinical signs for the survivors (20, 30 and 60 dpi). Horizontal lines indicate the median. Kruskal-Wallis followed by the Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann-Whitney test (20, 30 and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman r = -0.768) with the clinical score at 8 dpi. *P<0.05, **P<0.01, ***P<0.001. Treatments description= NI: non-infected (n=5); NT: infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).


A . Cumulative Kaplan - Meier survival curves of mice treated at different time - points post - infection . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * P < 0 . 01 , * * * P < 0 . 001 . Treatments description = infected , non - treated ( n = 5 ) ; infected , treated at 6 dpi ( 2 + 2 mg / kg ) ( n = 4 ) ; infected , treated at 7 dpi ( 2 + 2 mg / kg ) ( n = 9 ) ; infected , treated at 8 dpi ( 2 + 2 mg / kg ) ( n = 15 ) ; infected , treated at 8 dpi ( 10 + 10 mg / kg ) ( n = 4 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cumulative Kaplan-Meier survival curves of mice treated at different time-points post-infection. Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. **P<0.01, ***P<0.001. Treatments description= infected, non-treated (n=5); infected, treated at 6 dpi (2+2 mg/kg) (n=4); infected, treated at 7 dpi (2+2 mg/kg) (n=9); infected, treated at 8 dpi (2+2 mg/kg) (n=15); infected, treated at 8 dpi (10+10 mg/kg) (n=4).


B . Cumulative Kaplan - Meier survival curves of all the treated animals that presented with clinical signs ( n = 31 , all treatments combined ) . Log - rank ( Mantel - Cox ) test to compare treated groups with the infected , non - treated group . * * * * P < 0 . 0001 .

B. Cumulative Kaplan-Meier survival curves of all the treated animals that presented with clinical signs (n=31, all treatments combined). Log-rank (Mantel-Cox) test to compare treated groups with the infected, non-treated group. ****P<0.0001.


C . Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups .

C. Virus neutralizing antibodies detected in the serum of surviving mice from different experimental groups.


D . Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi ( LOD : limit of detection = 0 . 02 µg / mL ) .

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D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 µg/mL).


E . Viral load in the brain of mice from different experimental groups . The samples were either collected at the time of death , or at 100 dpi for the survivors .

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E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors.


Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : ( F ) IFN - β1 , ( G ) ISG15 , ( H ) Mx1 , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene .

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Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.


Cytokines and innate immune mediators ' profile in the brain of mice from different experimental groups : , ( I ) IFN - γ , ( J ) CXCL10 , ( K ) CCL5 , ( L ) IL - 6 , ( M ) TNF - α and ( N ) IL - 1β , detected in one brain hemisphere of mice from different experimental groups . The expression of the genes of interest was normalized to the GAPDH housekeeping gene .

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Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: , (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene.


A - R . Histopathological analysis of the brain in Tha - RABV - infected non - treated mice ( at 11 dpi ) , versus surviving mice ( at 100 dpi ) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail . The detection of Tha - RABV , using immunohistochemistry ( IHC ) on brain sections , revealed a heterogeneous invasion between the non - treated mice , ranging from extensive ( A ) to multifocal ( B ) . Higher magnification of extensive invasion in the cortex ( F ) , hippocampus ( G ) , brain stem ( H ) and cerebellum ( I ) . ( C , J , K , L , M ) In contrast , even if a non - specific IHC signal was sometimes observed , no virus was detected on the brain sections of treated mice at 100 days post - infection . Only rare signs of neuro - inflammation were observed , as perivascular cuffing ( D ) ( 2 / 7 mice ) and minimal meningitis ( E ) ( 3 / 7 mice ) . Data information : A - C and F - M : Tha - RABV IHC D - E : HE staining . F - I are high magnifications of A ; J - M are high magnifications of

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A-R. Histopathological analysis of the brain in Tha-RABV-infected non-treated mice (at 11 dpi), versus surviving mice (at 100 dpi) treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibodies cocktail. The detection of Tha-RABV, using immunohistochemistry (IHC) on brain sections, revealed a heterogeneous invasion between the non-treated mice, ranging from extensive (A) to multifocal (B). Higher magnification of extensive invasion in the cortex (F), hippocampus (G), brain stem (H) and cerebellum (I). (C,J,K,L,M) In contrast, even if a non-specific IHC signal was sometimes observed, no virus was detected on the brain sections of treated mice at 100 days post-infection. Only rare signs of neuro-inflammation were observed, as perivascular cuffing (D) (2/7 mice) and minimal meningitis (E) (3/7 mice). Data information: A-C and F-M: Tha-RABV IHC D-E: HE staining. F-I are high magnifications of A; J-M are high magnifications of C


( N , O ) Microglial cells were in general not reactive , but ( P ) small foci of reactive microglia ( hyperplasia with thicker cell processes ) could randomly be detected . As for control non - treated mice , reactive microglial cells ( Q ) ( 2 / 7 mice ) or resting microglia ( R ) were observed in treated mice . Representative images . Data information : N - R : Iba1 IHC to detect microglial cells O - P are high magnifications of N .

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(N,O) Microglial cells were in general not reactive, but (P) small foci of reactive microglia (hyperplasia with thicker cell processes) could randomly be detected. As for control non-treated mice, reactive microglial cells (Q) (2/7 mice) or resting microglia (R) were observed in treated mice. Representative images. Data information: N-R: Iba1 IHC to detect microglial cells O-P are high magnifications of N.


( B ) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . unparasitized hosts . Day - 4 of last instar larvae were used for this assay . Test larvae were parasitized by the wasp on day 0 of last instar .

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(B) Survival of parasitized and unparasitized host larvae 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. unparasitized hosts. Day-4 of last instar larvae were used for this assay. Test larvae were parasitized by the wasp on day 0 of last instar.


( C ) Survival of armyworm larvae injected with PBS , unparasitized , and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . * * P < 0 . 01 vs . PBS . Test larvae were exposed to the heat stress 4 h after injection of each sample .

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(C) Survival of armyworm larvae injected with PBS, unparasitized, and parasitized larval hemolymph 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. PBS. Test larvae were exposed to the heat stress 4 h after injection of each sample.


( D ) Time - dependent survival of armyworm larvae injected with 0 . 8 µmol / larva N - acetyl - L - tyrosine ( NAT ) : post NAT - injection 2 d after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . * * P < 0 . 01 vs . zero time .

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(D) Time-dependent survival of armyworm larvae injected with 0.8 µmol/larva N-acetyl-L-tyrosine (NAT): post NAT-injection 2 d after heat stress at 44oC for 50 min (data are means ± SEM; n=12). **P<0.01 vs. zero time.


( E ) Dose - dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 12 ) . Injection of more than 10 nmol NAT / larva caused a significant difference compared with zero ( log rank test using R version 3 . 2 . 2 ) .

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(E) Dose-dependent survival of armyworm larvae injected with NAT after heat stress at 44oC for 50 min (data are means ± SEM; n=12). Injection of more than 10 nmol NAT/larva caused a significant difference compared with zero (log rank test using R version 3.2.2).


( F ) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . PBS . Each chemical ( 0 . 5 µmol / larva ) was dissolved in PBS ; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine .

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(F) Survival of armyworm larvae injected with indicated chemical solution 1 day after heat stress at 44oC for 50 min (data are means ± SEM; n=10). **P<0.01 vs. PBS. Each chemical (0.5 µmol/larva) was dissolved in PBS; a minimal volume of dimethyl sulfoxide was used for poorly soluble chemicals such as tyrosine.


( A ) HPLC chromatogram and L - MS spectrogram of N - acetyltyrosine purified from pooled human serum ( # 12181201 , Cosmo Bio Co . , Japan ) . The peak fraction purified by HPLC was confirmed to be N - acetyltyrosine by LC - MS ( inserted spectrogram ) .

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(A) HPLC chromatogram and L-MS spectrogram of N-acetyltyrosine purified from pooled human serum (#12181201, Cosmo Bio Co., Japan). The peak fraction purified by HPLC was confirmed to be N-acetyltyrosine by LC-MS (inserted spectrogram).


( B ) NAT concentrations in the serum of mice ( Slc : ddY , 18 wks old , male ) exposed to heat ( 40oC ) and restraint stress ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time .

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(B) NAT concentrations in the serum of mice (Slc:ddY, 18 wks old, male) exposed to heat (40oC ) and restraint stress (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. zero time.


( C ) Effects of NAT on corticosterone concentrations in the serum of mice ( Slc : ddY , 9 - 10 wks old , male ) exposed to restraint stress for 60 min ( data are means ± SEM ; n = 6 ) . * * P < 0 . 01 vs . without stress . Test mice drank 1 . 5 mg / ml NAT solution for 24 h before exposure to stress .

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(C) Effects of NAT on corticosterone concentrations in the serum of mice (Slc:ddY, 9-10 wks old, male) exposed to restraint stress for 60 min (data are means ± SEM; n=6). ** P<0.01 vs. without stress. Test mice drank 1.5 mg/ml NAT solution for 24 h before exposure to stress.


( D ) Effect of NAT on stress - induced peroxidized lipid in the serum of mice ( Slc : ddY , 9 wks old , male ) forced to swim for 60 min ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . PBS .

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(D) Effect of NAT on stress-induced peroxidized lipid in the serum of mice (Slc:ddY, 9 wks old, male) forced to swim for 60 min (data are means ± SEM; n=10). *P<0.05 vs. PBS.


( A ) Survival of Drosophila melanogaster ( W1118 ) larvae fed with 2 µmol NAT / g - diet 2 d after heat stress at 38 . 5oC for 60 min ( data are means ± SEM ; n = 10 ) . * * P < 0 . 01 vs . zero time .

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(A) Survival of Drosophila melanogaster (W1118) larvae fed with 2 µmol NAT/g-diet 2 d after heat stress at 38.5oC for 60 min (data are means ± SEM; n=10). **P<0.01 vs. zero time.


( B ) Effects of NAT feeding on expression of antioxidant enzymes , catalase , SOD1 , and SOD2 ( data are means ± SEM ; n = 10 ) . * P < 0 . 05 vs . zero time .

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(B) Effects of NAT feeding on expression of antioxidant enzymes, catalase, SOD1, and SOD2 (data are means ± SEM; n=10). *P<0.05 vs. zero time.


( C ) Representative images of nuclei ( left ) and FoxO ( middle ) in the fat body of Drosophila larvae before and after NAT feeding for 12 h . White signals in the merged image ( right ) show the overlap of both signals . Scale bar : 50 µm . ( D ) Relative whiteness intensities before and after NAT feeding ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . zero time .

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(C) Representative images of nuclei (left) and FoxO (middle) in the fat body of Drosophila larvae before and after NAT feeding for 12 h. White signals in the merged image (right) show the overlap of both signals. Scale bar: 50 µm. (D) Relative whiteness intensities before and after NAT feeding (data are means ± SEM; n=15). **P<0.01 vs. zero time.


( E ) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae ( data are means ± SEM ; n = 8 ) . * P < 0 . 05 , * * P < 0 . 01 vs . zero time .

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(E) Effects of NAT feeding on FoxO expression in the fat body of Drosophila larvae (data are means ± SEM; n=8). *P<0.05, **P<0.01 vs. zero time.


( F ) Effects of N - acetyltransferase ( Dat ) knockdown in gut enteroendocrine cells ( TK - Gut - Gal4 > UAS - dsDat ) on survival of Drosophila larvae ( data are means ± SEM ; n = 15 ) . * * P < 0 . 01 vs . without NAT .

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(F) Effects of N-acetyltransferase (Dat) knockdown in gut enteroendocrine cells (TK-Gut-Gal4>UAS-dsDat) on survival of Drosophila larvae (data are means ± SEM; n=15). **P<0.01 vs. without NAT.


( A ) Survival of Drosophila S2 cells treated with preheating , 100 µM NAT , and 100 µM NAT with 10 mM 2 - aminobicyclo - ( 2 , 2 , 1 ) - heptane - 2 - carboxylic acid ( BCH ) after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , those were removed by changing the medium . * P < 0 . 05 , * * P < 0 . 01 vs . PBS .

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(A) Survival of Drosophila S2 cells treated with preheating, 100 µM NAT, and 100 µM NAT with 10 mM 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, those were removed by changing the medium. *P<0.05, **P<0.01 vs. PBS.


( B ) Survival of Drosophila S2 cells pretreated with 100 µM NAT , 100 µM NAT with 30 µg / ml antimycin A , and preheating with or without 30 µg / ml antimycin A 48 h after heat stress at 42oC for 60 min ( data are means ± SEM ; n = 10 ) . After pretreatment with chemicals for 12 h , they were removed by changing the medium . * P < 0 . 05 vs . PBS .

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(B) Survival of Drosophila S2 cells pretreated with 100 µM NAT, 100 µM NAT with 30 µg/ml antimycin A, and preheating with or without 30 µg/ml antimycin A 48 h after heat stress at 42oC for 60 min (data are means ± SEM; n=10). After pretreatment with chemicals for 12 h, they were removed by changing the medium. *P<0.05 vs. PBS.


( C ) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells . Upper : representative images of time - dependent changes in MitoRed localization in mitochondria after adding NAT . Scale bar : 20 µm . Lower : relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader . Different letters above bars represent significant differences [ P < 0 . 05 ( data are means ± SEM , n = 12 ) ] .

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(C) Effects of 100 µM NAT on MitoRed localization in mitochondria of S2 cells. Upper: representative images of time-dependent changes in MitoRed localization in mitochondria after adding NAT. Scale bar: 20 µm. Lower: relative intensities of MitoRed fluorescent signals in mitochondria were quantified by microplate reader. Different letters above bars represent significant differences [P <0.05 (data are means ± SEM, n=12)].


( D ) ROS concentrations in S2 cells after adding 100 µM NAT ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 vs . zero time .

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(D) ROS concentrations in S2 cells after adding 100 µM NAT (data are means ± SEM; n=12). *P<0.05 vs. zero time.


( F ) Effects of dsRNA targeting of FoxO on NAT - dependent expression of Keap1 ( data are means ± SEM ; n = 12 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies .

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(F) Effects of dsRNA targeting of FoxO on NAT-dependent expression of Keap1 (data are means ± SEM; n=12). *P<0.05, **P<0.01 vs. without NAT. Note that NAT feeding did not increase Keap1 expression in FoxO knockdown flies.


( G ) Effect of NAT on growth of HCT116 colon cancer cells in mice ( BALB / cSlc - nu / nu , 6 wks old , female ) ( data are means ± SEM ; n = 5 ) . * P < 0 . 05 , * * P < 0 . 01 vs . without NAT . Test mice began to freely drink 5 mg / ml NAT solution for 7 d before transplantation of the tumor cells on day 0 : the average amount of NAT consumed by each mouse was about 8 . 8 mg / day ( 0 . 4 - 0 . 5 g / kg weight / day ) . HCT116 cells ( 5 . 0 x 106 cells ) were transplanted into each test mouse using a 1 ml syringe with a 27G needle .

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(G) Effect of NAT on growth of HCT116 colon cancer cells in mice (BALB/cSlc-nu/nu, 6 wks old, female) (data are means ± SEM; n=5). *P<0.05, **P<0.01 vs. without NAT. Test mice began to freely drink 5 mg/ml NAT solution for 7 d before transplantation of the tumor cells on day 0: the average amount of NAT consumed by each mouse was about 8.8 mg/day (0.4 - 0.5 g/kg weight/day). HCT116 cells (5.0 x 106 cells) were transplanted into each test mouse using a 1 ml syringe with a 27G needle.


A Levels of ULK1 in MEFs exposed to hypoxia ( 1 % O2 ) for the indicated times . actin , Western

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A Levels of ULK1 in MEFs exposed to hypoxia (1% O2) for the indicated times.actin, Western blot


B Quantification of the ratio of ULK1 to actin in ( A ) .

B Quantification of the ratio of ULK1 to actin in (A).


C Levels of ULK1 in MEFs treated with FCCP ( 20 μM ) for the indicated times .

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C Levels of ULK1 in MEFs treated with FCCP (20 μM) for the indicated times.actin


D Quantification of the ratio of ULK1 to actin in ( C ) .

D Quantification of the ratio of ULK1 to actin in (C).


E MEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 6 h and then fixed by 4 % paraformaldehyde . Cells were stained with anti ‐ TIM23 ( mouse ) and anti ‐ ULK1 ( rabbit ) primary antibodies , then Alexa Fluor ‐ 488 ‐ labeled donkey anti ‐ rabbit IgG and Alexa Fluor ‐ 555 ‐ labeled donkey anti ‐ mouse IgG secondary antibodies before analysis by immunofluorescence microscopy . Scale bar , 10 μm .

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E MEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 6 h and then fixed by 4% paraformaldehyde. Cells were stained with anti‐TIM23 (mouse) and anti‐ULK1 (rabbit) primary antibodies, then Alexa Fluor‐488‐labeled donkey anti‐rabbit IgG and Alexa Fluor‐555‐labeled donkey anti‐mouse IgG secondary antibodies before analysis by immunofluorescence microscopy. Scale bar, 10 μm.


F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia ( 1 % O2 ) for 12 h . PNS , post ‐ nuclear supernatant ; Cyto , cytosol ; Mito , mitochondria .

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F Immunoblots of subcellular fractions from control MEFs or MEFs that were exposed to hypoxia (1% O2) for 12 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP ( 20 μM ) for 6 h . PNS , post ‐ nuclear supernatant ; Cyto , cytosol ; Mito , mitochondria .

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G Immunoblots of subcellular fractions from control MEFs or MEFs that were treated with FCCP (20 μM) for 6 h. PNS, post‐nuclear supernatant; Cyto, cytosol; Mito, mitochondria.ULK1


A HeLa cells were co − transfected with FUNDC1 − Myc and FLAG − ULK1 . Cells were lysed and immunoprecipitated with anti − Myc antibody 24 h after transfection .

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A HeLa cells were co−transfected with FUNDC1−Myc and FLAG−ULK1. Cells were lysed and immunoprecipitated with anti−Myc antibody 24 h after transfection.


B FLAG − FUNDC1 was co − transfected with GFP − vector or GFP − ULK1 for 24 h in HeLa cells , and then , cell lysates were immunoprecipitated with anti − GFP antibody .

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B FLAG−FUNDC1 was co−transfected with GFP−vector or GFP−ULK1 for 24 h in HeLa cells, and then, cell lysates were immunoprecipitated with anti−GFP antibody.


CMEFs were exposed to hypoxia ( 1 % O2 ) for 12 h or FCCP ( 20 μM ) for 3 h , followed by immunoprecipitation using anti ‐ ULK1 antibody .

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CMEFs were exposed to hypoxia (1% O2) for 12 h or FCCP (20 μM) for 3 h, followed by immunoprecipitation using anti‐ULK1 antibody.


D The expression of FUNDC1 is induced by 10 ng / ml tetracycline ( Tet ) in FUNDC1 ‐ inducible HeLa cells . Cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) for 24 h . Then , the cell lysates were prepared for immunoblotting using indicated antibodies .

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D The expression of FUNDC1 is induced by 10 ng/ml tetracycline (Tet) in FUNDC1‐inducible HeLa cells. Cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N) for 24 h. Then, the cell lysates were prepared for immunoblotting using indicated antibodies.


EULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 - / - cells were transfected with FUNDC1 ‐ Myc alone , FUNDC1 ‐ Myc and HA ‐ ULK1 , or FUNDC1 ‐ Myc and HA ‐ ULK1 ( K46N ) . Cells were harvested and lysed for immunoblots24 h after transfection .

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EULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1-/- cells were transfected with FUNDC1‐Myc alone, FUNDC1‐Myc and HA‐ULK1, or FUNDC1‐Myc and HA‐ULK1 (K46N). Cells were harvested and lysed for immunoblots24 h after transfection.


F Purified GST ‐ FUNDC1 and GST ‐ FUNDC1 ( S17A ) were subjected to an in vitro kinase assay with Myc ‐ ULK1 immunoprecipitated by anti ‐ Myc antibody from Myc ‐ ULK1 ‐ transfected cells about 24 h post ‐ transfection . Phosphorylated FUNDC1 was detected by anti ‐ p ‐ S17 ‐ FUNDC1 antibody . The red asterisks mark the target bands .

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F Purified GST‐FUNDC1 and GST‐FUNDC1 (S17A) were subjected to an in vitro kinase assay with Myc‐ULK1 immunoprecipitated by anti‐Myc antibody from Myc‐ULK1‐transfected cells about 24 h post‐transfection. Phosphorylated FUNDC1 was detected by anti‐p‐S17‐FUNDC1 antibody. The red asterisks mark the target bands.


G Western blotULK1 , FUNDC1 , and the phosphorylated FUNDC1 in MEFs exposed to hypoxia ( 1 % O2 ) for the indicated times .

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G Western blotULK1, FUNDC1, and the phosphorylated FUNDC1 in MEFs exposed to hypoxia (1% O2) for the indicated times.


HWestern blot analysis of the kinetics of ULK1 , FUNDC1 , and the phosphorylated in MEFs treated with FCCP ( 20 μM ) for the indicated times .

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HWestern blot analysis of the kinetics of ULK1, FUNDC1, and the phosphorylated in MEFs treated with FCCP(20 μM) for the indicated times.actin


I , J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified . HeLa cells were co ‐ transfected with Flag ‐ ULK1 and the indicated constructs . 24 h after transfection , cells were lysed for immunoprecipitation by anti ‐ Flag antibody .

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I, J The single mutant which can abolish the ULK1 and FUNDC1 interaction was identified. HeLa cells were co‐transfected with Flag‐ULK1 and the indicated constructs. 24 h after transfection, cells were lysed for immunoprecipitation by anti‐Flag antibody.


KMEFs were transfected with FUNDC1 ‐ Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA . 36 h after transfection , cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) and then lysed for immunoblotting with the indicated antibodies .

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KMEFs were transfected with FUNDC1‐Myc and its mutants after the endogenous FUNDC1 was knocked down by siRNA. 36 h after transfection, cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1) and then lysed for immunoblotting with the indicated antibodies.


L MEFs were co ‐ transfected with FUNDC1 ‐ Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA . 24 h post ‐ transfection , cells were treated with hypoxic ( 1 % O2 ) conditions for 12 h or FCCP ( 20 μM ) for 6 h before fixed by 4 % paraformaldehyde and stained with indicated antibodies . Scale bar , 10 μm .

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L MEFs were co‐transfected with FUNDC1‐Myc or its mutants and MitoDsred after the endogenous FUNDC1 was knocked down by siRNA. 24 h post‐transfection, cells were treated with hypoxic (1% O2) conditions for 12 h or FCCP (20 μM) for 6 h before fixed by 4% paraformaldehyde and stained with indicated antibodies. Scale bar, 10 μm.


AULK1 + / + and ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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AULK1+/+ and ULK1−/− cells were cultured under hypoxic (1% O2) condition for 12 h or 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


B ULK1 + / + and ULK1 − / − cells were treated with FCCP ( 20 μM ) for the indicated times in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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B ULK1+/+ and ULK1−/− cells were treated with FCCP (20 μM) for the indicated times in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


C ULK1 + / + and ULK1 − / − cells were transfected with FUNDC1 − Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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C ULK1+/+ and ULK1−/− cells were transfected with FUNDC1−Myc for 24 h and then harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


D ULK1 − / − cells were transfected with HA ‐ ULK1 or HA ‐ ULK1 ( K46N ) , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells lysates were immunoblotted .

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D ULK1−/− cells were transfected with HA‐ULK1 or HA‐ULK1 (K46N), then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells lysates were immunoblotted.


EULK1 − / − cells were transfected with HA − ULK1 or HA − ULK1 and then treated with FCCP ( 20 μM ) for 24 h . Cells were harvested in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cell lysates were immunoblotted .

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EULK1−/− cells were transfected with HA−ULK1 or HA−ULK1 and then treated with FCCP (20 μM) for 24 h. Cells were harvested in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cell lysates were immunoblotted.


F FUNDC1 ‐ knockdown MEFs were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17A ) for the indicated times . Cell lysates were used for immunoblotting .

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F FUNDC1‐knockdown MEFs were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17A) for the indicated times. Cell lysates were used for immunoblotting.


G ULK1 + / + cells were transfected with FUNDC1 ‐ Myc , and ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cells were fixed with 4 % paraformaldehyde and stained with anti ‐ Myc ( mouse ) and anti ‐ LC3 ( rabbit ) primary antibodies for immunofluorescence microscopy . Scale bar , 10 μm .

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G ULK1+/+ cells were transfected with FUNDC1‐Myc, and ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D). 24 h after transfection, cells were fixed with 4% paraformaldehyde and stained with anti‐Myc (mouse) and anti‐LC3 (rabbit) primary antibodies for immunofluorescence microscopy. Scale bar, 10 μm.


H ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were immunoblotted .

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H ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were immunoblotted.


I ULK1 − / − cells were transfected with FUNDC1 ‐ Myc or FUNDC1 - Myc ( S17D ) for 24 h in the absence or presence of 50 nm bafilomycin A1 ( BAF1 ) for an additional 6 h before harvesting . Cell lysates were detected with the citrate synthase activity kit .

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I ULK1−/− cells were transfected with FUNDC1‐Myc or FUNDC1-Myc (S17D) for 24 h in the absence or presence of 50 nm bafilomycin A1 (BAF1) for an additional 6 h before harvesting. Cell lysates were detected with the citrate synthase activity kit.


J HeLa cells were transfected with FUNDC1 ‐ Myc , FUNDC1 - Myc ( S17A ) , or FUNDC1 - Myc ( S17D ) . 24 h after transfection , cell lysates were immunoprecipitated with anti ‐ LC3 antibody .

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J HeLa cells were transfected with FUNDC1‐Myc, FUNDC1-Myc (S17A), or FUNDC1-Myc (S17D). 24 h after transfection, cell lysates were immunoprecipitated with anti‐LC3 antibody.


A ULK1 + / + or ULK1 − / − cells were cultured under hypoxic ( 1 % O2 ) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 , and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc , then cultured under hypoxic ( 1 % O2 ) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays .

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A ULK1+/+ or ULK1−/− cells were cultured under hypoxic (1% O2) or normoxic conditions in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1, and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc, then cultured under hypoxic (1% O2) conditions for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


B ULK1 + / + or ULK1 − / − cells were treated with or without FCCP ( 20 μM ) in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . ULK1 − / − cells were transfected with HA ‐ ULK1 and ULK1 − / − FUNDC1 ‐ KD cells were transfected with HA ‐ ULK1 and FUNDC1 ‐ Myc and then treated with FCCP ( 20 μM ) for 24 h in the absence or presence of 50 nM bafilomycin A1 ( BAF1 ) . Cells were harvested and lysed for immunoblotting assays .

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B ULK1+/+ or ULK1−/− cells were treated with or without FCCP (20 μM) in the absence or presence of 50 nM bafilomycin A1 (BAF1). ULK1−/− cells were transfected with HA‐ULK1 and ULK1−/− FUNDC1‐KD cells were transfected with HA‐ULK1 and FUNDC1‐Myc and then treated with FCCP (20 μM) for 24 h in the absence or presence of 50 nM bafilomycin A1 (BAF1). Cells were harvested and lysed for immunoblotting assays.


C ULK1 + / + , ULK1 − / − FUNDC1 ‐ KD , or ULK1 − / − FUNDC1 ‐ KD cells rescued by introduction of HA ‐ ULK1 and FUNDC1 ‐ Myc were cultured under hypoxic ( 1 % O2 ) conditions for 24 h or with FCCP ( 20 μM ) for 24 h . Samples were analyzed by electron microscopy . Scale bar , 2 μm . The yellow asterisks mark mitochondria . The red arrows indicate double ‐ membraned autophagic structures . The yellow arrow denotes autolysosomes .

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C ULK1+/+, ULK1−/− FUNDC1‐KD, or ULK1−/− FUNDC1‐KD cells rescued by introduction of HA‐ULK1 and FUNDC1‐Myc were cultured under hypoxic (1% O2) conditions for 24 h or with FCCP (20 μM) for 24 h. Samples were analyzed by electron microscopy. Scale bar, 2 μm. The yellow asterisks mark mitochondria. The red arrows indicate double‐membraned autophagic structures. The yellow arrow denotes autolysosomes.


D - E The mitochondrial coverage and mitochondrial − containing AVs shown in ( C ) were quantified . Recon , reconstitution .

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D-E The mitochondrial coverage and mitochondrial−containing AVs shown in (C) were quantified. Recon, reconstitution.


B . Representative immunoblot of transfected missense ROS1 TKD mutations in transfected HEK - 293A lysates .

B. Representative immunoblot of transfected missense ROS1 TKD mutations in transfected HEK-293A lysates.


C . Densitometry analysis of immunoblots ( N = 3 , biological replicates ) as represented in ( B ) . Fold - change in relative ROS1 phosphorylation is calculated by first calculating the ratio of phospho - ROS1 to total ROS1 protein for each variant and then normalizing to ROS1WT . One - way ANOVA and Sidak ' s test were used to control for multiple comparisons in C Error bars in figure represent mean ± SEM .

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C. Densitometry analysis of immunoblots (N = 3, biological replicates) as represented in (B). Fold-change in relative ROS1 phosphorylation is calculated by first calculating the ratio of phospho-ROS1 to total ROS1 protein for each variant and then normalizing to ROS1WT. One-way ANOVA and Sidak's test were used to control for multiple comparisons in C Error bars in figure represent mean ± SEM.


D . Representative immunoblot of ROS1 D2113 position substitutions ( E , G , Y , and Q as indicated ) in HEK - 293A cells . E . Densitometry of immunoblots ( N = 3 , biological replicates ) as represented in ( D ) . pROS1 - phospho - ROS1 , tROS1 - total - ROS1 . One - way ANOVA and Sidak ' s test were used to control for multiple comparisons in E . Error bars in figure represent mean ± SEM .

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D. Representative immunoblot of ROS1 D2113 position substitutions (E, G, Y, and Q as indicated) in HEK-293A cells. E. Densitometry of immunoblots (N = 3, biological replicates) as represented in (D). pROS1- phospho-ROS1, tROS1- total-ROS1. One-way ANOVA and Sidak's test were used to control for multiple comparisons in E. Error bars in figure represent mean ± SEM.


A . Immunoblot analysis of phospho - ROS1 and total ROS1 protein expression of NIH - 3T3 cells stably transduced with empty vector ( EV ) or indicated ROS1 TKD

A. Immunoblot analysis of phospho-ROS1 and total ROS1 protein expression of NIH-3T3 cells stably transduced with empty vector (EV) or indicated ROS1 TKD mutants


C . Soft - agar assay of NIH - 3T3 ROS1D2113N compared with NIH - 3T3 SLC - ROS1 fusion cells ( positive control ) or ROS1K1980E ( kinase - dead , negative control ) . + / - lorlatinib treated samples were compared for significance using two - way RM ANOVA ( matching across rows , N = 3 , biological replicates ) with Šídák ' s multiple comparisons test ( P values are indicated in figure )

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C. Soft-agar assay of NIH-3T3 ROS1D2113N compared with NIH-3T3 SLC-ROS1 fusion cells (positive control) or ROS1K1980E (kinase-dead, negative control). +/- lorlatinib treated samples were compared for significance using two-way RM ANOVA (matching across rows, N = 3, biological replicates) with Šídák's multiple comparisons test (P values are indicated in figure)


D . Immunoblot analysis of phospho - ROS1 and total ROS1 protein expression in MCF10A cells stably transduced with indicated ROS1

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D. Immunoblot analysis of phospho-ROS1 and total ROS1 protein expression in MCF10A cells stably transduced with indicated ROS1 variants


Cell proliferation data showing effects of treatment with DMSO ( Vehicle ) , crizotinib ( 250nM ) , lorlatinib ( 250 nM ) , and NVL - 520 ( 250 nM ) in MCF10A ROS1 wildtype ( WT ) ( G ) , D2113N ( H ) , D2113G ( I ) and SLC34A2 - ROS1 ( SLC - ROS1 ) ( J ) as indicated . Ordinary One - way ANOVA with Dunnett ' s multiple comparisons test was used to determine significant differences . N = 6 wells , biological replicates ( H - I ) . Error bars in figure represent mean ± SEM .

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Cell proliferation data showing effects of treatment with DMSO (Vehicle), crizotinib (250nM), lorlatinib (250 nM), and NVL-520 (250 nM) in MCF10A ROS1 wildtype (WT) (G), D2113N (H), D2113G (I) and SLC34A2-ROS1 (SLC-ROS1) (J) as indicated. Ordinary One-way ANOVA with Dunnett's multiple comparisons test was used to determine significant differences. N = 6 wells, biological replicates (H-I). Error bars in figure represent mean ± SEM.


A - B . Volcano plots of statistical discoveries from IMAC - enriched ( A ) and phospho - tyrosine - enriched ( B ) phosphoproteomic spectral counts comparing ROS1D2113N data to ROS1WT . Select phosphosites linked to indicated cellular functions are annotated within the graph . Unpaired t tests of log2 transformed counts from ROS1WT and ROS1D2113N cells were conducted using Graphpad Prism with variance assumption of individual variance for each role ( N = 3 , biological replicates ) . Multiple Comparison tests used False Discovery Rate ( FDR ) with two - stage step - up ( Benjamini , Krieger , and Yekutieli ) with desired FDR of 5 % . X axis features log2 - transformed ratio of normalized intensities of ROS1D2113N relative to ROS1WT while y axis features corresponding log10 ( q ) values .

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A-B. Volcano plots of statistical discoveries from IMAC-enriched (A) and phospho-tyrosine-enriched (B) phosphoproteomic spectral counts comparing ROS1D2113N data to ROS1WT. Select phosphosites linked to indicated cellular functions are annotated within the graph. Unpaired t tests of log2 transformed counts from ROS1WT and ROS1D2113N cells were conducted using Graphpad Prism with variance assumption of individual variance for each role (N=3, biological replicates). Multiple Comparison tests used False Discovery Rate (FDR) with two-stage step-up (Benjamini, Krieger, and Yekutieli) with desired FDR of 5%. X axis features log2-transformed ratio of normalized intensities of ROS1D2113N relative to ROS1WT while y axis features corresponding log10(q) values.


A . Immunoblotting was performed on cell lysates generated via transient transfection of the following plasmids : pCX4 ROS1 wildtype ( WT ) , ROS1D2113G ( D2113G ) , ROS1D2113N ( D2113N ) and the ROS1 fusions , CD74 - ROS1 , EZR - ROS1 and SLC34A2 - ROS1 ( SLC - ROS1 ) . The targets interrogated are indicated in the labels on the left side of the image panels and the catalog numbers as well as dilution for antibodies are reported in Methods . B . Immunoblot densitometry graphs to quantitatively compare activation of ROS1 , SHP2 , ERK1 / 2 and STAT3 ( N = 2 , biological replicates ) . Fusions are listed only by fusion partner : CD74 - ROS1 ( CD74 ) , EZR - ROS1 ( EZR ) , SLC34A2 - ROS1 ( SLC ) .

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A. Immunoblotting was performed on cell lysates generated via transient transfection of the following plasmids: pCX4 ROS1 wildtype (WT), ROS1D2113G (D2113G), ROS1D2113N (D2113N) and the ROS1 fusions, CD74-ROS1, EZR-ROS1 and SLC34A2-ROS1 (SLC-ROS1). The targets interrogated are indicated in the labels on the left side of the image panels and the catalog numbers as well as dilution for antibodies are reported in Methods. B. Immunoblot densitometry graphs to quantitatively compare activation of ROS1, SHP2, ERK1/2 and STAT3 (N = 2, biological replicates). Fusions are listed only by fusion partner: CD74-ROS1 (CD74), EZR-ROS1 (EZR), SLC34A2-ROS1 (SLC).


C . Immunoblotting was performed on cell lysates generated from stable HEK293A ROS1D2113N , HEK293A CD74 - ROS1 and NIH ROS1D2113N and NIH3T3 CD74 - ROS1 after treatment with ROS1 TKI . HEK293A stable cell lines were treated with DMSO , 100 nM entrectinib and 100 nM lorlatinib , and NIH3T3 stable cell lines were treated with 25 nM and 100 nM lorlatinib . The targets interrogated are indicated in the labels on the left side of the image panels in HEK293 images and on the right side of image panels in NIH3T3 images . D . Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1 , SHP2 , ERK1 / 2 and STAT3 and AKT1 phosphorylation from two independent replicate experiments . Pixel density of phosphorylated protein signal was divded by total protein signal , and this ratio was internally normalized to DMSO ( Vehicle ) treated cell ratio . Note that only the 100 nM lorlatinib condition that was common to the HEK293A and NIH3T3 experiments is depicted in graphs .

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C. Immunoblotting was performed on cell lysates generated from stable HEK293A ROS1D2113N, HEK293A CD74-ROS1 and NIH ROS1D2113N and NIH3T3 CD74-ROS1 after treatment with ROS1 TKI. HEK293A stable cell lines were treated with DMSO, 100 nM entrectinib and 100 nM lorlatinib, and NIH3T3 stable cell lines were treated with 25 nM and 100 nM lorlatinib. The targets interrogated are indicated in the labels on the left side of the image panels in HEK293 images and on the right side of image panels in NIH3T3 images. D. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2, ERK1/2 and STAT3 and AKT1 phosphorylation from two independent replicate experiments. Pixel density of phosphorylated protein signal was divded by total protein signal, and this ratio was internally normalized to DMSO (Vehicle) treated cell ratio. Note that only the 100 nM lorlatinib condition that was common to the HEK293A and NIH3T3 experiments is depicted in graphs.


A - C . Cell proliferation effects after treatment with SHP2 inhibitors ( TNO155 , RMC4550 ) , MEK1 / 2 inhibitors ( selumetinib , trametinib ) , and JNK1 / 2 / 3 inhibitor , SP600125 as indicated ( N = 6 , biological replicates ) .

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A-C. Cell proliferation effects after treatment with SHP2 inhibitors (TNO155, RMC4550), MEK1/2 inhibitors (selumetinib, trametinib), and JNK1/2/3 inhibitor, SP600125 as indicated (N = 6, biological replicates).


D - F . Cell proliferation effects after treatment with AKT1 / 2 / 3 inhibitor afuresertib , and MTOR1 inhibitor , everolimus , as indicated ( N = 6 , biological replicates ) .

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D-F. Cell proliferation effects after treatment with AKT1/2/3 inhibitor afuresertib, and MTOR1 inhibitor, everolimus, as indicated (N = 6, biological replicates).


G - I . Cell proliferation effects after treatment with JAK1 / 2 inhibitor ruxolitinib , and TGFBR1 inhibitor galunisertib ( N = 6 , biological replicates ) . All inhibitors were used at 250 nM final concentration , with the exception of galunisertib , which was used at 500 nM final concentration . Note that the DMSO data are the same in for each cell line when compared to different class of inhibitors . Error bars in figure represent mean ± SEM .

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G-I. Cell proliferation effects after treatment with JAK1/2 inhibitor ruxolitinib, and TGFBR1 inhibitor galunisertib (N = 6, biological replicates). All inhibitors were used at 250 nM final concentration, with the exception of galunisertib, which was used at 500 nM final concentration. Note that the DMSO data are the same in for each cell line when compared to different class of inhibitors. Error bars in figure represent mean ± SEM.


B . Kaplan - Meier survival curve of mice described in ( A ) . Comparison of survival curves using Log - rank ( Mantel - Cox ) test was used to compare difference between EV ( N = 5 ) and D2113N ( N = 5 ) , and EV and SLC34A2 - ROS1 ( N = 4 ) ; significance is indicated as P values within graph .

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B. Kaplan-Meier survival curve of mice described in (A). Comparison of survival curves using Log-rank (Mantel-Cox) test was used to compare difference between EV (N = 5) and D2113N (N = 5), and EV and SLC34A2-ROS1 (N = 4); significance is indicated as P values within graph.


F . Kaplan - Meier survival curve of mice described in ( C ) .

F. Kaplan-Meier survival curve of mice described in (C).


G . Immunoblot analysis of the phosphorylated ( p ) and total ( t ) proteins from NIH - 3T3 ROS1D2113N P1 cell lysates prepared from cells treated with vehicle ( DMSO ) or 10 , 100 , or 500 nM of crizotinib , entrectinib , or lorlatinib for 4 hours .

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G. Immunoblot analysis of the phosphorylated (p) and total (t) proteins from NIH-3T3 ROS1D2113N P1 cell lysates prepared from cells treated with vehicle (DMSO) or 10, 100, or 500 nM of crizotinib, entrectinib, or lorlatinib for 4 hours.


H - I . Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1 , SHP2 and ERK1 / 2 phosphorylation ( panel H data ) after treatment with indicated inhibitors from two independent biological replicate experiments .

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H-I. Immunoblot densitometry graphs to quantitatively compare extent of inhibition ROS1, SHP2 and ERK1/2 phosphorylation (panel H data) after treatment with indicated inhibitors from two independent biological replicate experiments.


Confocal laser scanning fluorescence microscopy ( CLSM ) images showing DNA staining ( blue in the merged images ) , rDNA FISH ( green ) and NCL immunofluorescence ( red ) in untreated HeLa cells or in cells treated with α ‐ amanitin ( 50 μg / ml ) , AMD ( 50 ng / ml ) or the Pol III inhibitor ML ‐ 60218 ( 200 μM ) for 5 h .

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Confocal laser scanning fluorescence microscopy (CLSM) images showing DNA staining (blue in the merged images), rDNA FISH (green) and NCL immunofluorescence (red) in untreated HeLa cells or in cells treated with α‐amanitin (50 μg/ml), AMD (50 ng/ml) or the Pol III inhibitor ML‐60218 (200 μM) for 5 h.


CLSM images of propidium iodide ‐ stained RNA after microinjection of buffer or nucleolar RNA into α ‐ amanitin ‐ treated HeLa cells . Nucleoli were visualized by immunofluorescence of nucleophosmin ( NPM ) . The graph represents the average number of nucleolar domains ( ± 95 % CI ) based on the analysis of 90 , 87 , 80 and 86 cells , respectively . * * P ‐ value 0 . 01 , t ‐ test .

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CLSM images of propidium iodide‐stained RNA after microinjection of buffer or nucleolar RNA into α‐amanitin‐treated HeLa cells. Nucleoli were visualized by immunofluorescence of nucleophosmin (NPM). The graph represents the average number of nucleolar domains (± 95% CI) based on the analysis of 90, 87, 80 and 86 cells, respectively. **P‐value 0.01, t‐test.


CLSM images showing nucleolar co ‐ localization of NCL ( immunofluorescence ) with aluRNA ( RNA FISH ) but not with L1 ‐ repeat RNA . Cells were pre ‐ treated in situ with RNase A or an RNase inhibitor . Nuclei were counterstained with DAPI . The signal intensity of nucleolar aluRNA was two ‐ fold higher compared to nucleoplasmic signal ( n = 92 , P ‐ value 0 . 05 , t ‐ test ) .

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CLSM images showing nucleolar co‐localization of NCL (immunofluorescence) with aluRNA (RNA FISH) but not with L1‐repeat RNA. Cells were pre‐treated in situ with RNase A or an RNase inhibitor. Nuclei were counterstained with DAPI. The signal intensity of nucleolar aluRNA was two‐fold higher compared to nucleoplasmic signal (n = 92, P‐value 0.05, t‐test).


Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre ‐ treated with α ‐ amanitin ( 50 μg / ml ) for 5 h or left untreated ( control ) ( ± 95 % CI . * * P ‐ value 0 . 01 , n = 90 , 87 , 86 , 83 or 86 cells , respectively ) . Representative CLSM images of propidium iodide ‐ stained RNA are shown on the right side .

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Graphs representing the average number of nucleolar bodies after microinjection of in vitro transcribed RNA into HeLa cells that were pre‐treated with α‐amanitin (50 μg/ml) for 5 h or left untreated (control) (± 95% CI. **P‐value 0.01, n = 90, 87, 86, 83 or 86 cells, respectively). Representative CLSM images of propidium iodide‐stained RNA are shown on the right side.


CLSMimages showing NPM ( immunofluorescence , red ) in HeLacells transfected with control ASO , aluRNAASO , or treated with transfection reagent only ( mock ) . Nuclei were counterstained with DAPI ( blue ) . A fraction of 80 ± 8 % of the cells treated with aluRNAASO ( n = 143 ) showed irregular nucleoli or dispersed nucleoli ( 53 % of the cells ) . Only 26 ± 9 % of the cells treated with control ASO presented abnormal nucleoli , with none of them showing a dispersion of nucleolar domains ( n = 123 ) , P ‐ value 0 . 01 , t ‐ test .

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CLSMimages showing NPM (immunofluorescence, red) in HeLacells transfected with control ASO, aluRNAASO, or treated with transfection reagent only (mock). Nuclei were counterstained with DAPI (blue). A fraction of 80 ± 8% of the cells treated with aluRNAASO (n = 143) showed irregular nucleoli or dispersed nucleoli (53% of the cells). Only 26 ± 9% of the cells treated with control ASO presented abnormal nucleoli, with none of them showing a dispersion of nucleolar domains (n = 123), P‐value 0.01, t‐test.


CLSM images showing NCL localization ( immunofluorescence ) , aluRNA ( RNA FISH ) and DNA ( DAPI ) . Transfection of HeLa cells was done as in ( A ) .

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CLSM images showing NCL localization (immunofluorescence), aluRNA (RNA FISH) and DNA (DAPI). Transfection of HeLa cells was done as in (A).


HeLa cells were transfected as in ( A ) , and nascent RNAs were visualized by pulse labeling with ethynyl uridine ( EU ) for 30 min . Nuclei were counterstained with DAPI ( DNA ) .

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HeLa cells were transfected as in (A), and nascent RNAs were visualized by pulse labeling with ethynyl uridine (EU) for 30 min. Nuclei were counterstained with DAPI (DNA).


Levels of 47Spre ‐ rRNA in mock ‐ , control ASO ‐ and aluRNAASO ‐ treated HeLacells . Top : Northern blot . Center : Agarose gel electrophoretic analysis of RNA . Bottom panel : Quantification from RT - qPCR levels of 47Spre ‐ rRNA normalized to 18SrRNA . Error bars represent the standard deviation ( n = 6 ) . * * P ‐ value 0 . 01 , t ‐ test .

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Levels of 47Spre‐rRNA in mock‐, control ASO‐ and aluRNAASO‐treated HeLacells. Top: Northern blot. Center: Agarose gel electrophoretic analysis of RNA. Bottom panel: Quantification from RT-qPCR levels of 47Spre‐rRNA normalized to 18SrRNA. Error bars represent the standard deviation (n = 6). **P‐value 0.01, t‐test.


HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1 ‐ repeat RNA and GFP from a separate promoter . Transfected cells were identified by GFP fluorescence , and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM . The graphs show that the volume of nucleoli is increased by 30 % in the presence of ectopic aluRNA ( ± 95 % CI , n = 20 ) . * * P ‐ value 0 . 01 , t ‐ test . Scale bar , 10 μm .

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HeLa cells were transfected with an empty vector or plasmids expressing aluRNA or L1‐repeat RNA and GFP from a separate promoter. Transfected cells were identified by GFP fluorescence, and nucleoli volumes were evaluated based on the immunofluorescence signal of the nucleolar marker NPM. The graphs show that the volume of nucleoli is increased by 30% in the presence of ectopic aluRNA (± 95% CI, n = 20). **P‐value 0.01, t‐test. Scale bar, 10 μm.


CLSM live ‐ cell imaging of RFP ‐ NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h . The arrows indicate nucleolar domains displaying time ‐ dependent increase in the nucleolar size or fusion of two domains into one . Scale bars , 10 μm .

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CLSM live‐cell imaging of RFP‐NCL in cells transfected with a plasmid expressing aluRNA and imaged for 10 h. The arrows indicate nucleolar domains displaying time‐dependent increase in the nucleolar size or fusion of two domains into one. Scale bars, 10 μm.


HeLa cells were transfected as in ( A ) and incubated for 24 h . Nascent RNA was pulse ‐ labeled for 30 min , and nucleoli were visualized by immunofluorescence of NPM . Scale bars , 10 μm .

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HeLa cells were transfected as in (A) and incubated for 24 h. Nascent RNA was pulse‐labeled for 30 min, and nucleoli were visualized by immunofluorescence of NPM. Scale bars, 10 μm.


Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27 % increase in the presence of ectopic aluRNA ( ± 95 % CI , n = 30 ) . * * P ‐ value 0 . 01 , * P ‐ value = 0 . 01 , t ‐ test .

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Graphs represent the intensity of nucleolar fluorescence signals as percentage of the intensity of nuclear signals and reveal a 27% increase in the presence of ectopic aluRNA (± 95% CI, n = 30). **P‐value 0.01, *P‐value = 0.01, t‐test.


CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α ‐ amanitin to inhibit Pol II transcription show the dispersion of nucleoli , as previously also imaged via RNAstaining ( Caudron ‐ Herger et al , ) .

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CLSMimages of UBFimmunofluorescence in mouseNIH 3T3cells treated for 5 h with α‐amanitin to inhibit Pol II transcription show the dispersion of nucleoli, as previously also imaged via RNAstaining (Caudron‐Herger et al, ).


CLSMimages showing the localization of NPM ( immunofluorescence , red ) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI ( blue ) .

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CLSMimages showing the localization of NPM (immunofluorescence, red) in mouse NIH 3T3 cells treated with ASO as indicated and counterstained with DAPI (blue).


CLSM images of MS2 ‐ tagged aluRNA or MS2 ‐ tagged control RNA ( a fragment from the transcript of the CDV3 gene ) . MS2 ‐ loop ‐ containing RNAs were visualized by RNA FISH using MS2 ‐ loop ‐ specific FISH probes ( green ) ; nucleoli were visualized by immunofluorescence of NCL ( red ) .

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CLSM images of MS2‐tagged aluRNA or MS2‐tagged control RNA (a fragment from the transcript of the CDV3 gene). MS2‐loop‐containing RNAs were visualized by RNA FISH using MS2‐loop‐specific FISH probes (green); nucleoli were visualized by immunofluorescence of NCL (red).


The top panel depicts the experimental approach used to tether MS2 ‐ loop ‐ containing RNAs to lacO arrays via a GFP ‐ tagged LacI ‐ MS2 coat fusion protein . The lacO arrays were stably integrated in the genome of U2OS cells . In the bottom panel , CLSM images of U2OS cells are shown that were transfected with MS2 ‐ aluRNAR . They reveal the localization of LacI ‐ GFP ‐ MS2 and NCL ( immunofluorescence , red ) . The arrow indicates the lacO array , which is associated with a nucleolar domain .

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The top panel depicts the experimental approach used to tether MS2‐loop‐containing RNAs to lacO arrays via a GFP‐tagged LacI‐MS2 coat fusion protein. The lacO arrays were stably integrated in the genome of U2OS cells. In the bottom panel, CLSM images of U2OS cells are shown that were transfected with MS2‐aluRNAR. They reveal the localization of LacI‐GFP‐MS2 and NCL (immunofluorescence, red). The arrow indicates the lacO array, which is associated with a nucleolar domain.


MS2 ‐ loop ‐ containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line . The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli ( ± 95 % CI ) and is plotted in the bar chart . Calculations are based on analysis of more than 100 cells . * * P ‐ value 0 . 01 , t ‐ test , from the analysis of two independent biological replicates . As controls , transcripts of the MS2 loops only , the MS2 ‐ CDV3 , MS2 ‐ RepA , MS2 ‐ STARD7 and MS2 ‐ CORO1C RNAs were used . The inset shows a CLSM image with MS2 ‐ GFP ‐ LacI ( green ) and NPM ( red , immunofluorescence ) after recruitment of MS2 ‐ aluRNA .

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MS2‐loop‐containing forward aluRNA or aluRNAL or aluRNAR were recruited to the single stably integrated lacO array in the U2OS F4IIB8 cell line. The propensity for nucleolar localization was evaluated as the average number of lacO arrays with tethered RNA detected in nucleoli (± 95% CI) and is plotted in the bar chart. Calculations are based on analysis of more than 100 cells. **P‐value 0.01, t‐test, from the analysis of two independent biological replicates. As controls, transcripts of the MS2 loops only, the MS2‐CDV3, MS2‐RepA, MS2‐STARD7 and MS2‐CORO1C RNAs were used. The inset shows a CLSM image with MS2‐GFP‐LacI (green) and NPM (red, immunofluorescence) after recruitment of MS2‐aluRNA.


Top : Scheme illustrating the experimental approach used to tether GFP ‐ tagged LacI fusion proteins via a GFP ‐ binding protein ( GBP ) to lacO arrays . The lacO arrays are stably integrated in the genome of U2OS cells . Bottom : CLSM images showing the localization of GBP ‐ LacI ‐ RFP , GFP ‐ NCL , GFP ‐ NPM or GFP ‐ TIP5 ‐ RBD fusion proteins relative to aluRNA that was visualized by RNA FISH . The insets contain an enlarged image of one of the lacO loci .

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Top: Scheme illustrating the experimental approach used to tether GFP‐tagged LacI fusion proteins via a GFP‐binding protein (GBP) to lacO arrays. The lacO arrays are stably integrated in the genome of U2OS cells. Bottom: CLSM images showing the localization of GBP‐LacI‐RFP, GFP‐NCL, GFP‐NPM or GFP‐TIP5‐RBD fusion proteins relative to aluRNA that was visualized by RNA FISH. The insets contain an enlarged image of one of the lacO loci.


CLSM images showing GBP ‐ LacI ‐ RFP and GFP ‐ NCL ( green ) recruited to lacO arrays as indicated by arrows . Immunofluorescence of endogenous NPM ( red ) marks nucleoli . The inset shows decondensation of lacO arrays in the nucleolus . The graph at the bottom depicts the average number of lacO loci tethered to nucleoli ( ± 95 % CI ) by the indicated proteins ( n = 72 ) . NCL ‐ ∆ RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding . * * P ‐ value 0 . 01 , t ‐ test .

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CLSM images showing GBP‐LacI‐RFP and GFP‐NCL (green) recruited to lacO arrays as indicated by arrows. Immunofluorescence of endogenous NPM (red) marks nucleoli. The inset shows decondensation of lacO arrays in the nucleolus. The graph at the bottom depicts the average number of lacO loci tethered to nucleoli (± 95% CI) by the indicated proteins (n = 72). NCL‐∆RNA is an NCL deletion mutant lacking the RBD and GAR domains required for RNA binding. **P‐value 0.01, t‐test.


CLSM images of UBF ( immunofluorescence , green ) or rDNA ( DNA FISH , green ) and DNA ( DAPI , blue ) after knockdown of NCL and NPM by siRNA in HeLa cells .

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CLSM images of UBF (immunofluorescence, green) or rDNA (DNA FISH, green) and DNA (DAPI, blue) after knockdown of NCL and NPM by siRNA in HeLa cells.


V . cholerae wild - type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase ( OD600 of 1 . 5 ) and induced with L - arabinose ( 0 . 2 % final conc . ) . Expression of MicV and VrrA was monitored on Northern blots . 5S rRNA served as loading control .

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V. cholerae wild-type and ΔrpoE strains carrying the indicated plasmids were grown to early stationary phase (OD600 of 1.5) and induced with L-arabinose (0.2% final conc.). Expression of MicV and VrrA was monitored on Northern blots. 5S rRNA served as loading control.


V . cholerae wild - type , ΔvchM and ΔvchM ΔrpoE strains harboring PmicV : : mKate2 plasmids were grown in M9 medium . Samples were collected during various growth phases and analyzed for fluorescence .

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V. cholerae wild-type, ΔvchM and ΔvchM ΔrpoE strains harboring PmicV::mKate2 plasmids were grown in M9 medium. Samples were collected during various growth phases and analyzed for fluorescence.


V . cholerae wild - type and ΔrpoE strains carrying PmicV : : mKate2 plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PmicV::mKate2 plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type and ΔrpoE strains carrying PvrrA : : mKate2 ( E ) plasmids were cultivated in LB to exponential phase ( OD600 of 0 . 4 ) and treated with ethanol ( 3 . 5 % final conc . ) , or water . Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock - treated samples were set to 1 .

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V. cholerae wild-type and ΔrpoE strains carrying PvrrA::mKate2 (E) plasmids were cultivated in LB to exponential phase (OD600 of 0.4) and treated with ethanol (3.5% final conc.), or water. Fluorescence was determined 180 min after ethanol treatment and mKate2 levels of the mock-treated samples were set to 1.


V . cholerae wild - type , ΔmicV , ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0 . 2 and treated with ethanol ( 3 . 5 % final conc . ) . After 5h of treatment , serial dilutions were prepared , recovered on agar plates and CFU / ml were determined .

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V. cholerae wild-type, ΔmicV, ΔvrrA or ΔvrrA ΔmicV strains were grown in LB to OD600 of 0.2 and treated with ethanol (3.5% final conc.). After 5h of treatment, serial dilutions were prepared, recovered on agar plates and CFU / ml were determined.